Nanopatterned Polymer Coatings for Marine Antifouling Applications

Nanopatterned Polymer Coatings for Marine Antifouling Applications

by

Claudia Madalina Grozea

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Chemistry University of Toronto

© Copyright by Claudia Madalina Grozea 2012

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Nanopatterned Polymer Coatings for Marine Antifouling

Applications

Claudia Madalina Grozea

Doctor of Philosophy

Department of Chemistry University of Toronto

2012

Abstract

Marine biofouling is the accumulation of marine species on surfaces submerged in seawater

leading to unwanted problems for man-made surfaces such as hulls of ships and aquaculture nets.

Historically, the amount of biofouling was regulated using metal based coatings whose usage

have been disused lately due to adverse toxic effects. Alternative environmentally friendly

coatings are currently avidly being pursued. Nanopatterned polymer thin films were investigated

as potential candidates for marine antifouling coatings. Polystyrene-block-poly(2-vinyl pyridine)

and polystyrene-block-poly(methyl methacrylate) diblock copolymer thin films self-assembled

using vapor solvent annealing into cylinders perpendicular to the substrate composed of poly(2-

vinyl pyridine) or poly(methyl methacrylate) respectively with diameters between 30 nm to 82

nm and center-to-center spacing between 46 nm to 113 nm in a polystyrene matrix on various

substrates such as silicon or nylon. Polystyrene-block-poly(2-vinyl pyridine) copolymers were

also mixed with the photoinitiator benzophenone and irradiated with ultraviolet light to crosslink

the polymer chains and decrease the surface hydrophobicity. In the case of polystyrene-block-

poly(methyl methacrylate), the yield of these nanopatterned films increased with the

modification of the vapor annealing method. A low temperature vapor annealing technique was

developed in which the annealing occurs at 2 °C. In another strategy, polystyrene and poly(2-

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vinyl pyridine) homopolymers were nanopatterned with alternating lines and grooves with

widths between 200 nm and 900 nm and depths between 15 nm to 100 nm using Thermal

Nanoimprint Lithography. Poly(2-vinyl pyridine) films were synthesized as brushes using

surface initiated Atom Transfer Radical Polymerization to produce robust polymer films. The

chemical and/or the topographical heterogeneity of the polymer surfaces influenced the

settlement of Ulva linza algae zoospores. Overall, the incorporation of nanoscale features

enhanced the antifouling properties of the samples. Further exploration of these types of

coatings is highly encouraged.

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Acknowledgments

First and foremost, I would like to thank my thesis supervisor Prof. Gilbert C. Walker. Without

him none of this would be possible. A brilliant mentor and supervisor, his incredible knowledge

and insight, approach to science, and concern for my success have guided me during the most

difficult times in my academic career.

I had a great graduate experience as part of the Walker lab, both professionally and outside the

lab, whether at picnics or at the camping trip… Thus, I would like to thank Adrienne, Melissa,

Christina, Isaac, Weiqing, Shell, James, Ruby, Nikhil and Mandy. In particular, I would like to

thank Nikhil for his mechanical properties measurements using nanoindentation, as well as all of

his help, advice and discussions over the years. Isaac was indispensible computer programming

skills in analyzing part of my data and discussions. I would also like to thank our former

postdoctoral fellows: Shan, for introducing me to diblock copolymers and AFM; and Zahra, for

her ellipsometry measurements. I gratefully acknowledge our collaborators Dr. Daniel Grozea

and Prof. Zheng-Hong Lu for the XPS measurements.

I am very thankful to our collaborators at the University of Birmingham, Prof. Maureen E.

Callow, Prof. James. A. Callow and Dr. John A. Finlay. The great experiments on algae

zoospores and diatom settlement would not have been possible without their dedication. Their

helpful discussions in planning and analyzing experiments, always including our samples for

testing in their schedule even at the last minute, taking the time to explain in detail how

everything was done and even showing me their procedure first-hand, made our collaboration a

pleasure.

I would also like to thank Prof. G. Julius Vancso at the University of Twente for welcoming me

into his laboratory. The time I spent there was very enriching and productive. I had the

opportunity to see how science is done across the ocean. Particularly, I am thankful to Dr.

Edmondo M. Benetti for sharing his expertise in polymer synthesis, and Michel Klein

Gunnewiek for sharing his workspace with me and helping me with ellipsometry and all else I

required. Many thanks to the rest of the Vancso group for making me feel welcome, sharing

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resources and helping me adjust in unfamiliar surroundings. I would like to also gratefully

acknowledge Prof. J. Huskens for the use of his Nanoimprint Press.

I would like to thank my committee members, Prof. M. Cynthia Goh and Prof. Dwight S. Seferos

for taking the time to review my thesis, and their participation in supervising my work. I would

like to gratefully acknowledge Prof. Mitchell A. Winnik for his interesting discussions and

advice over the years.

Last but not least, I would like to thank my family, in particular my husband Ivan, for their

nurture and support amidst the demanding schedule of graduate life.

Claudia M. Grozea

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Table of Contents

Abstract ........................................................................................................................................... ii

Acknowledgments .......................................................................................................................... iv

Table of Contents ........................................................................................................................... vi

List of Abbreviations ..................................................................................................................... xi

List of Tables ............................................................................................................................... xiii

List of Figures .............................................................................................................................. xvi

1 Introduction ................................................................................................................................ 1

1.1 Marine Biofouling ............................................................................................................... 1

1.1.1 Marine Biofouling Formation ................................................................................. 2

1.1.2 Factors Influencing Marine Biofouling .................................................................. 3

1.2 Antifouling Coatings ........................................................................................................... 4

1.3 Diblock Copolymers ........................................................................................................... 6

1.3.1 Diblock Copolymer Self-Assembly ........................................................................ 7

1.3.2 Diblock Copolymer Pattern Ordering ..................................................................... 9

1.4 Overview of Thesis ............................................................................................................. 9

1.5 References ......................................................................................................................... 10

2 Instrumental Techniques .......................................................................................................... 14

2.1 Atomic Force Microscopy ................................................................................................ 15

2.2 Scanning Electron Microscopy ......................................................................................... 17

2.3 X-ray Photoelectron Spectroscopy ................................................................................... 18

2.4 Contact Angle Method ...................................................................................................... 20

2.5 Ellipsometry ...................................................................................................................... 21

2.6 Fluorescence Microscopy ................................................................................................. 22

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2.7 References ......................................................................................................................... 23

3 Diblock Copolymers Polystyrene-block-poly(2-vinyl pyridine) and Polystyrene-block-poly(methyl methacrylate) Cylindrical Patterned Surfaces for Algae Zoospores Settlement Study ........................................................................................................................................ 25

3.1 Overview ........................................................................................................................... 25

3.2 Introduction ....................................................................................................................... 25

3.3 Materials and Methods ...................................................................................................... 26

3.3.1 Materials ............................................................................................................... 26

3.3.2 Characterization of Morphology in Thin Films .................................................... 28

3.3.3 Ulva Zoospore Settlement Assay .......................................................................... 29

3.4 Results and Discussion ..................................................................................................... 29

3.4.1 Ordering in Block Copolymer Thin Films ............................................................ 29

3.4.2 Block Copolymer Behavior Underwater .............................................................. 41

3.4.3 Settlement of Zoospores of the Green Alga Ulva ................................................. 47

3.5 Conclusions ....................................................................................................................... 50

3.6 References ......................................................................................................................... 50

4 Polystyrene-block-poly(2-vinyl pyridine) and Polystyrene-block-poly(methyl methacrylate) Patterned Nylon Surfaces for Inhibition of Algae Zoospores and Sporelings .. 52

4.1 Overview ........................................................................................................................... 52

4.2 Introduction ....................................................................................................................... 52


4.3.1 Materials ............................................................................................................... 54

4.3.2 Characterization of Morphology in Thin Films .................................................... 56

4.3.3 Ulva Zoospore Settlement Assay .......................................................................... 57

4.3.4 Ulva Sporelings Growth and Removal ................................................................. 57


4.4.1 Nylon Substrates ................................................................................................... 58

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4.4.2 Polymer Coated Nylon Films on Silicon .............................................................. 59

4.4.3 Polymer Coated Nylon Films on Glass Slides ...................................................... 66

4.4.4 Settlement of Algae Zoospores ............................................................................. 68

4.4.5 Growth and Removal of Algae Sporelings ........................................................... 72

4.5 Conclusions ....................................................................................................................... 74

4.6 References ......................................................................................................................... 75

5 Microdomain Orientation of Diblock Copolymer Polystyrene-block-poly(methyl methacrylate) Solvent Annealed at Low Temperatures ........................................................... 76

5.1 Overview ........................................................................................................................... 76

5.2 Introduction ....................................................................................................................... 76


5.3.1 Materials ............................................................................................................... 77

5.3.2 Characterization .................................................................................................... 78


5.4.1 Low-Temperature Solvent Annealing ................................................................... 79

5.4.2 Pattern Spacing and Dimensions .......................................................................... 93

5.5 Conclusions ....................................................................................................................... 95

5.6 References ......................................................................................................................... 96

6 Polystyrene-block-poly(2-vinyl pyridine) and Polystyrene-block-poly(methyl methacrylate) Surfaces with a Range of Nanopatterns for Inhibition of Algae Zoospores and Diatoms ............................................................................................................................. 98

6.1 Overview ........................................................................................................................... 98

6.2 Introduction ....................................................................................................................... 99

6.3 Materials and Methods .................................................................................................... 100

6.3.1 Materials ............................................................................................................. 100

6.3.2 Characterization of Morphology in Thin Films .................................................. 103

6.3.3 Ulva Zoospore Settlement Assay ........................................................................ 103

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6.3.4 Navicula Diatom Settlement Assay .................................................................... 103

6.4 Results and Discussion ................................................................................................... 104

6.4.1 PS-b-P2VP Diblock Copolymers on Si Substrates ............................................. 104

6.4.2 PS-b-PMMA Diblock Copolymers on Si Substrates .......................................... 109

6.4.3 PS-b-PMMA Diblock Copolymers on Nylon Coated on Si Substrates .............. 113

6.4.4 PS-b-PMMA Diblock Copolymers on Nylon Coated Glass Substrates ............. 115

6.4.5 Settlement of Algae Zoospores on PS-b-P2VP Diblock Copolymers ................ 120

6.4.6 Settlement of Algae Zoospores on PS-b-PMMA Diblock Copolymers ............. 123

6.4.7 Settlement of Diatoms on PS-b-PMMA Diblock Copolymers ........................... 127

6.5 Conclusions ..................................................................................................................... 130

6.6 References ....................................................................................................................... 130

7 Thermal Nanoimprint Lithography Nanopatterned Polystyrene and Surface-Initiated Polymerized Poly(2-vinyl pyridine) Surfaces for Algae Zoospores Assays .......................... 132

7.1 Overview ......................................................................................................................... 132

7.2 Introduction ..................................................................................................................... 132

7.3 Materials and Methods .................................................................................................... 135

7.3.1 Materials ............................................................................................................. 135

7.3.2 Surface-Initiated ATRP of P2VP ........................................................................ 136

7.3.3 Thermal Nanoimprint Lithography ..................................................................... 137

7.3.4 Characterization of Morphology in Thin Films .................................................. 139

7.3.5 Ulva Zoospore Settlement Assay ........................................................................ 139

7.4 Results and Discussion ................................................................................................... 140

7.4.1 PS and P2VP Polymer Films Nanoimprinting .................................................... 140

7.4.2 PS and P2VP Polymer Films Behavior Underwater ........................................... 145

7.4.3 Settlement of Algae Zoospores on PS and P2VP Nanopatterned Polymer Films ................................................................................................................... 146

7.5 Conclusions ..................................................................................................................... 149

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7.6 References ....................................................................................................................... 149

8 Conclusions and Future Directions ........................................................................................ 151

8.1 Conclusions ..................................................................................................................... 151

8.2 Future Directions ............................................................................................................ 154

8.3 References ....................................................................................................................... 156

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List of Abbreviations

AFM Atomic Force Microscopy

ATRP Atom Transfer Radical Polymerization

BP Benzophenone

CuCl Copper (I) chloride

CuBr2 Copper (II) bromide

EDTA Ethylenediaminetetraacetic acid disodium salt dihydrate

Me6TREN Tris[2-(dimethylamino)ethyl]amine

NIL Nanoimprint Lithography

P2VP Poly(2-vinyl pyridine)

PB-b-PS Polybutadiene-block-polystyrene

PI-b-PS Polyisoprene-block-polystyrene

PMMA Poly(methyl methacrylate)

PS Polystyrene

PS-b-P2VP Polystyrene-block-poly(2-vinyl pyridine)

PS-b-PB Polystyrene-block-polybutadiene

PS-b-PMMA Polystyrene-block-poly(methyl methacrylate)

P(S-r-MMA) Polystyrene-co-methyl methacrylate

P(S-r-VP) Polystyrene-co-2-vinyl pyridine

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SEM Scanning Electron Microscopy

TNIL Thermal Nanoimprint Lithography

UV Ultraviolet

XPS X-ray Photoelectron Spectroscopy

xiii

List of Tables

Table 2.1 Overview of analysis techniques and conditions used throughout this thesis.6-9 .......... 14

Table 3.1 Properties of homopolymers and a random copolymer. Mn is the number average

molecular weight and Mw is the weight average molecular weight. The unit of measure is g/mol.

PDI is the polydispersity index. .................................................................................................... 28

Table 3.2 Advancing Water Contact Angles. ............................................................................... 32

Table 3.3 XPS analysis of three different samples. ...................................................................... 38

Table 3.4 Advancing Water Contact Angles after water immersion. ........................................... 43

Table 4.1 PS-b-PMMA diblock copolymers properties. Mn is the number average molecular

weight and the unit of measure is g/mol. PDI is the polydispersity index. .................................. 55

Table 4.2 Properties of homopolymers and random copolymers. Mn is the number average

molecular weight and Mw is the weight average molecular weight. The unit of measure is g/mol.

PDI is the polydispersity index. .................................................................................................... 56

Table 4.3 Advancing water contact angles of polymers on nylon coated silicon. ........................ 61

Table 4.4 PS-b-PMMA nanopattern dimensions. ......................................................................... 63

Table 4.5 Characteristics of the PS-b-PMMA diblock copolymers. ƒPS is the volume fraction of

the PS block calculated from ƒPS = (wPS/ρPS) / ( (wPS/ρPS) + (1 - wPS)/ρPMMA) by using the

following densities for the PS and PMMA block: ρPS = 1.05 g cm-3 and ρPMMA = 1.18 g cm-3.17-18

χ is determined from χ = 0.0282 + 4.46/T as reported in literature for PS-b-PMMA. 16 The

experiments were performed at room temperature, 24 °C. ........................................................... 64

Table 4.6 PS-b-PMMA nanopattern dimensions. ......................................................................... 67



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following densities for the PS and PMMA block: ρPS = 1.05 g cm-3 and ρPMMA = 1.18 g cm-3.17-18

χ is determined from χ = 0.0282 + 4.46/T as reported in literature for PS-b-PMMA. 16 The

experiments were performed at room temperature, 24 °C. ........................................................... 68

Table 4.8 Advancing water contact angles of polymers on nylon coated glass slides. ................ 68


weight and the unit of measure is g/mol. PDI is the polydispersity index. .................................. 78

Table 5.2 Polymer-solvent interaction parameters (χP-S) calculated for different temperatures and

polymer-solvent pairs. ................................................................................................................... 84

Table 5.3 XPS analysis of samples with cylindrical and no pattern morphologies over time. ..... 86



following densities for the PS and PMMA block: ρPS = 1.05 g cm-3 and ρPMMA = 1.18 g cm-3.17, 24

χ is determined from χ = 0.0282 + 4.46/T as reported in literature for PS-b-PMMA.23 ............. 87

Table 5.5 Example of characteristics of the 130-133 PS-b-PMMA diblock copolymers when the

height threshold value yields 50 % PMMA surface coverage at 24 °C. ....................................... 90

Table 6.1 PS-b-P2VP diblock copolymers properties. Mn is the number average molecular

weight and the unit of measure is g/mol. PDI is the polydispersity index. ................................ 100


weight and the unit of measure is g/mol. PDI is the polydispersity index. ................................ 101

Table 6.3 Properties of homopolymers and random copolymers. Mn is the number average

molecular weight and Mw is the weight average molecular weight. The unit of measure is

kg/mol. PDI is the polydispersity index. .................................................................................... 102

Table 6.4 PS-b-P2VP nanopattern dimensions. .......................................................................... 107

Table 6.5 Advancing water contact angles of polymers on silicon. ........................................... 107

Table 6.6 PS-b-PMMA nanopattern dimensions. ....................................................................... 111

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Table 6.7 Advancing water contact angles of polymers on silicon. ........................................... 111

Table 6.8 Advancing water contact angles of polymers on nylon coated silicon. ...................... 114

Table 6.9 Advancing water contact angles of polymers on nylon coated glass slides. .............. 117

Table 7.1 Silicon Templates Parameters. .................................................................................... 138

Table 7.2 Advancing water contact angles of PS polymers on silicon. ...................................... 142

Table 7.3 Advancing water contact angles of P2VP polymers on silicon. ................................. 144

xvi

List of Figures

Figure 1.1 Marine biofouling of a) Aquaculture nets and b) Pier pillars. ....................................... 1

Figure 1.2 Linear block copolymer architectures a) AB diblock copolymer, b) ABA triblock

copolymer, and c) ABC triblock copolymer. .................................................................................. 6

Figure 1.3 AB type diblock copolymer which is configured in a) lamellar pattern and b)

cylindrical pattern. The A block of one copolymer chain associate with the A block of another

copolymer chain, while the B block of a copolymer chain associate with the B block of another

copolymer chain. ............................................................................................................................. 8

Figure 2.1 Atomic Force Microscope operation schematic. ......................................................... 16

Figure 2.2 AFM images of PS-b-P2VP a) Height image, and b) 3-D view of the height image. 16

Figure 2.3 Scanning Electron Microscope operation schematic. .................................................. 18

Figure 2.4 Examples of SEM images in cross-section a) Nanopatterned polystyrene, and b) Glass

prism coated with alternating metal layers. .................................................................................. 18

Figure 2.5 X-ray Photoelectron Spectroscopy operation schematic. ............................................ 19

Figure 2.6 Example of XPS spectra of PS-b-P2VP + BP UV films a) survey, and b) high

resolution C 1s. ............................................................................................................................. 20

Figure 2.7 Advancing water contact angle of a) Hydrophobic sample, and b) Hydrophilic sample.

....................................................................................................................................................... 20

Figure 2.8 Ellipsometry operation schematic. .............................................................................. 21

Figure 2.9 Epifluorescence microscopy operation schematic. ...................................................... 22

Figure 3.1 Diblock copolymers a) PS-b-P2VP and b) PS-b-PMMA, and Photoinitiator c) BP. .. 27

Figure 3.2 a) PS, and b) P2VP. ..................................................................................................... 28

xvii

Figure 3.3 AFM height images of a) solvent annealed PS-b-P2VP film, b) PS film, c) P2VP film,

and d) P(S-r-VP) film. Image sizes: 1 μm x 1 μm. Z range: 20 nm. ........................................... 31

Figure 3.4 AFM height images of a) solvent annealed PS-b-P2VP and BP film, b) solvent

annealed and UV irradiated PS-b-P2VP and BP film, and c) solvent annealed PS-b-PMMA film.

Image sizes: 1 μm x 1 μm. Z range: 20 nm. ................................................................................. 34

Figure 3.5 Oxidative photodegradation of PS in air. .................................................................... 35

Figure 3.6 Oxidative photodegradation of PS and BP in air. ........................................................ 37

Figure 3.7 XPS survey spectrum of PS-b-P2VP film after solvent annealing, PS-b-P2VP and BP

after solvent annealing, and PS-b-P2VP and BP after UV in air. ................................................. 38

Figure 3.8 High-resolution XPS of a) C 1s, b) O 1s, and c) N 1s spectra of a) PS-b-P2VP film

after solvent annealing, dotted line and b) PS-b-P2VP and BP after UV in air, solid line. .......... 39

Figure 3.9 a) Force-indentation curve of the PS-b-P2VP film. Dots are the data point, while the

solid line is the fit by a paraboloidal tip shape. b) the elastic modulus distribution for PS-b-P2VP

film, and c) the elastic modulus distribution for PS-b-P2VP and BP film after UV irradiation. . 40

Figure 3.10 Force-extension curve of a) PS-b-P2VP film, and b) PS-b-P2VP and BP film after

UV irradiation. The grey curve is the trace data, while the black curve is the retract data. ........ 41

Figure 3.11 AFM height images of solvent annealed PS-b-P2VP films a) in water after 2 hours,

Image size: 0.5 µm x 0.5 µm b) in air after 2 hours in water, c) in air after 24h hours in water,

and d) in air after 8 days in water. Image sizes: 1 µm x 1 µm. Z range: 20 nm. ........................ 42

Figure 3.12 AFM height images in water after 2 hours of a) PS, b) P2VP, and c) P(S-r-VP).

Image size: 1 µm x 1 µm. Z range: 40 nm. .................................................................................. 44

Figure 3.13 AFM height images of cross-linked PS-b-P2VP and BP films a) in water after 2

hours, b) in air after 2 hours in water, c) in air after 24 hours in water, d) in air after 8 days in

water, e) in air after 5 weeks in water, and f) in air after 3 weeks in seawater. Image size: 1 µm x

1 µm. Z range: 40 nm. .................................................................................................................. 45

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Figure 3.14 AFM height images of solvent annealed PS-b-PMMA films a) 2 hours in water, b) in

air after 2 hours in water, c) in air after 2 hours in water, and d) in air after 8 days in water.

Image size: 1 µm x 1 µm. Z range: 40 nm. .................................................................................. 46

Figure 3.15 a) The density of attached Ulva spores on polymers on silicon wafers. Each point is

the mean from 90 counts on 3 replicate slides (30 on each wafer). Bars show 95% confidence

limits. b) advancing water contact angle for polymers on silicon wafers, and c) Average grain

size analysis. ................................................................................................................................. 49

Figure 4.1 Aquaculture nylon nets a) clean, and b) after one month seawater immersion showing

marine biofouling. ......................................................................................................................... 53

Figure 4.2 Nylon 6, 6. ................................................................................................................... 54

Figure 4.3 Diblock copolymers a) PS-b-P2VP and b) PS-b-PMMA, and Photoinitiator c) BP. .. 55

Figure 4.4 a) PS, b) P2VP, and c) PMMA. ................................................................................... 56

Figure 4.5 AFM height images of a) nylon piece, Z range: 200 nm. b) nylon coated on silicon,

and c) nylon coated on glass slide. Z range: 30 nm. Image sizes: 1 μm x 1 μm. ......................... 59

Figure 4.6 AFM height images of polymer films on nylon coated silicon a) solvent annealed PS-

b-P2VP, b) solvent annealed PS-b-P2VP and BP after UV irradiation, and c) UV PS-b-P2VP and

BP after 2 hours water immersion. Image sizes: 1 μm x 1 μm. Z range: 20 nm. ....................... 60

Figure 4.7 AFM height images of solvent annealed PS-b-PMMA films on nylon coated silicon a)

105-106, b) 130-133, and c) 160-160. Image sizes: 1 μm x 1 μm. Z range: 30 nm. .................. 63

Figure 4.8 AFM height images of polymer films on nylon coated silicon a) PS, b) P2VP, c)

PMMA, d) P(S-r-2VP), and e) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm. ....... 65

Figure 4.9 AFM height images of solvent annealed PS-b-PMMA polymer films on nylon coated

glass slides a) 52-52, b) 66-63.5, c) PS, d) PMMA, and e) P(S-r-MMA). Image sizes: 1 μm x 1

μm. Z range: 30 nm. ..................................................................................................................... 67

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Figure 4.10 a) The density of attached Ulva spores on polymers on nylon coated silicon. Each

point is the mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95%

confidence limits. and b) advancing water contact angle for polymers on nylon coated silicon.. 70

Figure 4.11 The density of attached Ulva spores on polymers on nylon. Each point is the mean

from 90 counts on 3 replicate slides (30 on each wafer). Bars show 95% confidence limits. ..... 71

Figure 4.12 a) The density of attached Ulva spores on polymers on nylon coated glass slides.

Each point is the mean from 90 counts on 3 replicate slides (30 on each slide). Bars show 95%

confidence limits. b) The biomass of Ulva sporelings on polymers mounted on nylon coated

glass slides after 7 days. Each point is the mean biomass from 6 replicate slides measured using a

fluorescence plate reader (RFU; relative fluorescence unit). Bars show standard error of the

mean. and c) advancing water contact angle for polymers on nylon coated glass slide. .............. 73

Figure 4.13 Images showing a) Ulva sporelings on polymer coatings on nylon coated glass

slides. From left: Glass-Nylon, PS, PMMA, P(S-r-MMA), PS-b-PMMA 66-63.5, PS-b-PMMA

52-52, b) Effect of water pressure of 18 kPa on the polymer films. and c) Close up image of PS-

b-PMMA 66-66 and 52-52 (right) films sheared off at 26 kPa. ................................................... 74

Figure 5.1 PS-b-PMMA diblock copolymer. ................................................................................ 78

Figure 5.2 AFM height images of 130-133 PS-b-PMMA films a) after spin coating, b) annealed

at 24 °C, pattern typical for 73 % of samples, c) annealed at 24 °C, pattern typical for 6 % of the

samples, d) annealed at 24 °C, pattern typical for 21 % of the samples, e) annealed at 2 °C,

pattern typical for 38 % of the samples, and f) annealed at 2 °C, pattern typical for 62 % of

samples. Image sizes: 1 μm x 1 μm. Z range: 30 nm. In the inset in all parts is a Fourier

transform of the corresponding AFM image. ............................................................................... 80

Figure 5.3 Type of structures present after solvent annealing at 24 °C and 2 °C as a percent of

samples. ......................................................................................................................................... 82

Figure 5.4 Images of 130-133 PS-b-PMMA films a) AFM height image showing the bottom of

the polymer film. Image size: 1 μm x 1 μm. Z range: 10 nm, b) AFM height image showing the

top of the film after oxygen plasma treatment. Image size: 1 μm x 1 μm. Z range: 30 nm, c)

SEM image showing a cross-sectional view of the film interior at a 20º angle after oxygen

xx

plasma treatment and d) SEM image showing a zoom in area of cross-sectional view of the film

interior at a 20º angle after oxygen plasma treatment. .................................................................. 83

Figure 5.5 High-resolution C 1s spectra with fitted curves for a) films with a cylindrical pattern

after 2 h annealing, b) films with no surface structure after 2 h annealing, c) films with a

cylindrical pattern after 22 h annealing, and d) films with no surface structure after 22 h

annealing. ...................................................................................................................................... 85

Figure 5.6 130-133 PS-b-PMMA films a) Surface height histogram, and b) Normalized

accumulative histogram of the height topography. T – Temperature, R – Room. ....................... 89

Figure 5.7 AFM height images of PS-b-PMMA film solvent annealed at room temperature, 24ºC,

a) Cylinders perpendicular to the substrate after 2 h to 5 days of annealing, b) Cylinders both

perpendicular and parallel to the substrate after 6 days to 8 days of annealing, c) Lamellae

perpendicular to the substrate after 8.5 days of annealing, d) Lamellae both perpendicular and

parallel to the substrate after 9.5 days of annealing, and e) Lamellae parallel to the substrate after

10.5 days of annealing. Image sizes: 1 μm x 1 μm. Z range: 30 nm. ......................................... 91

Figure 5.8 AFM height image of PS-b-PMMA film solvent annealed at low temperature, 2ºC, a)

Cylinders perpendicular to the substrate after 2 h to 5 days of annealing, b) Spheres after 6.5 days

to 8.5 days of annealing, c) Spheres and lamellae perpendicular to the substrate after 9.5 days of

annealing, d) Lamella perpendicular to the substrate after 10 days of annealing, e) Lamellae both

perpendicular and parallel to the substrate after 11 days to 12.5 days of annealing, and f) Lamella

parallel to the substrate after 13 days of annealing. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

....................................................................................................................................................... 92

Figure 5.9 AFM height images of PS-b-PMMA films annealed at 2°C a) 160-160, b) 105-106, c)

66-63.5, and d) 52-52. Image sizes: 1 μm x 1 μm. Z range: 30 nm. ........................................... 94

Figure 5.10 a) Type of structures present after solvent annealing at 24 °C and 2 °C for different

PS-b-PMMA copolymer as a function of PS Mn and as a percent of samples; b) D, center-to-

center cylinder spacing, and d, cylinder diameter, as a function of PS Mn for different PS-b-

PMMA copolymers. ...................................................................................................................... 95

Figure 6.1 a) Diblock copolymer PS-b-P2VP, and b) Photoinitiator BP. ................................... 100

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Figure 6.2 a) Diblock copolymer PS-b-PMMA, and b) Nylon 6, 6. ........................................... 101

Figure 6.3 a) PS, b) P2VP, and c) PMMA. ................................................................................. 102

Figure 6.4 AFM height images of PS-b-P2VP films on silicon a) 75-21, b) 75-21 and BP after

UV irradiation and 2 hours water immersion, c) 172-42, d) 172-42 and BP after UV irradiation

and 2 hours water immersion, e) 325-92, and f) 325-92 and BP after UV irradiation and 2 hours

water immersion. Image sizes: 1 μm x 1 μm. Z range: 30 nm. ................................................. 106

Figure 6.5 AFM height images of polymer films on silicon a) PS, b) P2VP, and c) P(S-r-2VP).

Image sizes: 1 μm x 1 μm. Z range: 30 nm. ............................................................................... 108

Figure 6.6 AFM height images of solvent annealed PS-b-PMMA films on silicon a) 52-52, b) 66-

63.5, c) 105-106, d) 130-133, and e) 160-160. Image sizes: 1 μm x 1 μm. Z range: 30 nm. ... 110

Figure 6.7 AFM height images of polymer films on silicon a) PS, b) PMMA, and c) P(S-r-

MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm. ................................................................ 112

Figure 6.8 AFM height images of PS-b-PMMA films on nylon coated silicon a) 52-52, b) 66-

63.5, and c) 105-106. Image sizes: 1 μm x 1 μm. Z range: 30 nm. .......................................... 113

Figure 6.9 AFM height images of polymer films on nylon coated silicon a) Nylon, b) PS, c)

PMMA, and d) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm. ............................. 115

Figure 6.10 AFM height images of PS-b-PMMA films on nylon coated glass slides a) 52-52, b)

66-63.5, c) 105-106, d) 130-133, and e) 160-160. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

..................................................................................................................................................... 116

Figure 6.11 AFM height images of PS-b-PMMA 130-133 films on nylon coated glass slides a)

Before solvent annealing, b) Cylinders orientated parallel and perpendicular to the substrate (Cyl

+ Lam surface), c) Spheres, and d) Lamellae. Image sizes: 1 μm x 1 μm. Z range: 30 nm. .... 119

Figure 6.12 AFM height images of polymer films on nylon coated glass slides a) Nylon, b) PS, c)

PMMA, and d) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm. ............................. 120

xxii

Figure 6.13 a) The density of attached Ulva spores on polymers on silicon. Each point is the

mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95% confidence

limits. and b) advancing water contact angle for polymers on silicon. ....................................... 122

Figure 6.14 a) The density of attached Ulva spores on polymers on silicon. Each point is the

mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95% confidence

limits. and b) advancing water contact angle for polymers on silicon. ....................................... 124

Figure 6.15 a) The density of attached Ulva spores on polymers on nylon coated silicon. Each

point is the mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95%

confidence limits. and b) advancing water contact angle for polymers on nylon coated silicon.126

Figure 6.16 a) The density of attached Navicula diatoms on polymers on nylon coated glass

slides. Each point is the mean from 90 counts on 3 replicate slides (30 on each substrate). Bars

show 95% confidence limits. and b) advancing water contact angle for polymers on nylon coated

glass slides. ................................................................................................................................. 129

Figure 7.1 PS. .............................................................................................................................. 135

Figure 7.2 Surface-initiated ATRP polymerization of P2VP procedure a) immobilization of

initiator, b) brush synthesis, and c) catalyst removal. ................................................................. 136

Figure 7.3 Polymer Thermal Nanoimprint Lithography a) release layer coated silicon template

and the polymer film coated on a silicon substrate, b) imprinting at a temperature above the Tg

and high pressure, c) template demolding from imprinted polymer film. .................................. 138

Figure 7.4 AFM height images of silicon templates with dimensions a) 200 nm wide line x 200

nm wide groove x 100 nm deep, b) 300 nm wide line x 700 nm wide groove x 30 nm deep, and

c) 500 nm wide line x 900 nm wide groove x 200 nm deep. Image sizes: 5 μm x 5 μm. Z range:

100 nm. ....................................................................................................................................... 141

Figure 7.5 AFM height images of PS films a) flat unmodified surface, b) 200 nm wide line x 200

nm wide groove x 50 nm deep nanopatterned surface, c) 700 nm wide line x 300 nm wide groove

x 15 nm deep nanopatterned surface, and d) 900 nm wide line x 500 nm wide groove x 100 nm

deep nanopatterned surface. Image sizes: 5 μm x 5 μm. Z range: 50 nm. ................................ 142

xxiii

Figure 7.6 AFM height images of P2VP films a) flat unmodified surface, b) 200 nm wide line x

200 nm wide groove x 50 nm deep nanopatterned surface, c) 700 nm wide line x 300 nm wide

groove x 15 nm deep nanopatterned surface, and d) 900 nm wide line x 500 nm wide groove x

100 nm deep nanopatterned surface. Image sizes: 5 μm x 5 μm. Z range: 50 nm. ................... 144

Figure 7.7 AFM height images of nanoimprinted films of PS left side and P2VP right side with

dimensions 900 nm wide line x 500 nm wide groove x 100 nm deep in water a) and b) 10 min

immersion, c) and d) 2 h immersion. Image sizes: 5 μm x 5 μm. Z range: 50 nm. .................. 145

Figure 7.8 a) The density of attached Ulva spores on polymers on silicon. Each point is the mean

from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95% confidence limits.

and b) advancing water contact angle for polymers on silicon. .................................................. 147

Figure 7.9 Typical epifluorescence microscopy images of zoospores on polymers on silicon of

PS films left side and P2VP right side of a) and b) unmodified films, c) and d) 200 nm wide line

x 200 nm wide groove x 50 nm deep nanopattern, e) and f) 700 nm wide line x 300 nm wide

groove x 15 nm deep nanopattern, g) and h) 900 nm wide line x 500 nm wide groove x 100 nm

deep nanopattern Images width: 450 μm. .................................................................................. 148

1

1 Introduction

Part of this chapter is adapted from Grozea, C. M.; Walker, G. C. Soft Matter 2009, 5, 4088-

4100. DOI: 10.1039/B910899H. Reproduced by permission of The Royal Society of Chemistry

(RSC).

1.1 Marine Biofouling Marine biofouling has been major problem for more than 2000 years for man-made surfaces

submerged in seawater such as ships’ hulls, aquaculture nets or pier support pillars, Figure 1.1.1

Unwanted living organisms such as diatoms, algae, barnacles or tubeworms that accumulate on

the submerged surfaces cause biofouling. The effects of biofouling on ships include an increase

in surface roughness which causes a high hydrodynamic frictional resistance leading to higher

fuel consumption, up to 40 %. Additional issues include an increase in the frequency of dry-

docking, corrosion of the coating and the introduction of invasive or non-native marine species

to new environments.1–4 In the case of aquaculture farming, an increase in cleaning of

equipment due to biofouling leads to an estimated 20 % increase in the cost of fish production.5

a) b)

Figure 1.1 Marine biofouling of a) Aquaculture nets and b) Pier pillars.

2

1.1.1 Marine Biofouling Formation

More than 4000 marine species have been documented on fouled structures and many of these

can adapt to changing environments.6 Previous studies have focused on studying cell

morphology, composition of adhesive or mechanism of adhesion for various marine organisms

such as diatoms,7-11 algae12–17 and barnacles.18–23 Studies have also elucidated how marine

biofouling occurs, and four stages have been proposed.24 The first stage involves the creation of

a conditioning film within a minute of water submersion made from the settlement of organic

molecules such as polysaccharides, proteins and inorganic molecules. The formation of the

conditioning film is due to physical factors such as electrostatic interactions and van der Waals

forces. Next, within a day, bacteria and single-cell diatoms settle on the surface. Within a week,

bacteria and diatoms along with protozoa and rotifers form a microbial film which provides

protection from predators and changes in the environment as well as provides stimuli for

macrofoulers, more complex and larger species, to settle on the surface. The third stage involves

the attachment of macrofoulers such as algal spores, barnacle cyprids and marine fungi to the

surface. In the final stage, larger marine invertebrates and macroalgae settle and grow.

Biofouling arises from both physical factors such as electrostatic interaction and biochemical

reactions such as permanent adhesion. However, in practice this sequence of events holds true

for only a number of organisms.25 The presence of a biofilm is not always necessary and marine

species can settle at the same time rather than in sequence. Species such as zoospores of the alga

Ulva linza25 and cyprids of the barnacle Balanus amphitrite26 can settle on pristine surfaces,

surfaces without a biofilm layer.

Algae are one of the most common ship biofouler.27 The algal zoospore is around 11-12 µm in

length and pyriform in shape.12 The four anterior flagella are involved in the zoospore

movement through the water column. The algae are motile up to the point at which they commit

to settlement; hence, they can “choose” whether to settle on a surface. The forward part of the

zoospore ends in an apical dome or papilla of about 250 nm to 1 µm at the widest part. Small

vesicles in the range of 45 – 65 nm are found right at the top end of the apical papilla. Larger

vesicles, 150 – 300nm, are also found in this anterior region. These vesicles are believed to be

involved in the attachment process due to their location in the algae zoospores and their absence

in attached zoospores. Video microscopy has shown that a swimming algae zoospore senses a

surface by rotating on the apical papilla.28 During this phase, the zoospore can swim away from

3

the surface or release a small deposit of elastic material on the surface. If the surface is not

suitable after the zoospore released this elastic material, the zoospore can still detach and swim

away. The zoospore attaches permanently by stopping its rotation and discharging the contents

of its vesicles. The zoospore is then surrounded by an adhesive pad and it adopts a more

rounded morphology. The flagella are withdrawn inside the body and eventually a new cell wall

is produced. The same video microscopy study also revealed that attached zoospores attract

swimming zoospore to the surface resulting in adhesion and formation of zoospore groups.

1.1.2 Factors Influencing Marine Biofouling

Marine biofouling is affected by a number of factors which can depend on water conditions.1

The water temperature varies across the oceans with latitude from below zero at the poles to high

20s at the equator. In locations with high water temperatures such as tropical areas, biofouling is

heavy due to the potential of species to reproduce and grow year round.29 On the other hand, in

areas with low temperatures, biofouling is not as heavy and might only be present in the summer

months when temperatures are higher. Salinity also influences the growth rate and maximum

species size. Seawater has a high content of salts such as chloride and sodium ions with a global

average of 3.5 wt%.1 The salinity content varies with geographical position as well as depth. A

low salinity environment is unfavorable to some of the common fouling species; nonetheless,

algae can withstand this effect. Temperature and salinity are both affected by the amount of

solar radiation.

Water pollution can have either a beneficial or harmful effect on marine species.1 Contaminants

present in water may increase the nutrient supply. On the other hand, contaminants may be toxic

to the marine species or deplete the oxygen content in the area. Another factor which influences

the abundance of marine species is location in the sea. In coastal waters there are more species

present and hence heavier fouling. The interaction between the various fouling species and

themselves will affect the amount of biofouling. However, the factors mentioned above in

practical contexts may not be possible to modify. The factors that can be easily controlled and

modified are the properties of the substrate. This can be achieved by changing the surface with

various coatings.1

4

1.2 Antifouling Coatings The most effective coatings to date have been based on compounds such as tin and copper

biocides. In particular, tributyltin self-polishing copolymer paints have had widespread use.

Studies have shown that these paints are toxic to the environment, 3,30-32 for example they cause

defective growth in the shell of the oyster Crassostrea gigas.1 A larger problem is the

accumulation of these compounds in mammals and fish and the associated health problems.

Thus, regulations passed at the Antifouling Systems Convention by the International Maritime

Organization in October 2001 banned the application of tributyltin paints on ships from 2003 and

banned the presence of tributyltin paints on ships from 2008.1 In addition, the use of biocidal

paints such as copper paints is becoming more regulated, with limits on copper release. Thus,

considerable effort has gone into researching and developing alternative materials that are non-

toxic.

New materials for combating marine biofouling must have antifouling properties such as

inhibition of the settlement of marine species and/or fouling release properties such as low

adhesion of the fouling marine species. Antifouling materials deter settlement by hindering the

formation of strong surface bonds to natural marine adhesives.33 After the adhesive secreted by

the marine species has spread over the surface, adhesion occurs by one of or a combination of

chemical bonding, electrostatic interactions, mechanical interlocking or diffusion. Materials

designed for fouling release properties promote the failure of the adhesive joint that forms. The

major approaches in designing nontoxic polymer coatings involve the use of homogeneous

surfaces such as hydrophobic, hydrophilic or amphiphilic surfaces, heterogeneous surfaces such

as patterned or mixed surfaces, and 3D surfaces such as microtopographic patterned surfaces.34-35

Hydrophobic homogenous surfaces are effective in foul release. These include ω-substituted

alkanethiolates,36-37 fluorinated polymers38 and poly(dimethyl siloxane) polymers (PDMS).1,39

PDMS is widely used as a non-toxic foul release coating. PDMS surfaces have low surface

energy and low microroughness, except they are mechanically weak. Biofouling is reduced

under hydrodynamic conditions,40 however, under static and low flow, biofouling still

develops.41 On the other hand, hydrophilic surfaces such as polymers based on poly(ethylene

glycol)38, 42 are effective in preventing settlement of marine species such as algae zoospores.

Amphiphilic surfaces can be designed to have both foul release and antifouling properties, as in

5

the case of polymers with both fluorinated and poly(ethylene glycol) areas in their chain.43 In

such a system utilized by Krishnan et al, the mechanism for zoospore removal was different than

for pure hydrophobic surfaces.43 Water infiltrated between the coating and the zoospore biofilm

and caused the biofilm to detach in pieces. The zoospores have a stronger bond with each other

than with the surface.

Another approach focuses on heterogeneous surfaces, which includes patterned surfaces or

surfaces with a mixed character. These surfaces are usually designed to have both hydrophobic

and hydrophilic areas. An example of a mixed surface is one made of phase separated

hydrophobic poly(dimethyl siloxane) domains and hydrophilic quaternary ammonium salts

groups.44 A patterned surface can have alternating stripes of hydrophobic, fluorinated polymer,

and hydrophilic polymers, poly(ethylene glycol).45 Zoospores settlement was affected by stripe

size on a PEGylated background with low settlement on the 2 µm and 5µm alternating stripes.

The zoospores could not discern the different chemistries present and thus consider the surface to

be pure PEG. The complexity of these surfaces with different chemistry and domain sizes may

further enhance the deterring of marine biofouling properties.

Finally, 3D surfaces include microtopographic-patterned surfaces usually designed using

photolithographic techniques. An effective antifouling coating, the Sharklet AFTM, was inspired

from the skin of the shark.46-47 A poly-(dimethyl siloxane) polymer Sharklet AFTM pattern in

Schumacher et al. consists of 3 µm high and 2 µm wide ribs of lengths 4, 8, 12, and 16 µm.48

Most of the zoospores that settled were observed around the edges of the diamond repeat unit.

The Sharklet AFTM with its unique geometric features is much better as an antifouling surface

than topographies with similar proportions such as circular pillars or long ridges.

Amphiphilic systems that combine antifouling and foul release properties as well as physically

patterned polymers show promise as effective coatings. Nanoscale structures can target directly

the sensing apparatus of some marine species such as algae or even the organisms themselves if

they are on the same scale such as bacteria or they can inhibit the attachment due to the small

available surface area. However, these highly ordered structures have not been investigated yet.

6

1.3 Diblock Copolymers Patterned polymer surfaces with nanoscale domains show promise for many applications

involving cell-surface interactions,49-50 and also as scaffolds for making nanowires,51 quantum

dots,52 and organic optoelectronic applications.53 Self-assembly is becoming a powerful

“bottom-up” method for engineering nanostructured materials. In particular, block copolymers

can self-assemble in a variety of nanostructures.54-56

Block copolymers are macromolecules of two or more segments of chemically distinct polymers,

each polymer a sequence of identical repeat units.54 The simplest architecture is the AB diblock

copolymer consisting of two polymer chains, block A and B, covalently attached to each other.

In the case of triblock copolymers, two block sequences are possible, ABA, made of only two

distinct polymer chains, and ABC, made of three distinct polymer chains. Diblock and triblock

copolymers are linear block sequences; however, higher number block copolymers have the

blocks joined in the center and are called starblocks.

a)

b)

c)

Figure 1.2 Linear block copolymer architectures a) AB diblock copolymer, b) ABA triblock copolymer, and c) ABC triblock copolymer.

7

1.3.1 Diblock Copolymer Self-Assembly

Diblock copolymers are one of the most studied block copolymer architectures.54 In bulk, the

phase behavior of diblock copolymers depends on the volume fraction of each block (ƒ), the

degree of polymerization (N) and the segment-segment (Flory-Huggins) interaction parameter

(χ). The interaction parameter can be approximated by

χ ≈ αT-1 + β Equation 1.1

where T is the absolute temperature and α > 0 and β are constants for known values of ƒ and a

parameter n. For the case of diblock copolymers n is 1, and this value is determined by the

functionality of the coupling agent used to synthesize the copolymers.

The value of χ is controlled by the degree of incompatibility between the copolymer blocks, thus,

the selection of the monomers for the A and B blocks. When A and B are incompatible or the

temperature of the system is lowered, χ is large and positive, or N is large, free energy can be

minimized by decreasing the contacts between the A and B monomers by losing some entropy

through ordering. The blocks are covalently attached and chain movement is restricted leading

to a microphase separation. On the other hand, if χ or N is decreased such that the entropy gain

is more important, the chains will mix leading to a disordered state. Thus, it is the product χN

which determines phase separation. For example, the order-disorder transition occurs when χN ~

10 in the case of a symmetric diblock copolymer, ƒ = 0.5. When the product χN » 10, the

equilibrium morphology of this copolymer is lamellae. Other simple patterns include spheres

and hexagonal packed cylinders, while more complex morphologies include gyroid and

perforated lamella.

8

a)

b)

Figure 1.3 AB type diblock copolymer which is configured in a) lamellar pattern and b) cylindrical pattern. The A block of one copolymer chain associate with the A block of another copolymer chain, while the B block of a copolymer

chain associate with the B block of another copolymer chain.

In thin films, additional factors must be taken into account when predicting the final

morphology. The diblock copolymer is confined to a surface between two boundaries, substrate

and atmosphere. The differences in interfacial energies between the copolymer blocks and the

boundaries can lead to reorientation of the equilibrium pattern such as cylinders orientated

parallel instead of perpendicular to the surface or the formation of hybrid structures such as

hexagonally perforated layers.57-58 There is also an effect on the final pattern induced by the film

thickness.59 The block copolymer needs to conform to a thickness that may not always

correspond to the bulk repetition length leading to a variety of structures such as cylinders

orientated perpendicular to the surface to cylinders orientated parallel to the surfaces to

perforated lamella as the film thickness increases.

9

1.3.2 Diblock Copolymer Pattern Ordering

External fields have been used extensively to improve the orientation and lateral ordering of

domains such as temperature gradients or electric fields.60-62 For example, Bodycomb et al. used

temperature gradients to induce a lamella orientation in polystyrene-block-polyisoprene films.60

Alternatively, a chemically patterned surface approach has been used by Kim et al. to produce

lamella orientated polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA).62 Recently,

solvent vapor annealing has become a widespread method to manipulate patterns. The

environment of the copolymer film surface is filled with solvent vapor. The solvent vapor swells

the copolymer and imparts mobility to the polymer chains. Kim et al. showed that nearly defect-

free cylinder orientated polystyrene-block-poly(ethylene oxide) can be obtained by controlling

the rate of solvent annealing.63 Peng et al. examined pattern quality of PS-b-PMMA as a

function of annealing time as well as of the amount of solvent.64 In the case of a small annealing

vessel, as time increases, the surface morphology changed from spherical micelles to well-

ordered flowerlike patterns and then to straight lines. Another study by Peng et al. examined the

effect of solvent selectivity on the morphology of PS-b-PMMA films.65 Only the films that were

annealed in acetone vapor, a PMMA selective solvent, showed cylinders perpendicular to the

substrate. A neutral solvent, THF, resulted in a terraced morphology with no surface structures.

1.4 Overview of Thesis

The motivation behind this thesis is to investigate non-toxic alternative antifouling polymer

coatings, provide insights in the factors that affect marine organisms’ settlement as well as

determine the length-scale relevant to sensing.

Nanopatterned self-assembled diblock copolymer surfaces were fabricated, characterized and

tested for antifouling properties in a marine environment. These surfaces provide both physical

and chemical nanopatterning, thus, they are an optimal surface for probing biological responses

on specific length scale in the nanometer range.

After an introduction in Chapter 1 on marine biofouling and block copolymer, Chapter 2 follows

with an introduction to the principles behind the instrumentation used throughout these studies.

10

In particular, the Atomic Force Microscope (AFM) was an indispensable tool in surface pattern

characterization both in air and fluid.

Chapter 3 explores the development and suitability of the diblock copolymers polystyrene-block-

poly(2-vinyl pyridine) (PS-b-P2VP) and polystyrene-block-poly(methyl methacrylate) (PS-b-

PMMA) as coatings to inhibit the settlement of algae zoospores.

Chapter 4 shows the application of diblock copolymers on nylon coated silicon substrates

designed to mimic the surface of an aquaculture net. The algae settlement assay is performed on

these surfaces as well as flat nylon substrates.

In Chapter 5 a novel method developed to improve the yield of desired pattern orientation is

presented. Vapor solvent annealing is combined with temperature control to pattern polystyrene-

block-poly(methyl methacrylate) copolymer films.

The effect of pattern size on algae zoospores and diatoms is investigated in Chapter 6. Surfaces

with varying nanopattern dimensions are fabricated for both diblock copolymers polystyrene-

block-poly(2-vinyl pyridine) and polystyrene-block-poly(methyl methacrylate).

Chapter 7 presents a study on algae settlement with nanopatterned homopolymers in order to

separate the effect of nanoscale patterning and surface hydrophobicity. Poly(2-vinyl pyridine)

(P2VP) and polystyrene (PS) polymer films were imprinted with nanopatterns using lithography.

Finally, chapter 8 completes the thesis with a brief summary and conclusions on polymer

coatings for marine antifouling applications. Future experimental directions are also considered.

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(43) Krishnan, S.; Ayothi, R.; Hexemer, A.; Finlay, J. A.; Sohn, K. E.; Perry, R.; Ober, C. K.; Kramer, E. J.; Callow, M. E.; Callow, J. A.; Fischer, D. A. Langmuir 2006, 22, 5075–5086.

(44) Majumdar, P.; Lee, E.; Patel, N.; Ward, K.; Stafslien, S. J.; Daniels, J.; Chisholm, B. J.; Boudjouk, P.; Callow, M. E.; Callow, J. A.; Thompson, S. E. M. Biofouling 2008, 24, 185–200.

(45) Finlay, J. A.; Krishnan, S.; Callow, M. E.; Callow, J. A.; Dong, R.; Asgill, N.; Wong, K.; Kramer E. J.; Ober, C. K. Langmuir 2008, 24, 503–510.

(46) Bechert, D. W.; Bruse, M.; Hage, W. Exp. in Fluids 2000, 28, 403-412.

(47) Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Schumacher, J. F.; Wilkerson, W.; Wilson, L. H.; Callow, M. E.; Callow, J. A.; Brennan, A. B. Biofouling 2006, 22, 11–21.

(48) Schumacher, J. F.; Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Wilson, L. H.; Callow, M. E.; Callow, J. A.; Finlay, J. A.; Brennan, A. B. Biofouling, 2007, 23, 55–62.

(49) Tsai, I. Y.; Kimura, M.; Stockton, R.; Green, J. A.; Puig, R.; Jacobson, B.; Russell, T. P. J. Biomed. Mater. Res. Part A 2004, 71, 462–469.

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(51) Fan, H. J.; Werner, P.; Zacharias, M. Small 2006, 2, 700–717.

13

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(55) Fredrickson, G. H.; Bates, F. S. Annu. Rev. Mater. Sci. 1996, 26, 501–550.

(56) Hamley, I. W. The Physics of Block Copolymers; Oxford University Press: New York, 1998.

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14

2 Instrumental Techniques

The efficacy of a coating to inhibit the settlement of marine species will determine its suitability

as an antifouling protector for man-made surfaces immersed in seawater, potentially leading to

numerous benefits both economic and environmental. The coating must be designed, fabricated,

characterized and tested for marine antifouling properties. Characterization of the coating’s

surface properties is critical in understanding the interaction between the coating and marine

species. For example, Ulva linza algae zoospores behavior was shown to be affected by a

number of surface properties including surface chemistry,1-2 topography3 and wettability.4-5 In

this chapter the operation principles behind the instrumental techniques used to evaluate the

surface properties of the polymer coatings throughout this thesis are presented. In addition, the

method used to determine the number of attached marine species representing the antifouling

efficacy is also explained. Table 2.1 is a brief overview of these techniques and their application

in this thesis.

Table 2.1 Overview of analysis techniques and conditions used throughout this thesis.6-9

Technique Resolution Environment Information

Atomic Force Microscopy 0.1 nm Air, Solution Surface topography

Scanning Electron Microscopy 5 nm Vacuum Cross-sectional topography

X-ray Photoelectron Spectroscopy 150 µm Vacuum Chemical composition and

structure

Contact Angle Meter 1 mm Air Wettability

Ellipsometry 1 mm Air Thickness

Fluorescence Microscopy 220 nm Air Marine species density

15

2.1 Atomic Force Microscopy The versatility of the atomic force microscope (AFM) has made it an indispensible tool in many

fields of science concerned with determination of surface structure and properties since its

invention in 1986 by Binnig et al.6, 10 AFM has a very high resolution, 0.1 nm, and can be used in

any environment, vacuum,11 air12-14 and even in solution.15-16 Solution imaging makes it an ideal

tool for monitoring surface behavior underwater to determine coating performance in actual

conditions. AFM is a surface technique, thus, it has a very poor depth of field. AFM is used in

this thesis mainly as a surface imaging technique;12-14, 17-18 however, it can also measure other

surface properties such as adhesion. 19-20 A large variety of samples can be examined, from soft

polymers12-14, 17-18 to hard ceramic surfaces.21

A schematic of the operation of an AFM is shown in Figure 2.1. The AFM uses a sharp tip

attached to a cantilever to scan over the sample surface and the interaction force between the tip

and sample are monitored.6, 22 In this case, the piezoelectric scanner moves the tip in the x, y and

z directions as it scans the sample. The scanner is made of piezoelectric materials, a material

that converts electrical potential into mechanical motion. The x and y piezo electrodes are used

to scan the tip over the surface. The force sensor monitors the force between the tip and sample

and is made of a cantilever attached to a tip and an optical lever. The optical lever is made of a

laser focused onto the back of the cantilever which reflects the beam to a split photodiode

detector. The tip interaction with the surface will affect the position of the reflected laser beam

resulting in a change in the light measured by the photodiodes of the detector. Finally, the

feedback control feeds the signal from the force sensor back into the piezoelectric scanner to

control the expansions of the z piezo electrode in order to maintain a constant set level for the

interaction force, thus, highly increasing the sensitivity of the instrument. The z piezo electrode

moves up and down and the amount of this movement directly correlates to the topography of the

sample. A height map of the sample, a three dimensional view, is constructed based on the

voltage applied to the z piezo electrode as the tip moves across the sample. An example of a

height map is shown in Figure 2.2.

16

Figure 2.1 Atomic Force Microscope operation schematic.

a) b)

Figure 2.2 AFM images of PS-b-P2VP a) Height image, and b) 3-D view of the height image.

The pertinent AFM modes used throughout this thesis are Contact Mode and Tapping Mode.22

In Contact Mode, as the name implies, the tip is in contact with the surface. The deflection of

the cantilever is kept constant by the feedback control. The piezoelectric scanner is moved

vertically at each xy position on the sample to keep the set deflection constant resulting in a

constant force between the tip and the sample. This mode is the fastest and has very high

resolution; however, lateral forces can distort features. Both later forces and the high forces

applied can result in sample damage for soft samples. Nevertheless, soft samples have been

imaged in this mode without problems23-24 and in addition this mode works very well in solution

imaging.6 The forces in solutions are lower due to the absence of the water capillary forces

between the tip and sample present in air.

17

In Tapping Mode, the cantilever oscillates at or near its resonance frequency as it scans the

sample.22 A constant oscillation amplitude is kept by the feedback control. The amplitude is

affected by the tip and the force field of the sample. Similar to Contact Mode, the z height of the

piezoelectric scanner is moved to keep the set amplitude constant resulting in a constant tip

sample interaction.

AFM tips are typically made of silicon nitride or silicon.22 Silicon nitride tips have low force

constants, thus, they can be deflected by small forces, and have high frequencies, thus, they can

withstand vibrational instabilities. They are ideal for Contact Mode. Silicon tips are stiffer and

they have high force constants and have high frequencies. These tips are usually used for

Tapping Mode.

2.2 Scanning Electron Microscopy

Another widespread method of determining the surface morphology uses a Scanning Electron

Microscope (SEM).25 In this technique, a focused beam of electrons is scanned across the

surface of a sample. The electron beam interacts with the surface leading to the formation of

various photons and electrons as shown in Figure 2.3. The electron beam has energy between 1

to 30 keV and the spot size has a 2 to 10 nm diameter, thus, SEM has a good resolution but not

as high as AFM. Samples must be electrically conductive to minimize imaging errors caused by

charge buildup; however, insulators can also be measured by coating them with a thin

electrically conductive film. The sample holders are small, a few cm, though the samples can be

also mounted on an edge in order to image the cross section and the stage can be moved in the x,

y and z directions as well as rotated. This particular feature was used to image the cross section

of polymer films in this thesis. Figure 2.4 shows examples of SEM cross section imaging of a

nanopatterned polymer and metal layers deposited on a glass prism. In this thesis, SEM

experiments are performed in vacuum, thus, solution imaging is not possible on this particular

machine. The common detection of secondary electrons is used to construct the images in this

thesis. Secondary electrons are weak electrons, less than 50 eV, which are ejected due to

inelastic scattering when an incident beam of several keV is used.25 These electrons are

produced from the top of the sample, 50 Å to 500 Å deep.

18

Figure 2.3 Scanning Electron Microscope operation schematic.

a) b)

Figure 2.4 Examples of SEM images in cross-section a) Nanopatterned polystyrene, and b) Glass prism coated with alternating metal layers.

2.3 X-ray Photoelectron Spectroscopy

X-ray Photoelectron Spectroscopy (XPS), historically known as Electron Spectroscopy for

Chemical Analysis, is used to determine the chemical composition of the sample as well as the

structure and oxidation state of the compounds.25 A schematic of the working principle of XPS

is shown in Figure 2.5. A monochromatic X-ray beam of known energy, hυ where h is Planck’s

constant and υ is frequency, impinges upon the surface of the samples and it displaces an

electron from the inner shells with an energy Eb, binding energy. Typical X-ray resolution is 150

19

µm, however, better resolution instruments are available.7, 25 Experiments are done in vacuum to

deter beam attenuation and contamination from the environment. Emitted electrons cannot travel

more than 1 to 5 nm thorough the sample, thus, XPS is a surface sensitive technique. The kinetic

energy, Ek, of the emitted electron is detected by an electron spectrometer, the analyzer, which

has an electrostatic correction factor, w, the work function. The binding energy of the displaced

electron is

Eb = hυ – Ek – w Equation 2.1

A spectrum of the number of emitted electrons as a function of the emitted electron binding

energy is generated. The atom and orbital of the emitting electron correlates to the binding

energy. A survey spectrum as shown in Figure 2.6A has a low resolution; however, it

encompasses a wide area and provides the chemical composition of the sample. When the peaks

present in the survey spectrum is measured with a high resolution, additional information such as

oxidation state becomes apparent as shown in Figure 2.6B. In both cases, the spectra also

provide the relative number of each atom type.

Figure 2.5 X-ray Photoelectron Spectroscopy operation schematic.

20

a) b)

Figure 2.6 Example of XPS spectra of PS-b-P2VP + BP UV films a) survey, and b) high resolution C 1s.

2.4 Contact Angle Method

Contact angle measurement is a surface sensitive technique with a 1 mm spatial resolution which

measures the contact angle of a liquid droplet with the surface.8 This technique is important in

analyzing the interactions between solids and liquids to better understand surface wettability,

biocompatibility and adhesion.26 The contact angle, θ, is the angle between the surface and a

tangent to the liquid drop profile at the intersection point. Three boundaries intersect at this

point, the solid sample, the liquid drop and the air environment. When the contact angle is high,

usually above 90°, the liquid does not wet the surface and the surface is called hydrophobic,

Figure 2.7A. When the contact angle is low, the liquid wets the surface and the surface is called

hydrophilic, Figure 2.7B. Contact angle measurement is a straight forward technique

accomplished using goniometry. The sample is placed on a sample stage and illuminated using a

light source. The liquid drop is dispensed and a zoom microscope lens attached to a camera

captures the image of the droplet. The angle formed between the sample and the tangent to the

drop profile is then calculated usually by a computer analysis program. Purity of the liquid,

surface roughness and heterogeneity can affect measurements.

a) b)

Figure 2.7 Advancing water contact angle of a) Hydrophobic sample, and b) Hydrophilic sample.

21

2.5 Ellipsometry Ellipsometry uses polarized light to investigate surface properties of very thin films such as

thickness, refractive index or optical anisotropy.25 A polarized incident beam is reflected (or

transmitted) from the surface of the sample and the change in the state of polarization, ψ, the

elliptical angle, and Δ, the phase shift, between the s- and p- polarized light, of this reflected (or

transmitted) light is measured. These measured values can then be correlated to the sample

thickness and other properties. The surface roughness of the samples must be small and the

measurement is done at oblique incidence.27 The measurement is fast and non-destructive with a

high precision in thickness determination, 0.1 Å; however, the spatial resolution is low:

millimeters. In addition, the data can be complicated to analyze since it requires dielectric

function modeling, the creation of an optical model and fitting the measured spectra. The

thickness of the amplitude and phase values that match the experimental values is assumed to be

the thickness of the sample; hence, ellipsometry is an indirect method.

A general schematic of an ellipsometer is shown in Figure 2.8. In a nulling ellipsometer, a

circular polarized incident laser beam is reflected off the sample and then linear polarized

reflected light reaches the analyzer.25 The polarizer and compensator rotate until this linear

polarization in the reflected beam is obtained. The analyzer is also rotated until a minimum light

intensity is observed at the detector. On the other hand, in a rotating compensator ellipsometer, a

spectroscopic type ellipsometer, the spectra is measured by changing the wavelength of light.27

Measurements are usually performed in the UV-visible region. In this configuration, a

compensator or retarder is positioned between the sample and analyzer to improve measurement

sensitivity. A compensator introduces a 90° phase shift in the reflected beam by slowing down

one of the two orthogonal light parts.

Figure 2.8 Ellipsometry operation schematic.

22

2.6 Fluorescence Microscopy Fluorescence microscopy is a technique used in particular in biology to observe organisms, either

alive or fixed.28 Molecules can be detected with good selectivity and specificity with good

signal-to-background noise ratio. When organic molecules absorb photons of energy hυ,

electrons are excited from the ground electronic state to the excited single states.9 Electrons can

descend from one energy level to another by releasing energy as heat. Fluorescence occurs when

the electrons return to the ground state from the first excited single state and these electrons are

remitted as photons. The fluorescence light has a longer wavelength, less energy, than the

absorbed wavelength. An epifluorescence microscope was used in this thesis to visualize and

count the number of attached marine species on the polymer samples. A schematic of an

epifluorescence microscope is shown in Figure 2.9. The beam from a fluorescence light source

reflects of a dichromatic mirror, which reflects shorter wavelengths and transmits longer

wavelengths, and is focused by the objective onto the sample.9 The sample is excited and the

fluorescence light, at a longer wavelength, passes through the objective and dichromatic mirror

to the detector. For example, algae zoospores will fluoresce because of the presence of

chlorophyll in the chloroplast.29

Figure 2.9 Epifluorescence microscopy operation schematic.

23

2.7 References (1) Krishnan, S.; Ayothi, R.; Hexemer, A.; Finlay, J. A.; Sohn, K. E.; Perry, R.; Ober, C. K.;

Kramer, E. J.; Callow, M. E.; Callow, J. A.; Fischer, D. A. Langmuir 2006, 22, 5075-5086.

(2) Krishnan, S.; Wang, N.; Ober, C. K.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Hexemer, A.; Kramer, E. J.; Sohn, K. E.; Fischer, D. A. Biomacromolecules 2006, 7, 1449-1462.

(3) Schumacher, J. F.; Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Wilson, L. H.; Callow, M. E.; Callow, J. A.; Finlay, J. A.; Brennan, A. B. Biofouling 2007, 23, 55-62.

(4) Ista, L. K.; Callow, M. E.; Finlay, J. A.; Coleman, S. E.; Nolasco, A. C.; Simons, R. H.; Callow, J. A.; Lopez, G. P. Appl. Environ. Microbiol. 2004, 70, 4151-4157.

(5) Schilp, S.; Kueller, A.; Rosenhahn, A.; Grunze, M.; Pettitt, M. E.; Callow, M. E.; Callow, J. A. Biointerphases 2007, 2, 143-150.

(6) Eaton, P.; West, P. Atomic Force Microscopy; Oxford University Press: New York, 2010.

(7) Brundle, C. R.; Evans, C. A. Jr.; Wilson. S. Encyclopedia of materials characterization: surfaces, interfaces, thin films; Butterworth-Heinemann: Stoneham, 1992.

(8) Ratner, B. D.; Chilkoti, A.; Castner, D. G. Clinical Materials 1992, 11, 25‐36. (9) Chiarini-Garcia, H.; Melo, R. C. N. Light Microscopy Methods and Protocols; Springer

Science + Business Media: New York, 2011.

(10) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930-933.

(11) Hoffmann, R.; Baratoff, A.; Hug, H. J.; Hidber, H. R.; von Lohneysen, H.; Guntherodt, H. J. G. Nanotechnology 2007, 18, 395503.

(12) Peng, J.; Wei, Y.; Wang, H.; Li, B.; Han, Y. Macromol. Rapid Commun. 2005, 26, 738–743.

(13) Zhao, B.; Brittain, W. J.; Zhou, W.; Cheng, S. Z. D. Macromolecules 2000, 33, 8821–8827.

(14) Park, S.; Wang, J.-Y.; Kim, B.; Chen, W.; Russell, T. P. Macromolecules 2007, 40, 9059–9063.

(15) Yokokawa, M.; Yoshimura, S. H.; Naito, Y.; Ando, T.; Yagi, A.; Sakai, N.; Takeyasu, K. IEE Proceedings-Nanobiotechnology 2006, 153, 60–66.

(16) Doktycz, M. J.; Sullivan, C. J.; Hoyt, P. R.; Pelletier, D. A.; Wu, S.; Allison, D. P. Ultramicroscopy 2003, 97, 209–216.


(18) Yang, J.; Wang, Q.; Yao, W.; Chen, F.; Fu, Q. Appl. Surf. Sci. 2011, 257, 4928–4934.

(19) Eaton, P.; Smith, J.; Graham, P.; Smart, J.; Nevell, T.; Tsibouklis, J. Langmuir 2002, 18, 3387-3389.

(20) Santore, M. M.; Kozlova, N. Langmuir 2007, 23, 4782-4791.

(21) Uematsu, K.; Moriyoshi, Y.; Saito, Y.; Nowotny, J. Key Eng. Mater. 1995, 111-112, 57-70.

(22) Training Manual; Digital Instruments Veeco Metrology Group: Santa Barbara, 1999.

24

(23) Müller, D. J.; Schoenenberger, C. A.; Schabert, F.; Engel, A. J. Struct. Biol. 1997, 119, 149-157.

(24) Murphy, M. F.; Lalor, M. J.; Manning, F. C. R.; Lilley, F.; Crosby, S. R.; Randall, C.; Burton, D. R. Microsc. Res. Tech. 2006, 69, 757–765.

(25) Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis; 6th ed., Thomson Brooks/Cole: Belmont, 2007.

(26) Contact Angle and Surface Angle Meter Operation Manual; KSV Instruments: Monroe, 2006.

(27) Fujiwara, H. Spectroscopic ellipsometry: principles and applications; John Wiley & Sons: Chichester, England, 2007.

(28) Yuste, R. Nature Methods 2005, 2, 902-904.

(29) Ederth, T.; Pettitt, M. E.; Nygren, P.; Du, C.-X.; Ekblad, T.; Zhou, Y.; Callow, M. E.; Callow, J. A.; Liedberg, B. Langmuir 2009, 25, 9375–9383.

http://books1.scholarsportal.info.myaccess.library.utoronto.ca/search.html?searchTerm=Fujiwara%2C+Hiroyuki&searchField=Author

25

3 Diblock Copolymers Polystyrene-block-poly(2-vinyl pyridine) and Polystyrene-block-poly(methyl methacrylate) Cylindrical Patterned Surfaces for Algae Zoospores Settlement Study

Content in this chapter is adapted with permission from Grozea, C. M.; Gunari, N.; Finlay, J. A.;

Grozea, D.; Callow, M. E.; Callow, J. A.; Lu, Z.-H.; Walker, G. C. Biomacromolecules 2009, 10,

1004–1012. Copyright 2009 American Chemical Society.

3.1 Overview

Nanopatterned surfaces with hydrophobic and hydrophilic domains were produced using the

diblock copolymer polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) and polystyrene-

block-poly(methyl methacrylate) (PS-b-PMMA). The PS-b-P2VP diblock copolymer, mixed

with the photoinitiator benzophenone and spin-coated onto silicon wafers, showed self-

assembled cylindrical structures, which were retained after ultraviolet (UV) treatment for

crosslinking. The thin films displayed cylindrical domains after immersion in water. This study

shows that pattern retention in water is possible for a long period of time, at least for five weeks

in pure water and three weeks in artificial seawater. The PS-b-PMMA diblock showed self-

assembled cylindrical structures. PS-b-P2VP and PS-b-PMMA cylindrical patterned surfaces

showed reduced settlement of zoospores of the green alga Ulva compared to unpatterned

surfaces. The copolymers were investigated using Atomic Force Microscopy (AFM) and X-ray

photoelectron spectroscopy (XPS).

3.2 Introduction Marine biofouling of surfaces is a problem for structures immersed in seawater such as ship hulls

and aquaculture nets.1 In order to prevent biofouling without the use of biocides, materials must

be developed that have antifouling properties i.e. inhibit the settlement of the colonizing stages

26

such as spores and larvae, and/or have fouling release properties i.e. exhibit low adhesion of the

fouling organisms so that they are ‘released’ by hydrodynamic forces. Zoospores of the fouling

macroalga Ulva respond to a number of surface properties including wettability2-3, charge4,

surface chemistry5-6 and topography7-8. The response of spores to both nanoscale topographic

features and variation in local chemistry has not been explored.

In biological studies, in situ measurements are preferred in order to mimic the actual

environment of the species of interest and minimize species destruction. For applications such as

antifouling coatings, studies should therefore be conducted in an aqueous environment since it is

the hydrated surface with which the settling cell, spore or larva interacts. Thus, nanoscale

patterns geared towards these studies must be stable for a long period submerged in water. A

regular spacing of the features is desirable to probe for biological responses on specific length

scales, which is a promise of diblock copolymer systems. Diblock copolymers can self-assemble

in a variety of ordered structures such as cylinders and lamella at the nanoscale.9-12 However, to

the best of our knowledge no diblock system durable to long-term exposure to water has been

reported. A method of obtaining cylindrical nanopatterned surfaces of polystyrene-block-poly(2-

vinyl pyridine) (PS-b-P2VP) that are stable in water is presented. These surfaces provide both

physical and chemical patterning due to the hydrophobic and hydrophilic nature of the domains.

By photocrosslinking the cylindrical domains, a more durable material that can withstand

immersion in water for extended periods of time is produced. We also show the response of the

patterned surface to a biological system, by quantifying the settlement (i.e. initial attachment) of

zoospores of the green alga Ulva.

3.3 Materials and Methods

3.3.1 Materials

Hydroxy terminated PS-b-P2VP diblock copolymer (Polymer Source) and the photoinitiator

benzophenone (BP; Sigma Aldrich) were used in this experiment. PS-b-P2VP polymer has a

polydispersity index of 1.06 and a number average molecular weight for PS of 75000 g/mol and

for P2PV of 21000 g/mol. Thin films were prepared by spin coating 0.3 wt% toluene solutions

of the diblock copolymer or the diblock mixed with BP (1:1 w/w) on silicon substrates at 2000

27

rpm for 45 s. The silicon substrates were prepared by cleaning in piranha solution (3:1 v/v

concentrated H2SO4 : 30 % H2O2) for 10 min. Caution: Piranha is a very strong oxidant. The

thin films were solvent vapor annealed using toluene and chloroform (1/1 v/v) for 3 h. The

polymer films were placed on a glass Petri dish at the bottom of glass vessel of 450 cm3 volume

with a lid. A 10 mL glass vial filled with toluene and a 10 mL glass vial filled with chloroform

were placed beside the Petri dish in the glass vessel, the lid was placed on top and the system

was left at room temperature. After annealing, the PS-b-P2VP with BP films were UV irradiated

using a Mercury Arc Lamp (Pen-Ray, 90-0012-01) with an intensity of 15 mW/cm2 for 5 min in

air.

PS-b-PMMA diblock copolymer (Polymer Source) with a polydispersity index of 1.10 was used

in this experiment. The number average molecular weight for PS is 130000 g/mol and for

PMMA is 133000 g/mol. Thin films were prepared by spin coating 0.3 wt% toluene solutions of

the diblock copolymer on piranha-cleaned silicon substrates at 2000 rpm for 45 s. The thin films

were solvent vapor-annealed using acetone for 5 h.

a) b)

c)

Figure 3.1 Diblock copolymers a) PS-b-P2VP and b) PS-b-PMMA, and Photoinitiator c) BP.

Homopolymers PS (Polymer Source) and P2VP (Polymer Source) were used as received.

Polystyrene-co-2-vinyl pyridine random copolymer (P(S-r-VP); Polymer Source) was also used.

The properties of these polymers can be seen in Table 3.1. Thin films were prepared by spin

coating 0.3 wt% toluene solutions of PS, P2VP, or P(S-r-VP) on piranha cleaned silicon

substrates at 2000 rpm for 45 s.

28

Table 3.1 Properties of homopolymers and a random copolymer. Mn is the number average molecular weight and Mw is the weight average molecular weight. The unit of measure is g/mol. PDI is the polydispersity index.

Label Mn Mw PDI

PS 131000 138000 1.05

P2VP 22000 24000 1.09

P(S-r-2VP) 75000 128000 1.7

a) b)

Figure 3.2 a) PS, and b) P2VP.

3.3.2 Characterization of Morphology in Thin Films

All polymer thin films were imaged using AFM to examine the surface topography. The AFM

(Digital Instruments, Dimension 5000) operated in Tapping Mode was used to perform the

measurements in air. Rectangular shaped silicon probes (NanoWorld, NCH) with resonance

frequencies in the range 280-320 kHz and a spring constant of 40 N/m were used. All

measurements in solution were obtained using the Molecular Force Probe AFM (Asylum

Research, MFP-3D) operated in Contact Mode. V-shaped, silicon nitride cantilevers (Veeco,

DNP) exhibiting a nominal spring constant of 0.12 N/nm were used. The mechanical properties

of the thin film were measured by AFM imaging and nanoindentation using rectangular shaped

silicon tips (NanoWorld, NCH) with nominal a spring constant of 40 N/m. In short, a 1 x 1 μm

AFM image was obtained in tapping mode. After imaging, the AFM stylus was positioned over

the P2VP region and then the PS region and two indentation curves were obtained over each

specific region of interest. Similarly different areas of the thin film were scanned and

indentation plots were obtained. The resulting force-indentation curves were analyzed with

custom-programmed analysis software (Wavemetrics, Igor Pro).

29

X-ray photoelectron spectroscopy (XPS) was used to obtain the chemical composition of the

polymer films. An ESCA (Phi, 5500) system with an Al Kα (1486.7 eV) monochromated X-ray

source was used to obtain the spectra at a takeoff angle of 45°.

A contact angle meter (KSV Instruments, Cam101) was used to measure the advancing contact

angle of the films using ultrapure water (Mili-Q 18 MΩ).

Ellipsometry was performed with a nulling ellipsometer (Waterloo Digital Electronics, Exacta

2000) at 60° to measure the thickness of the films.

3.3.3 Ulva Zoospore Settlement Assay

Attachment experiments were performed using zoospores released from mature Ulva linza plants

using standard methods.2-8 Samples were equilibrated in 0.22 µm filtered artificial seawater for

one hour before testing. Zoospores were settled in individual dishes containing 10 mL of

zoospore suspension, in the dark at ~ 20 °C. Each dish contained one silicon wafer (size 2.5 x

2.5 cm) coated in polymer. After 60 min the substrates were washed in seawater to remove

unsettled zoospores. Substrates were fixed using 2.5 % glutaraldehyde in seawater. The density

of zoospores attached to the surface was counted on each of the replicate silicon wafers using an

image analysis system (Imaging Associates Ltd.) attached to an epifluorescence microscope

(Zeiss, Aksioskop 2). Spores were visualized by autofluorescence of chlorophyll. Counts were

made for 30 fields of view (each 0.17 mm2) on each wafer.

3.4 Results and Discussion

3.4.1 Ordering in Block Copolymer Thin Films

The morphology of the copolymer thin films was characterized using AFM. In Figure 3.3A the

height image of a spin-cast and solvent annealed PS-b-P2VP film can be seen. The lattice

spacing of the hexagonal packing domains is 48 ± 2 nm. The weight ratio between PS and P2VP

is about 3:1, thus, in this system it is expected that the matrix would be PS and the cylinders

would be P2VP. The brighter areas in the image correspond with the PS matrix, while the darker

30

areas correspond to the P2VP domains. The solubility of the blocks and the solvent evaporation

rate are responsible for the orientation of the cylinders normal to the surface. During annealing,

the copolymer swells with the solvent. The solvent imparts mobility to the copolymer, thus, the

block can reorganize. However, some defects such as holes can be present as can be seen in the

Figure 3.3A. The larger darker area in the image corresponds to a hole in the film. The

formation of holes is due to the mismatch between the thickness of the copolymer layers and the

periodicity of the bulk.13 The cylindrical domains are orientated normal to the surface, however,

the layer adjacent to the silicon surface has the domains orientated parallel to the surface because

the P2VP block is more polar than the PS block. Liu et al. found that the film thickness for an

asymmetric PS-b-P2VP block copolymer can be quantized by

t = (n + α)L + β Equation 3.1

where, t is the film thickness, n is the number of bulk cylindrical layers, α is the fractional

thickness of the top cylindrical layer, L is the layer period, and β is the thickness of the lamellar

layer adjacent to the surface.14 When the thickness of the film is different from t by L/2, holes

and islands will appear. In addition, these holes can also be produced by nucleation from small

impurities present on the surface. The advancing water contact angle of these surfaces is 94 ±

3°, as shown in Table 3.2, which corresponds to a hydrophobic surface. This contact angle for

the copolymer system is not surprising since the advancing water contact angle of pure PS is 95

± 3° and of pure P2VP is 65 ± 3°. Figure 3.3B shows a PS film, while part C shows a P2VP.

The films show no surface structure as expected from pure homopolymers. The advancing water

contact angle of the random copolymer P(S-r-VP) is 88 ± 3°. This random copolymer is made of

two monomer units, PS and P2VP, which are randomly distributed along the chain either as

single molecules or as alternating segment of various lengths. The random copolymer does not

phase segregate or shows nanoscale structures as seen in Figure 3.3D. The thickness of the films

used throughout this study is 20 ± 3 nm.

31

a) b)

c) d)

Figure 3.3 AFM height images of a) solvent annealed PS-b-P2VP film, b) PS film, c) P2VP film, and d) P(S-r-VP) film. Image sizes: 1 μm x 1 μm. Z range: 20 nm.

32

Table 3.2 Advancing Water Contact Angles.

Label Contact Angle Contact Angle Image

PS

95 ± 3°

P2VP

65 ± 3°

P(S-r-VP)

88 ± 3°

PS-b-P2VP

94 ± 3°

PS-b-P2VP + BP

94 ± 3°

PS-b-P2VP + BP UV

61 ± 3°

PS-b-PMMA

84 ± 3°

33

In Figure 3.4A, the height image of a spin-cast and solvent annealed film of PS-b-P2VP mixed

with BP is shown. The brighter areas in the image correspond to the PS matrix, while the darker

areas correspond to the P2VP domains, cylinders orientated normal to the surface. The

advancing water contact angle of the thin films remains 94 ± 3°, as shown in Table 3.2, which

implies that BP is either not present at the surface or it does not have a great effect on the surface

energy of the film. The height of the grains in this image is 3 ± 1 nm.

In Figure 3.4B the height image of a spin-cast and solvent annealed PS-b-P2VP mixed with BP

film after it was photocrosslinked is shown. The brighter areas in the image correspond to the

P2VP domains, while the darker areas correspond to the PS matrix. In this case, the matrix is

lower in height in the AFM image than the cylinders. The advancing water contact angle

decreases to 61 ± 3°, which indicates that these new films are more hydrophilic than the

noncrosslinked films. The UV irradiation helps to introduce more oxygen-containing surface

groups. The height of the grains is 3 ± 1 nm.

In Figure 3.4C the height image of a spin-cast and solvent annealed film of PS-b-PMMA is

shown. The brighter areas in the image correspond to the PS matrix, while the darker areas

correspond to the PMMA domains, cylinders orientated normal to the surface. The lattice

spacing of the hexagonal packing domains is 110 ± 2 nm. The advancing water contact angle of

this thin film is 84 ± 3° as shown in Table 3.2.

34

a) b)

c)

Figure 3.4 AFM height images of a) solvent annealed PS-b-P2VP and BP film, b) solvent annealed and UV irradiated PS-b-P2VP and BP film, and c) solvent annealed PS-b-PMMA film. Image sizes: 1 μm x 1 μm. Z range: 20 nm.

UV radiation has wavelengths shorter than visible light in the range of 10 nm to 400 nm of the

electromagnetic spectrum.15 The energy of a photon of light, E, is

E = hυ = hc / λ Equation 3.2

where h is Planck’s constant, υ is the frequency, c is the speed of light and λ is the wavelength.

When UV radiation is absorbed by a molecule it promotes electrons from the ground energy state

to an excited energy state. The electrons return to a lower energy state by dissipating the

absorbed energy as heat, light, or in chemical reactions. In particular, oxidative

photodegradation includes processes initiated by light in air such as crosslinking, chain

scissioning and the formation of oxygen-containing functional groups.16 In crosslinking,

polymer chains join together to form larger chains and even polymer networks. On the other

hand, chain scissioning leads to a breakdown of polymer chains resulting in shorter chains.

These processes take place simultaneously and competitively.

35

Figure 3.5 shows the oxidative photodegradation of PS upon UV irradiation in air.17 First, a PS

radical forms upon irradiation. This PS radical can react with another radical leading to a larger

polymer chain. The PS radical can also react with oxygen from the air to form a peroxy radical.

In turn, the peroxy radical can react to form an alkoxy radical, a carbonyl species and a shorter

chain PS radical. The PS radical and subsequent peroxy radicals can also undergo additional

reactions with other PS chains.16 Nevertheless, all these reactions lead to larger PS chains and

chain scissioning with the formation of carbonyl containing chains.

a)

b)

c)

d)

Figure 3.5 Oxidative photodegradation of PS in air.

36

Direct UV irradiation can cause thin film polymers to dewet, especially PS on Si wafers.18

Carroll et al. showed that by adding a BP containing molecule to the PS solution and then spin-

coating to obtain thin films, the dewetting process is inhibited. In addition, BP is a photoinitiator

with absorption bands at 253 nm, 280 nm and 330 nm.17 Figure 3.6 shows the oxidative

photodegradation of PS film containing BP upon UV irradiation in air. BP forms a ketyl radical,

which abstracts a hydrogen atom from PS. When the sample is exposed to atmospheric oxygen,

the BP radical forms a highly reactive hydroperoxide radical and BP. This hydroperoxide radical

reacts with a BP radical to form hydrogen peroxide and BP. Moreover, this hydroperoxide

radical can undergo further reaction with the PS radical to form a PS hydroperoxide.16 This can

further break down to produce a shorter chain polymer radical and carbonyl species, leading to

an increase in polarity of the polymer chains. The PS radical species can recombine to give rise

to crosslinked chains. UV irradiation can also produce radical centers on the PS or P2VP chains

without the use of BP.

37

a)

b)

c)

d)

Figure 3.6 Oxidative photodegradation of PS and BP in air.

XPS analysis was performed on three different samples, PS-b-P2VP film after solvent annealing,

PS-b-P2VP and BP after solvent annealing, and PS-b-P2VP and BP after UV in air as shown in

Figure 3.7. Table 3.3 shows the relative chemical composition of the films is carbon, oxygen

and nitrogen, which was expected for our phase separated block copolymer. The compositions

of the PS-b-P2VP film and the PS-b-P2VP and BP film are similar in C, O, and N. The

difference between these two samples can be attributed to variations between samples that occur

due to self-assembly because XPS has a finite depth of penetration into the surface of around 5-6

nm. Analysis of unpatterned PS-b-P2VP films shows that at the surface only PS is present; no N

peak was observed in the spectra. The UV treated sample has a larger fraction of O and a lower

38

fraction of C, which is due to oxidation. Nitrogen chemical deviations can be attributed to

sample variations.

Figure 3.7 XPS survey spectrum of PS-b-P2VP film after solvent annealing, PS-b-P2VP and BP after solvent annealing, and PS-b-P2VP and BP after UV in air.

Table 3.3 XPS analysis of three different samples.

Carbon 1s % Oxygen 1s % Nitrogen 1s %

PS-P2VP 93.9 4.9 1.2

PS-b-P2VP + BP 92.6 6.4 0.9

PS-b-P2VP + BP UV 65.2 33.1 1.7

High-resolution XPS analysis was performed on these three different samples for C, O, and N,

which are shown in Figure 3.8. The PS-b-P2VP and PS-b-P2VP with BP samples had similar

spectra, thus, for clarity only one was included in the figure. In Figure 3.8A the C 1s spectrum

shows another peak at about 289 eV binding energy for the UV irradiated sample. This peak

39

corresponds to the carbonyl group. The O 1s peak of the UV sample is broader, which indicates

an increase in the type of O bonds. In addition, more peaks are present in the N 1s spectrum for

the UV treated sample. These peaks that appear at a higher binding energy can be attributed to

pyridone and to pyridine-N-oxide. Both the matrix and the cylinders survive the UV irradiation

treatment.

a) b)

c)

Figure 3.8 High-resolution XPS of a) C 1s, b) O 1s, and c) N 1s spectra of a) PS-b-P2VP film after solvent annealing, dotted line and b) PS-b-P2VP and BP after UV in air, solid line.

The mechanical properties of the PS-b-P2VP copolymer film and the PS-b-P2VP and BP film

after UV treatment in air were investigated using AFM indentation measurements. Figure 3.9A

shows the force-indentation curve for PS-b-P2VP. The PS-b-P2VP and BP film after UV has a

similar force curve. The Young’s modulus was determined by considering load-indentation

dependence for a paraboloidal tip shape19 given by equation 3.3

2/32 )1(3

4 δvREF

−= Equation 3.3

40

where F is the loading force in nN, E is Young’s modulus in Pa, R is the radius of curvature of

the tip in nm, δ is the indentation in nm, and ν is the Poisson’s ratio (0.5). The force indentation

curves obtained on the darker and brighter regions did not show different moduli. PS and P2VP

have been previously shown to have similar mechanical properties such as glass transition

temperature and elastic modulus, thus the average elastic modulus of the PS-b-P2VP surface is a

good approximation.20-21 Figure 3.9B shows a histogram of the moduli obtained by fitting the

force-indentation curves using equation 3.3. The elastic modulus for PS-b-P2VP is 3.3 ± 1 GPa.

This value is in good agreement with the previously reported value for PS of 3.5 GPa.22 The

elastic modulus for the UV irradiated film is 4.1 ± 1 GPa. The UV irradiated film was not

statistically different than the regular PS-b-P2VP film due to the presence of some uncross-

linked chains. The role of finite sample thickness and the stiffness of the underlying substrate

has not been taken into detailed account in the modeling of the film modulus, and which could

lead to a ca. 50% error in the estimated moduli.23

a)

b) c)

Figure 3.9 a) Force-indentation curve of the PS-b-P2VP film. Dots are the data point, while the solid line is the fit by a paraboloidal tip shape. b) the elastic modulus distribution for PS-b-P2VP film, and c) the elastic modulus distribution

for PS-b-P2VP and BP film after UV irradiation.

41

Figure 3.10 shows the force vs. extension curves for PS-b-P2VP copolymer films and for PS-b-

P2VP and BP film after UV treatment in air. Figure 3.10A shows a large hysteresis between the

trace and retrace data. The PS-b-P2VP film shows a plastic deformation after the tip indents into

the surface. Also, there is a strong repulsive force present in the trace curve starting at about 20

nm. The onset of the repulsive force coincides with the thickness measured by ellipsometry.

The onset of the repulsive force ranges from 10 to 20 nm. Figure 3.10B shows a lower hysteresis

between the trace and retrace data. The crosslinked PS-b-P2VP film displays a decrease in this

plastic deformation behavior due to the crosslinking restraining the flow of the copolymer

chains.

a) b)

Figure 3.10 Force-extension curve of a) PS-b-P2VP film, and b) PS-b-P2VP and BP film after UV irradiation. The grey curve is the trace data, while the black curve is the retract data.

3.4.2 Block Copolymer Behavior Underwater

When the noncrosslinked PS-b-P2VP films were immersed in ultrapure water, the pattern was

preserved after 2 h, with the measurement performed in water. This can be seen from the AFM

height images in Figure 3.11A. The brighter areas in the image correspond with the PS matrix,

while the darker areas correspond to the P2VP domains. Hydrophobic surfaces such as PS can

develop nanobubbles at the interface with a polar solvent such as water which can affect the

properties of the surface.24 The diblock copolymer surface does not promote the formation of

nanobubbles, since the available PS surface between the cylinders is not enough for these

hundreds of nanometer sized features to develop. The advancing water contact angle of these

hydrated films is 72 ± 3°, which is much lower than the PS-b-P2VP film after solvent annealing

as shown in Table 3.4. The pKa of the conjugate acid for P2VP is 4.5, which is smaller than the

42

one for ultrapure water, pKa = 5.5.25 Thus, P2VP proton uptake will not affect the algae

settlement. When the films are taken out of the water, dried under nitrogen gas and measured in

air, a cylindrical pattern is visible after 2 h of water immersion as shown in Figure 3.11B. After

24 h, the pattern is no longer present; however, some isolated cylinders can still be seen, Figure

3.11C. When the film is immersed in water, the hydrophobic PS matrix tries to minimize the

contact area exposed to the water. The copolymer starts to move, until the pattern disappears

from the surface. The pattern does not reappear after the film is immersed in water for more than

8 days as shown in Figure 3.11D. The advancing water contact angle of these films is again

much lower than before, 63 ± 3°. When the film is immersed in water for a longer period of

time, water has time to penetrate into the film and cause restructuring of the copolymer. Drying

under nitrogen gas is not enough to cause the pattern to return.

a) b)

c) d)

Figure 3.11 AFM height images of solvent annealed PS-b-P2VP films a) in water after 2 hours, Image size: 0.5 µm x 0.5 µm b) in air after 2 hours in water, c) in air after 24h hours in water, and d) in air after 8 days in water. Image

sizes: 1 µm x 1 µm. Z range: 20 nm.

43

Table 3.4 Advancing Water Contact Angles after water immersion.

Contact angle

after solvent

annealing

Contact angle

after 2 h in

water

Contact angle

after 8 days in

water

PS 95 ± 3° 91 ± 3° -

P2VP 63 ± 3° 63 ± 3° -

P(S-r-VP) 88 ± 3° 80 ± 3° -

PS-b-P2VP 94 ± 3° 72 ± 3° 63 ± 3°

PS-b-P2VP + BP UV 61 ± 3° 64 ± 4° 62 ± 3°

PS-PMMA 84 ± 3° 80 ± 3° 70 ± 3°

Figure 3.12A shows the PS film in water after 2 h in water. The film again shows no surface

structure as expected from a pure homopolymer. The advancing water contact angle for PS

slightly decreases from 95 ± 3° to 91 ± 3°. The other homopolymer, P2VP, is shown in Figure

3.12B also after 2 h in water and no surface structure is present either. The advancing water

contact angle remains the same at 63 ± 2°. The surface of the random copolymer P(S-r-VP) is

shown in Figure 3.12C after 2 h water immersion. No surface pattern is detected, however, the

advancing water contact angle decreases from 88 ± 3° to 80 ± 3° due to P2VP units migrating to

the surface.

44

a) b)

c)

Figure 3.12 AFM height images in water after 2 hours of a) PS, b) P2VP, and c) P(S-r-VP). Image size: 1 µm x 1 µm. Z range: 40 nm.

The crosslinked PS-b-P2VP mixed with BP films were also immersed in water for various

periods of time. Cylinders orientated normal to the surface when placed in water can be seen in

the AFM height image after 2 h in water in Figure 3.13A. The darker areas in the image

correspond with the P2VP domains, while the brighter areas correspond to the PS matrix. After

the copolymer film was dried with nitrogen gas after 2 h, 24 h and after 8 days in water the

pattern reappeared. When the copolymer is crosslinked, it is harder for the water to infiltrate the

film. In addition, the oxidation of the film, which decreases the hydrophobicity of the film,

results in retention of the pattern. Figure 3.13B, C and D shows the copolymer film in air after 2

h, 24 h and 8 days in water respectively. The advancing water contact angle of these films is

about 64 ± 4° and 62 ± 3°, which indicates that water does not infiltrate significantly into the

film. Thus, the UV crosslinking stabilizes the surface groups. The pattern was also present upon

drying after five weeks in water as seen in Figure 3.13E. In addition, the copolymer film showed

pattern retention after immersion in artificial seawater for three weeks and then dried in nitrogen

gas, Figure 3.13F.

45

a) b)

c) d)

e) f)

Figure 3.13 AFM height images of cross-linked PS-b-P2VP and BP films a) in water after 2 hours, b) in air after 2 hours in water, c) in air after 24 hours in water, d) in air after 8 days in water, e) in air after 5 weeks in water, and f) in

air after 3 weeks in seawater. Image size: 1 µm x 1 µm. Z range: 40 nm.

46

When the PS-b-PMMA films were immersed in water, the pattern is still present after 2 h, with

the measurement performed in water. This can be seen from the AFM height image in Figure

3.14A. The brighter areas in the image correspond to the PMMA domains, while the darker

areas correspond to the PS matrix. After water immersion for 2 h, the film was dried under

nitrogen gas and measured in air the film shows a cylindrical pattern, Figure 3.14B. The

advancing water contact angle of these films is 80 ± 3°, which is slightly lower than the PS-b-

PMMA film after solvent annealing. After 24 h and 8 days in water, the film looks more swollen

and the pattern is not as clear as before as seen in Figure 3.14C and D respectively. The

advancing water contact angle of these films is again much lower than before, 70 ± 3°. As the

copolymer is immersed for longer periods of time in water, the film swells and the pattern is less

clear. However, because the PS-b-PMMA copolymer is more hydrophilic than the PS-b-P2VP

copolymer, it retains the pattern for a longer period of time.

a) b)

c) d)

Figure 3.14 AFM height images of solvent annealed PS-b-PMMA films a) 2 hours in water, b) in air after 2 hours in water, c) in air after 2 hours in water, and d) in air after 8 days in water. Image size: 1 µm x 1 µm. Z range: 40 nm.

47

3.4.3 Settlement of Zoospores of the Green Alga Ulva

The density of zoospores attached to a surface indicates how hospitable that surface is for

settlement. Permanent attachment is the consequence of the secretion of a hydrophilic self-

aggregating glycoprotein, which anchors the spore permanently to the substratum.25 In Figure

3.15A the density of zoospores settled (attached) on the polymers on silicon wafers is shown.

Visual observations of settled spores did not reveal any differences in the pattern of distribution

on any of the surfaces.

The settlement density of spores was greatest on the two control surfaces, that is, PS and P2PV;

however, it was slightly higher on P2VP. Although settlement density on smooth, uncharged

surfaces generally correlates with increasing hydrophobicity,2,3 this trend is not seen here.

However, neither of the control surfaces is entirely smooth, holes and small bumps are present at

the surface and are maintained when the surfaces are immersed in water. The advancing water

contact angle for PS decreases after 2 h immersion in water from 95 ± 3° to 91 ± 3°, while the

one for P2VP remains the same at 63 ± 2°; thus, no major reorganization of the polymers takes

place on this time scale.

The lowest settlement density was on the PS-b-P2VP and BP after UV treatment and PS-b-

PMMA surfaces, both of which are comprised of cylindrical patterns. The advancing water

contact angle after 2 h in water was 64 ± 4° for PS-b-P2VP and BP after UV treatment and 80 ±

4° for PS-b-PMMA. The PS-b-P2VP and BP after UV treatment film does not reorganize in

water. The density of spores was significantly lower on PS-b-PMMA and the high degree of

swelling associated with this surface after immersion in seawater may have enhanced its

efficacy. It is interesting to note that the cylindrical pattern appears to inhibit the settlement of

spores. The chemical and topographical heterogeneity of these surfaces may be working

independently or in tandem to discourage spore settlement. The different hydrophobicity

between the cylindrical portion of the pattern and the matrix may deter spores, which would have

settled on a surface with uniform hydrophobicity. The overall advancing water contact angle of

the surface after water immersion can be seen in Figure 3.15B. For example, the uniform P2VP

polymer surface has the same overall advancing water contact angle, but very different spore

settlement densities. Similarly, the nanoscale roughness may act as a deterrent if the spores

48

prefer a smoother surface. Alternatively, it is possible that all of this heterogeneity simply

confuses the spore by sending it conflicting signals.

On the PS-b-P2VP surface, which also had the patterning, the settlement density was similar to

that on the random copolymer surface, P(S-r-VP). The P(S-r-VP) copolymer does not phase

segregate into nanopatterned domains, thus grain size analysis gives a value of 0 for grain size as

shown in Figure 3.15C; however, at the film surface both PS and P2VP areas are present.

Settlement densities on these surfaces were intermediate between the high settlement on the PS

and P2VP and the low settlement on the PS-b-P2VP and BP after UV treatment and PS-b-

PMMA surfaces. In water, the P(S-r-VP) surface restructures slightly, with mobile P2VP units

migrating to the surface; the advancing water contact angle value of this film decreases from 88

± 3° to 80 ± 3° after 2 h in water. Similarly, the PS-b-P2VP block copolymer restructures

underwater to have more of the P2VP domains at the surface. The advancing water contact

angle value of this film after 2 h in water decreases from 94 ± 3° to 72 ± 3°.

Another factor that can influence the settlement of spores is the area of the surface that is

available for contact with the spores. This contact area is slightly larger for the homopolymers

and the random copolymers than for the patterned copolymer surfaces. The hardness of the

adhesive produced by the spores must also be taken into account. The modulus of the adhesive

is in the MPa range, 0.2 ± 0.1 MPa for the outer 600 nm thick layer and 3 ± 1 MPa for the inner

layer.27 The polymer surfaces are much harder than the adhesive, in the GPa range; thus, in

order to increase contact area the glue is required to conform to the surface. Roughness alone or

contact area alone does not explain settlement behavior, something beyond the contact area seem

to be at play.

49

a)

0

200

400

600

800

1000

PS

P2V

P

P(S

-r-V

P)

PS

-b-P

2VP

PS

-b-P

2VP

+B

P U

V

PS

-b-P

MM

A

Spo

re d

ensi

ty (n

o. m

m-2

)

b)

0

20

40

60

80

100

PS

P2VP

P(S-

r-VP

)

PS-b

-P2V

P

PS-b

-P2V

P +B

P U

V

PS-b

-PM

MA

Adva

ncin

g w

ater

con

tact

ang

le (°

)

c)

0

200

400

600

800

PS

-b-P

2VP

PS

-b-P

2VP

+B

P

UV

P(S

-r-V

P)

Gra

in S

ize

(nm

2 )

Figure 3.15 a) The density of attached Ulva spores on polymers on silicon wafers. Each point is the mean from 90 counts on 3 replicate slides (30 on each wafer). Bars show 95% confidence limits. b) advancing water contact angle

for polymers on silicon wafers, and c) Average grain size analysis.

50

3.5 Conclusions Thin films of PS-b-P2VP block copolymer mixed with BP show cylindrical domains. The

cylindrical domains can also be seen when the sample is dried after five weeks in water and three

weeks in artificial seawater. A simple route to obtain surfaces that retain their nanoscale patterns

after water immersion is presented in this chapter. Furthermore, these nanopatterned films were

also shown to affect the settlement response of zoospores of Ulva. Spore settlement density was

significantly reduced on the cylindrical patterned PS-b-P2VP and BP after UV cross-linking and

PS-b-PMMA surfaces.




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(24) Agrawal, A.; Park, J.; Ryu, D. Y.; Hammond, P. T.; Russell, T. P.; McKinley, G. H. Nano Lett. 2005, 5, 1751-1756.

(25) Callow, J. A.; Callow, M. E. In Biological Adhesives: Smith, A. M., Callow, J. A ., Eds.; Springer: Berlin, 2006, p 63-78.

(26) Tantavichet, N.; Pritzker, M. D.; Burns, C. M. J. Appl. Pol. Sci. 2001, 81, 1493-1497.

(27) Walker, G. C.; Sun, Y.; Guo, S.; Finlay, J. A.; Callow, M. E.; Callow, J. A. J. Adhesion 2005, 81, 1101-1118.

52

4 Polystyrene-block-poly(2-vinyl pyridine) and Polystyrene-block-poly(methyl methacrylate) Patterned Nylon Surfaces for Inhibition of Algae Zoospores and Sporelings

4.1 Overview

Polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) and polystyrene-block-poly(methyl

methacrylate) (PS-b-PMMA) diblock copolymer nanopatterned surfaces were fabricated on

nylon pieces, nylon coated silicon or glass substrates. Nylon substrates were used in order to

mimic aquaculture nets. The PS-b-P2VP, PS-b-P2VP UV crosslinked and PS-b-PMMA diblock

copolymers showed self-assembled cylindrical structures. PS-b-P2VP UV crosslinked, PS-b-

PMMA and nylon thin films showed reduced settlement of Ulva linza zoospores. In the case of

algae sporelings only PS-b-PMMA diblock copolymers and controls were tested. PMMA, PS-b-

PMMA and nylon thin films showed reduced settlement and growth. The surface structure of the

polymers films were investigated using Atomic Force Microscopy (AFM).

4.2 Introduction Marine aquaculture also known as fish farming is a rapidly growing food producing industry. In

1960, aquaculture provided only 5 % of the fishery output for consumption, while in 2000 this

value rose to 34 % and in 2010 to 47 %.1 Fish are grown in complex enclosures consisting of net

cages made of ropes and netting, cage collars and mooring systems.2 The netting for the cages is

can be made of a square mesh knotless nylon. The entire surface of the fish enclosures is

susceptible to marine biofouling. This can be seen in particular in Figure 4.1, which shows a

nylon net placed in seawater for one month. The fouling of the nets lead to damage of the cage

enclosure as well as a decline in fish health due to low levels of dissolved oxygen, decrease in

food availability and accumulation of waster products caused by low water flow.3 Cleaning of

equipment due to biofouling can lead to an estimated 20 % increase in the cost of fish

production.4

53

a) b)

Figure 4.1 Aquaculture nylon nets a) clean, and b) after one month seawater immersion showing marine biofouling.

The prevention of marine biofouling in aquaculture farming has also been based on heavy metals

antifouling paints as used in the shipping industry.3 The highly toxic tin and nickel coatings are

no longer used. Copper-based coatings are still widely used as paints despite their negative

effects on non-target species in and around the fish farms. Thus, this industry can benefit too

from new environmentally friendly non-toxic antifouling coatings.

The previous chapter presented a study on cylindrical nanopatterned surfaces of polystyrene-

block-poly(2-vinyl pyridine) (PS-b-P2VP) and polystyrene-block-poly(methyl methacrylate)

(PS-b-PMMA) and the response of algae zoospores, a common marine biofoulant, to these

patterns. This study was done on model silicon surfaces; however, real world applications such

as cage nets or ships’ hulls are not made of silicon. In this chapter, nylon surfaces are used

instead of silicon polymer supports. The nanopatterned diblock copolymers were coated on

nylon pieces or nylon coated silicon or glass support in order to mimic the nylon nets used in

aquaculture farming. The response of algae zoospores and sporelings to these coatings is

investigated to determine their suitability as antifouling coatings.

54


4.3.1 Materials

Nylon substrates were prepared by cutting 2.5 x 2.5 cm pieces from a nylon 6, 6 sheet

(Thyssenkrupp Materials) or dissolving a nylon piece and recasting it. Thin nylon films were

prepared by spin coating 0.3 wt% formic acid solutions on silicon or glass slides at 2000 rpm for

45 s. The silicon and glass substrates were prepared by cleaning in piranha solution (3:1 v/v

concentrated H2SO4 : 30 % H2O2) for 10 min. Caution: Piranha is a very strong oxidant.

Figure 4.2 Nylon 6, 6.

Hydroxy terminated PS-b-P2VP diblock copolymer (Polymer Source) and the photoinitiator

benzophenone (BP; Sigma Aldrich) were used as received. PS-b-P2VP polymer has a

polydispersity index of 1.06 and a number average molecular weight for PS of 75000 g/mol and

for P2PV of 21000 g/mol. Thin films were prepared by spin coating 0.3 wt% toluene solutions

of the diblock copolymer or the diblock mixed with BP (1:1 w/w) on nylon pieces and nylon

coated silicon substrates at 2000 rpm for 45 s. The thin films were solvent vapor annealed using

toluene and chloroform (1/1 v/v) for 3 h. The PS-b-P2VP with BP films were UV irradiated

using a Mercury Arc Lamp (Pen-Ray, 90-0012-01) with an intensity of 15 mW/cm2 for 5 min in

air.

PS-b-PMMA diblock copolymers (Polymer Source) were used as received. The properties of the

diblock copolymers are shown in Table 4.1. Thin films were prepared by spin coating 0.3 wt%

toluene solutions on various substrates at 2000 rpm for 45 s. The PS-b-PMMA 105-106, 130-

133 and 160-160 copolymers were coated on nylon pieces and nylon coated silicon substrates,

while the PS-b-PMMA 66-63.5 and 52-52 were coated on nylon coated glass slides. The thin

films were solvent vapor-annealed using acetone for 5 h.

55

Table 4.1 PS-b-PMMA diblock copolymers properties. Mn is the number average molecular weight and the unit of measure is g/mol. PDI is the polydispersity index.

Label for PS-b-PMMA PS Mn PMMA Mn PDI

160-160 160000 160000 1.09

130-133 130000 133000 1.10

105-106 105000 106000 1.13

66-63.5 66000 63500 1.08

52-52 52000 52000 1.09

a) b)

c)

Figure 4.3 Diblock copolymers a) PS-b-P2VP and b) PS-b-PMMA, and Photoinitiator c) BP.

Homopolymers PS (Polymer Source), P2VP (Polymer Source) and PMMA (Polymer Source)

were used as received. Random copolymers polystyrene-co-2-vinyl pyridine (P(S-r-VP);

Polymer Source) and polystyrene-co-methyl methacrylate (P(S-r-MMA); Polymer Source) were

also used. The properties of the polymers can be seen in Table 4.2. Thin films were prepared by

spin coating 0.3 wt% toluene solutions on nylon pieces and nylon coated silicon substrates at

2000 rpm for 45 s. In addition, PS, PMMA and P(S-r-MMA) were also spin coated on nylon

coated glass slides.

56

Table 4.2 Properties of homopolymers and random copolymers. Mn is the number average molecular weight and Mw is the weight average molecular weight. The unit of measure is g/mol. PDI is the polydispersity index.

Label Mn Mw PDI

PS 131000 138000 1.05

P2VP 22000 24000 1.09

PMMA 106000 114.5000 1.08

P(S-r-2VP) 75000 128000 1.7

P(S-r-MMA) 102000 173000 1.7

a) b)

c)

Figure 4.4 a) PS, b) P2VP, and c) PMMA.


All polymer films were imaged using AFM to examine the surface topography. The AFM



frequencies in the range 280-320 kHz and a spring constant of 40 N/m were used.

57







zoospore suspension in the dark at ~ 20°C, twice the standard volume and half the standard

concentration in order to ensure surface coverage of nylon pieces. Each dish contained one

polymer substrate on a nylon piece or nylon coated silicon or glass. After 45 min the substrates

were washed in seawater to remove unsettled zoospores. Substrates were fixed using 2.5%

glutaraldehyde in seawater. The density of zoospores attached to the surface was counted on

each of the replicate substrates using an image analysis system (Imaging Associates Ltd.)

attached to an epifluorescence microscope (Zeiss, Aksioskop 2). Spores were visualized by

autofluorescence of chlorophyll. Counts were made for 30 fields of view (each 0.17 mm2) on

each polymer.

4.3.4 Ulva Sporelings Growth and Removal

Algae zoospores were allowed to settle on the polymer coatings on nylon coated glass slides for

45 minutes and then gently washed as described above. The zoospores were cultured using

supplemented seawater medium for 7 days to produce sporelings (young plants) on 6 replicate

slides of each treatment. Sporeling growth medium was refreshed every 48 hours.

Sporeling biomass was determined in situ by measuring the fluorescence of the chlorophyll

contained within the sporelings in a Tecan fluorescence plate reader. Using this method the

biomass was quantified in terms of relative fluorescent units (RFU). The RFU value for each

slide is the mean of 70 point fluorescence readings taken from the central portion. The sporeling

growth data are expressed as the mean RFU of 6 replicate slides; bars show SEM (standard error

of the mean).

58

The strength of attachment of sporelings was assessed using the water jet apparatus. The

intention was that individual slides of each treatment would be exposed to increasing water

pressures.


4.4.1 Nylon Substrates

In Figure 4.5A the surface structure of a nylon piece cut from a nylon 6, 6 sheet is shown. The

surface shows irregular features and has a mean surface roughness of 64 ± 3 nm. This surface is

very rough compared to a silicon surface, which has a mean surface roughness of 0.2 ± 0.1 nm.

In addition this surface roughness is much higher than the diblock copolymer film thickness,

around 20 ± 3 nm. A nylon piece was dissolved in formic acid and recast from solution as a thin

polymer films using spin coating. The morphology of nylon polymer thin films on silicon

substrates is shown in Figure 4.5B, while the morphology of nylon on glass substrates is shown

in Figure 4.5C. These thin films have a lower roughness than the original nylon pieces. The

mean roughness is 2 ± 1 nm. These surfaces also have more regular nanoscale sized features.

The advancing water contact angles of nylon coated on silicon is 49 ± 2° and of nylon coated on

glass slides is 53 ± 2°.

59

a)

b) c)

Figure 4.5 AFM height images of a) nylon piece, Z range: 200 nm. b) nylon coated on silicon, and c) nylon coated on glass slide. Z range: 30 nm. Image sizes: 1 μm x 1 μm.

4.4.2 Polymer Coated Nylon Films on Silicon

The morphology of the PS-b-P2VP copolymer thin films on nylon coated on silicon is shown in

Figure 4.6A. The lattice spacing of the hexagonal packing domains also known as the center-to

center spacing is 48 ± 2 nm. The diameter of the cylinders is 31 ± 2 nm. The brighter areas in

the image correspond with the PS matrix, while the darker areas correspond to the P2VP

cylindrical domains. The advancing water contact angle is 86 ± 2°, which is lower than PS-b-

P2VP coated directly on silicon, 94 ± 3°. The diblock copolymer interacts with a more

hydrophobic surface in the case of the nylon coating, contact angle of 49 ± 2°, as opposed to a

silicon substrate, contact angle 11 ± 2°. The P2VP polar domains are less attracted to the

substrate and more of this block will be present at the surface. However, the overall pattern

remains the same with no major reorientation.

60

In the case of PS-b-P2VP mixed with BP and UV crosslinked, the surface morphology is seen in

Figure 4.6B. The brighter areas in the image correspond with the P2VP domains, while the

darker areas correspond to the PS matrix. The advancing water contact angle is lower at 43 ± 3°.

The surface is more hydrophilic due to oxygen incorporated groups during the UV irradiation

process. When these films are placed in water, the original cylindrical morphology orientated

perpendicular to the surface is observed as seen in Figure 4.6C.

a)

b) c)

Figure 4.6 AFM height images of polymer films on nylon coated silicon a) solvent annealed PS-b-P2VP, b) solvent annealed PS-b-P2VP and BP after UV irradiation, and c) UV PS-b-P2VP and BP after 2 hours water immersion.

Image sizes: 1 μm x 1 μm. Z range: 20 nm.

61

Table 4.3 Advancing water contact angles of polymers on nylon coated silicon.

Label Contact Angle

PS 87 ± 2°

P2VP 58 ± 2°

P(S-r-VP) 80 ± 2°

PS-b-P2VP 86 ± 2°

PS-b-P2VP + BP UV 43 ± 3°

PMMA 72 ± 2°

P(S-r-MMA) 72 ± 2°

PS-b-PMMA 105-106 71 ± 2°

PS-b-PMMA 130-133 71 ± 2°

PS-b-PMMA 160-160 71 ± 2°

Figure 4.7 shows the height images of spin-coated and solvent annealed films of PS-b-PMMA on

nylon coated silicon with various molecular weights. Figure 4.7A shows the morphology of PS-

b-PMMA 105-106, while Figure 4.7B shows the morphology of PS-b-PMMA 130-133 and

finally Figure 4.7C shows the morphology of PS-b-PMMA 160-160. The brighter areas in the

images correspond to the PS matrix, while the darker areas correspond to the PMMA domains

made of cylinders orientated perpendicular to the surface. The center-to-center distance between

the cylinders and the cylinder diameters for these patterns can be seen in Table 4.4. The pattern

size increases as the molecular weight of the copolymer increases as expected. The phase

behavior of diblock copolymers depends on the volume fraction of each block (ƒ), the degree of

polymerization (N) and the segment-segment (Flory-Huggins) interaction parameter (χ).12-15 The

62

volume fraction value does not change for the PS-b-PMMA copolymer, the 1:1 ratio between the

two block remains constant as the molecular weight is increased resulting in a ƒ = 0.5. The final

pattern will not change. The degree of polymerization value increases with an increase in

molecular weight, although, the value of χ stays the same since the selection of PS and PMMA

monomers for the two blocks in the copolymer does not change. The value of χ for PS-b-PMMA

with ƒPS = 0.5 can be approximated16 by

χ = (0.0282 ± 0.002) + (4.46 ± 0.6) / T Equation 4.1

Thus, the product χN will increase as the molecular weight of the copolymer increases as shown

in Table 4.5; however, this effect does not affect the final pattern given that the value is above

the value of the order-disorder transition, 10. The equilibrium morphology in bulk is lamella.

The PS-b-PMMA copolymer is spin cast as a thin polymer film and other effects come into play

in determining the final morphology such as the interfacial energy of the two boundaries, air and

annealing solvent.19-21 The polymer pattern is trapped in a cylindrical morphology orientated

perpendicular to the substrate. The advancing water contact angle of these thin films is 71 ± 2°.

This value is lower than for the PS-b-PMMA copolymer on silicon due to a decrease in attraction

of the PMMA block to the nylon surface.

63

a) b)

c)

Figure 4.7 AFM height images of solvent annealed PS-b-PMMA films on nylon coated silicon a) 105-106, b) 130-133, and c) 160-160. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

Table 4.4 PS-b-PMMA nanopattern dimensions.

Label for PS-b-PMMA Center-to-center spacing (nm) Cylinder diameter (nm)

105-106 90 ± 4 62 ± 4

130-133 109 ± 4 74 ± 4

160-160 113 ± 4 82 ± 4

64

Table 4.5 Characteristics of the PS-b-PMMA diblock copolymers. ƒPS is the volume fraction of the PS block calculated from ƒPS = (wPS/ρPS) / ( (wPS/ρPS) + (1 - wPS)/ρPMMA) by using the following densities for the PS and PMMA block: ρPS = 1.05 g cm-3 and ρPMMA = 1.18 g cm-3.17-18 χ is determined from χ = 0.0282 + 4.46/T as reported in literature for PS-b-

PMMA. 16 The experiments were performed at room temperature, 24 °C.

Label for PS-b-PMMA N ƒPS χN

105-106 2070 0.50 89

130-133 2580 0.49 111

160-160 3140 0.50 136

Figure 4.8 shows the height images of the homopolymers and random copolymers control

surfaces. Figure 4.8A shows a PS film, while part B shows a P2VP film and part C shows a

PMMA film. These films show no surface structure as expected from pure homopolymers. The

advancing water contact angle are 87 ± 2°, 58 ± 2° and 72 ± 2° respectively. Figure 4.8D shows

a P(S-r-2VP) film, whereas part E shows a P(S-r-MMA) film. A random copolymer is made of

two monomer units, PS and P2VP or PS and PMMA in the second case, which are randomly

distributed along the chain either as single molecules or as alternating segment of various

lengths. The random copolymer does not phase segregate or shows nanoscale structures. The

advancing water contact angle of the random copolymer P(S-r-VP) is 80 ± 2° and the contact

angle for P(S-r-MMA) is 72 ± 2°.

65

a) b)

c) d)

e)

Figure 4.8 AFM height images of polymer films on nylon coated silicon a) PS, b) P2VP, c) PMMA, d) P(S-r-2VP), and e) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm.

66

4.4.3 Polymer Coated Nylon Films on Glass Slides

The morphology of the thin polymer films, diblock copolymers, homopolymers and a random

copolymer, on nylon coated glass slides can be seen in Figure 4.9. Figure 4.9A shows the height

image of PS-b-PMMA 52-52, whereas part B shows the height image of PS-b-PMMA 66-63.5.

The PS-b-PMMA diblock copolymer show a cylindrical pattern orientated perpendicular to the

substrate similar to the one observed for larger molecular weight polymers. The brighter areas in

the images correspond to the PS matrix, while the darker areas correspond to the PMMA

domains made of cylinders orientated perpendicular to the surface. The pattern dimensions, the

center-to-center distance and the cylinder diameter, decrease with a decrease in molecular weight

as seen in Table 4.6. In this case too, the volume fraction of the PS-b-PMMA copolymers is

keep constant at ƒ = 0.5, a 1:1 ratio between the two block as the molecular weight is increased,

thus, the final pattern stays constant. The product χN decreases as the molecular weight of the

copolymer decrease as shown in Table 4.7, since the degree of polymerization, N, decreases with

a decrease in molecular weight. This new value does not affect the final pattern given that the

value is still above the value of the order-disorder transition. The advancing water contact angle

of these thin films is 78 ± 2° and 77 ± 2° respectively.

Figure 4.8 C shows the height image of a PS film, while part D shows the height image of a

PMMA film and part E shows the height image of a P(S-r-MMA) film. These polymer thin

films are spin coated on nylon coated glass. Both homopolymers and the random copolymer

control surfaces show no surface structure as expected. The advancing water contact angles are

95 ± 2°, 77 ± 2° and 81 ± 2° respectively.

67

a) b)

c) d)

e)

Figure 4.9 AFM height images of solvent annealed PS-b-PMMA polymer films on nylon coated glass slides a) 52-52, b) 66-63.5, c) PS, d) PMMA, and e) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm.



52-52 55 ± 4 34 ± 4

66-63.5 72 ± 4 45 ± 4

68

Table 4.7 Characteristics of the PS-b-PMMA diblock copolymers. ƒPS is the volume fraction of the PS block calculated from ƒPS = (wPS/ρPS) / ( (wPS/ρPS) + (1 - wPS)/ρPMMA) by using the following densities for the PS and PMMA block: ρPS = 1.05 g cm-3 and ρPMMA = 1.18 g cm-3.17-18 χ is determined from χ = 0.0282 + 4.46/T as reported in literature for PS-b-

PMMA. 16 The experiments were performed at room temperature, 24 °C.

Label for PS-b-PMMA N ƒPS χN

52-52 1020 0.50 44

66-63.5 1270 0.51 55

Table 4.8 Advancing water contact angles of polymers on nylon coated glass slides.

Label Contact Angle

PS 95 ± 2°

PMMA 77 ± 2°

P(S-r-MMA) 81 ± 2°

PS-b-PMMA 52-52 78 ± 2°

PS-b-PMMA 66-63.5 77 ± 2°

4.4.4 Settlement of Algae Zoospores

The settlement density of zoospores attached to the polymer thin films on nylon coated silicon

substrates is shown in Figure 4.10A. The lower the number of settled zoospores the more

inhospitable the surface is for zoospore adhesion and the better the antifouling properties. The

settlement density was greatest on the hydrophobic PS surface, which displayed no surface

structure. This trend corresponds with previous literature where settlement density on smooth

and uncharged surfaces generally correlates with increasing hydrophobicity.5-6 The other

homopolymer surfaces, P2VP and PMMA, showed a lower number of settled zoospores. These

69

surfaces are more hydrophilic than PS and PMMA is more hydrophobic than P2VP. The

hydrophobicity trend does not hold true for PMMA.

In the case of the random copolymers, P(S-r-VP) and P(S-r-MMA), the settlement density was

lower than PS. For the P(S-r-VP) surface the number of settled zoospores was slightly larger

than for P2VP. This intermediate settlement correlates well to the surface hydrophobicity as

well as the nature of the surface, both PS and P2VP areas are present on this surface. The

zoospore settlement density for P(S-r-MMA) was unexpectedly low, much lower than for

PMMA and similar to some of the nanopatterned diblock copolymers. The hydrophobicity of

this surface is similar to PMMA and there are no structures visible on the surface.

The PS-b-P2VP surfaces showed an intermediate zoospore settlement similar to P2VP films.

The hydrophobicity of these films is similar to PS, thus, higher than P2VP. However, these

surfaces are not smooth like the pure P2VP and PS homopolymers, they display cylindrical

nanosized domains of P2VP in a PS matrix. In this case, the hydrophobicity trend does not hold

true. The nanoscale features and / or the nanoscale differences in hydrophobicity between the

cylinders and matrix appear to influence the zoospores preference in attachment sites.

The lowest settlement density was on the PS-b-P2VP and BP after UV treatment, the PS-b-

PMMA surfaces and on the nylon coated silicon substrate. The PS-b-P2VP and BP after UV

treatment and nylon coated silicon substrates have the lowest water contact angles. The nylon

surface shows some nanoscale sized features, whereas the diblock copolymer displays cylindrical

nanosized P2VP domains in a PS matrix. Hydrophobicity appears to control the settlement

density of zoospores; however, the influence of nanopatterning cannot be dismissed. On the

other hand, the PS-b-PMMA films have a higher contact angle and still have a low number of

attached zoospores. These surfaces too display nanosized cylindrical domains, made of PMMA

instead of P2VP, in a PS matrix. There is a slight decrease in zoospore settlement with a

decrease in pattern dimension. In this case, the nanopatterning appears to influence the

settlement density more than the surface hydrophobicity.

Overall, the results in Figure 4.10 suggest that both hydrophobicity and nanoscale sized domains

influence zoospore settlement. The cylindrical nanopatterned diblock copolymer coated nylon

surfaces also show low zoospore attachment as seen for silicon coated surfaces. These surfaces

display chemical heterogeneity due to a difference in hydrophobicity between the cylindrical

70

domains and the matrix as well as ordered topographical features. The nanoscale patterning

adds a new dimension to the polymer surfaces. The surface patterning might be working

independently or together with the low surface hydrophobicity to discourage zoospore

settlement.

a)

b)

Figure 4.10 a) The density of attached Ulva spores on polymers on nylon coated silicon. Each point is the mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95% confidence limits. and b) advancing water

contact angle for polymers on nylon coated silicon.

71

The zoospore settlement density on thin polymer films coated directly on nylon pieces can be

seen in Figure 4.11. The PS-b-PMMA 130-133 and 160-160 polymers detached from the nylon

support in water, thus, they could not be measured. The trend in settlement is very different than

for nylon coated silicon surfaces. The highest settlement is on the P2VP polymer film, while PS

and PMMA have a lower settlement. There is an intermediate zoospore settlement for the nylon

piece, the random copolymers, P(S-r-2VP) and P(S-r-MMA), and the diblock copolymers PS-b-

P2VP and PS-b-PMMA 105-106. The lowest settlement is on the diblock copolymer PS-b-P2VP

and BP UV treated. The trend in settlement does not follow the hydrophobicity trend for most of

the surfaces. The nanoscale patterning has an effect on the settlement on the PS-b-P2VP, but not

on the settlement on the PS-b-PMMA surfaces as was seen in previous studies. The performance

of the PS-b-P2VP and BP UV treated sample maintains its antifouling efficacy. These

differences in settlement could be due to the high roughness of the nylon supports affecting the

polymer pattern. This roughness is greater than the polymer film thickness.

Figure 4.11 The density of attached Ulva spores on polymers on nylon. Each point is the mean from 90 counts on 3 replicate slides (30 on each wafer). Bars show 95% confidence limits.

72

4.4.5 Growth and Removal of Algae Sporelings

Thin polymer films were spin coated on nylon coated glass slides and assessed for antifouling

and foul release properties. Glass slides allow for zoospore settlement to be carried out in a

Quadriperm dish which has less area around the sample than the Petri dishes used for the

polymer coated silicon substrates, thus, reducing the choice of substrate for zoospore settlement.

In addition, the glass slides are the correct size to fit the water channel and water jet apparatus,

facilitating the measurement of attachment strength and fouling-release performance of the

coatings.

The density of algae zoospores settlement on polymer surfaces on nylon coated glass slides is

shown in Figure 4.12A. The highest zoospore attachment was on the hydrophobic PS surface

similar to previous studies on nylon coated silicon substrates. The P(S-r-MMA) surface and the

PS-b-PMMA 66-63.5 surfaces showed slightly higher settlement than nylon, PMMA and PS-b-

PMMA 52-52. The number of attached zoospores decreases with a decrease in nanopattern

dimensions for the PS-b-PMMA diblock copolymers as seen for the larger molecular weight

block copolymers on nylon coated on silicon substrates. The hydrophobicity of these surfaces is

similar except for nylon which is more hydrophilic. Hydrophobicity plays a larger effect on the

settlement in this case, while the nanoscale patterning only shows a slight influence on the PS-b-

PMMA 52-52 surface.

The algae zoospores with time grow into young plants also known as sporelings. The density of

the settled algae zoospores from Figure 4.12A which were allowed to mature into sporelings on

the polymer surfaces on nylon coated glass slides is quantified in Figure 4.12B and shown

visually in Figure 4.13A. The production of sporeling biomass follows the zoospore settlement

density trend. Although, the differences in biomass between the polymers is not as great as the

difference in zoospore numbers due to auto-inhibition, the inhibition of germination as a

consequence of high spore density, and competition for nutrients on the high settlement density

samples which limits the zoospore growth rate.22

The strength of attachment of sporelings was supposed to be assessed using a water jet apparatus.

However, when the polymer films were exposed to a water pressure of 18 kPa, the lowest

pressure attainable with the water jet, the films were torn and delaminated as can be seen in

Figure 4.13B and C.

73

a) b)

c)

Figure 4.12 a) The density of attached Ulva spores on polymers on nylon coated glass slides. Each point is the mean from 90 counts on 3 replicate slides (30 on each slide). Bars show 95% confidence limits. b) The biomass of Ulva sporelings on polymers mounted on nylon coated glass slides after 7 days. Each point is the mean biomass from 6 replicate slides measured using a fluorescence plate reader (RFU; relative fluorescence unit). Bars show standard

error of the mean. and c) advancing water contact angle for polymers on nylon coated glass slide.

74

a)

b) c)

Figure 4.13 Images showing a) Ulva sporelings on polymer coatings on nylon coated glass slides. From left: Glass-Nylon, PS, PMMA, P(S-r-MMA), PS-b-PMMA 66-63.5, PS-b-PMMA 52-52, b) Effect of water pressure of 18 kPa on

the polymer films. and c) Close up image of PS-b-PMMA 66-66 and 52-52 (right) films sheared off at 26 kPa.

4.5 Conclusions Thin polymer films of PS-b-P2VP and PS-b-PMMA diblock copolymers, respective

homopolymers and random copolymers were fabricated on a variety of structure, nylon pieces,

nylon coated silicon or glass substrates. The diblock copolymer films showed cylindrical

domains orientated perpendicular to the surface, P2VP or PMMA, in a PS matrix. The pattern

dimensions increased with an increase in molecular weight for the PS-b-PMMA polymer films.

Zoospore settlement density was reduced on the nanopatterned PS-b-P2VP and BP after UV

cross-linking on both nylon supports and nylon coated on silicon supports. PS-b-PMMA

surfaces showed reduced settlement on nylon coated silicon and glass slide supports, with a

decrease in settlement as the pattern dimensions decreased. PS-b-PMMA diblock copolymers as

well as PMMA and P(S-r-MMA) showed reduced zoospore settlement and sporelings growth.

The nylon thin films also showed antifouling properties against algae zoospores and sporelings.

Both polymer hydrophobicity and nanoscale polymer patterning influence zoospore settlement

and growth either by working independently or together.

75

4.6 References (1) FAO. 2012. The state of world fisheries and aquaculture, Food and Agriculture Organization

of the United Nations. Accessed July 2012.

(2) Moe, H.; Olsen, A.; Hopperstad, O. S.; Jensen, Ø.; Fredheim, A. Aquacult. Eng. 2007, 37, 252-265.

(3) Fitridge, I.; Dempster, T.; Guenther, J.; de Nys, R. Biofouling 2012, 28, 649-669.

(4) Enright, C. T.; Elner, R. W.; Griwold, A.; Borgese, E. M. World Aquacult. 1993, 24, 49–51.



(7) Ederth, T.; Nygren, P.; Pettitt, M. E.; Östblom, M.; Du, C.-X.; Broo, K.; Callow, M. E.; Callow, J. A,; Liedberg, B. Biofouling 2008, 24, 303-312.








(15) Matsen, M. W.; Bates, F. S. J. Chem. Phys. 1997, 107, 2436-2448.

(16) Yue, Z.; Sivaniah, E.; Hashimoto, T. Macromolecules 2008, 41, 9948-9951.

(17) Brandrup, J.; Immergut, E. H.; Grulke, E. A.; Abe, A.; Bloch, D. R. Polymer Handbook, 4th ed.; John Wiley & Sons: New York, 1999.

(18) Guo, R.; Huang, H.; Du, B.; He, T. J. Phys. Chem. B 2009, 113, 2712-2724.


(20) Park, I.; Park, S.; Park, H.-W.; Chang, T.; Yang, H.; Ryu, C. Y. Macromolecules 2006, 39, 315–318.


(22) Agrawal, S. C. Folia Microbiol., 2009, 54, 273-302.

76

5 Microdomain Orientation of Diblock Copolymer Polystyrene-block-poly(methyl methacrylate) Solvent Annealed at Low Temperatures

Content in this chapter is adapted with permission from Grozea, C. M.; Li, I. T. S.; Grozea, D.;

Walker, G. C. Macromolecules 2011, 44, 3901–3909. Copyright 2011 American Chemical

Society.

5.1 Overview The effect of temperature during solvent annealing on the microdomain orientation of block

copolymer ultrathin films was investigated using symmetric polystyrene-block-poly(methyl

methacrylate) (PS-b-PMMA) diblock copolymers. When acetone solvent annealing was

performed at low temperatures, 2 °C, 38 % of samples exhibited a pattern of cylinders

perpendicular to the substrate, while 62 % of the samples exhibited a mixture of cylinders

orientated parallel and perpendicular to the substrate. In the case of acetone solvent annealing at

room temperature, only 6 % of samples exhibited a pattern with cylinders perpendicular to the

substrate, while 73 % of the samples exhibited a mixture of cylinders orientated parallel and

perpendicular to the substrate. We discuss how the morphology is affected by temperature

during solvent annealing. The low-temperature method was used to pattern cylinders

perpendicular to the substrate using PS-b-PMMA copolymers with molecular weight ranging

from 52000 g/mol per block to 160000 g/mol per block.

5.2 Introduction Block copolymers can self-assemble in a variety of structures such as cylinders at the nanoscale.

The phase behavior of copolymers depends on ƒ, the volume fraction of each block, and on χN,

the product of the degree of polymerization and the segment-segment (Flory-Huggins)

77

interaction parameter.1-2 In thin films, boundary effects3 and film thickness4 also play an

important role in the final surface morphology.

Occasionally, for some applications, it is necessary to vary the dimensions of the domains. A

widespread approach is to use a blend made of a block copolymer and one of the corresponding

homopolymers.5-8 Otherwise, dimensional scaling can be achieved by changing the molecular

weight of the block copolymer. In this study, a method of fabricating cylindrically

nanopatterned PS-b-PMMA films using low temperature solvent annealing is presented. The

effect of lower temperatures on the annealing solvent has not been previously investigated.

Various approaches were evaluated to explain the effect of temperature on the film morphology.

Furthermore, this method is used to investigate the effect of molecular weight on the dimensions

of the cylindrical domains.


5.3.1 Materials

PS-b-PMMA diblock copolymers with various molecular weights as shown in Table 5.1 were

purchased from Polymer Source and used as received. Ultrathin films were prepared by spin-

coating 1 wt% toluene solutions of the diblock copolymers on silicon substrates at 2000 rpm for

45 s. The silicon substrates were prepared by cleaning in piranha solution (3:1 v/v concentrated

H2SO4 : 30 % H2O2) for 10 min. Caution: piranha is a very strong oxidant. The ultrathin films

were solvent vapor-annealed using acetone for 2 h at 2 °C. A glass vessel of 450 cm3 volume

with a lid and a 10 mL acetone layer at the bottom was used for the vapor annealing. The films

were placed on a glass Petri dish at the bottom of the vessel just above the acetone layer. The

films are in an air-acetone environment. The films were also solvent vapor-annealed using

acetone for 2 h at 24 °C, room temperature. Acetone exhibits roughly a 3-fold decrease in

saturated vapor pressure when temperature is decreased from 24 to 2 °C.9

In the case of the 130-133 PS-b-PMMA films, floated samples were obtained by immersion into

a water bath. Then, the copolymer film was transferred upside down onto a silicon substrate.

This transfer resulted in the placement of the bottom surface of the original film onto the top of

the new substrate. The 130-133 PS-b-PMMA films had also the PMMA block removed by

78

oxygen-based plasma etching (Electronetics Corp., Vacuum Ionization Cleaner 500) in order to

enhance the image contrast in SEM cross-sectional experiments.

Figure 5.1 PS-b-PMMA diblock copolymer.


Label PS Mn PMMA Mn PDI

160-160 160000 160000 1.09

130-133 130000 133000 1.10

105-106 105000 106000 1.13

66-63.5 66000 63500 1.08

52-52 52000 52000 1.09

5.3.2 Characterization

Atomic force microscopy (AFM, Digital Instruments, Dimension 5000) operated in Tapping

Mode was used to examine the surface topography. Rectangular-shaped silicon probes

(NanoWorld, NCH) with resonance frequencies in the range 280-320 kHz and a spring constant

of 40 N/m were used. The number of PMMA domains was counted using NIS-Elements

Advanced Research (Nikon). All other data processing such as cylinder diameter was done

using IGOR Pro 6.12 (WaveMetrics). Scanning electron microscopy (SEM, Hitachi, S-5200)

79

operated at 5 kV and 20 μA was used to analyze the cross sections of the plasma-treated samples.

The stage was tilted at a 20° angle.


angle of water on the films using ultrapure water (Mili-Q 18 MΩ).

X-ray photoelectron spectroscopy (XPS) was used to obtain the chemical composition of the

polymer films. An ESCA (Phi, 5500) system with an Al Kα (1486.7 eV) monochromated X-ray

source was used to obtain the spectra at a takeoff angle of 45°.


5.4.1 Low-Temperature Solvent Annealing

The 130-133 PS-b-PMMA films before solvent annealing display a wormlike morphology as

shown in Figure 5.1A. The inset of Figure 5.1A is a Fourier transform of the AFM image

showing that no ordered cylindrical pattern is present. The advancing water contact angle of

these films is 92 ± 3°. The copolymer chains do not have enough time to self-assemble into the

equilibrium morphology due to fast solvent evaporation during spin coating. The PS block is

attracted to the air interface due to a lower surface energy, while the more polar PMMA block

segregates to the SiOx interface.10 A typical pattern after solvent annealing in acetone at room

temperature is shown in Figure 5.1B, where cylinders are not very well-defined and ordered; the

cylinders are orientated both parallel and perpendicular to the substrate. The brighter areas in the

image correspond to the PS matrix, while the darker areas correspond to the PMMA domains as

shown in previous studies.11-13 The Fourier transform of the AFM image in the inset of Figure

5.1B shows that there is no ordered cylindrical pattern present. There were also samples that

self-assembled into cylinders perpendicular to the substrate after solvent annealing in acetone at

room temperature as shown in Figure 5.1C. The Fourier transform of the AFM image in the

inset of Figure 5.1C shows a pattern close to the six-point pattern of very ordered cylindrical

domains. The advancing water contact angle of these films is 84 ± 3°; more of the PMMA block

is present at the surface. The rest of the samples showed no features at the surface as shown in

Figure 5.1D. There is only one block present at the surface, indicative of lamella orientated

parallel to the surface. The Fourier transform of the AFM image in the inset of Figure 5.1D

80

shows that there is no ordered cylindrical pattern present. The Fourier transform of the AFM

image in the inset shows that there is no ordered pattern present.

a) b)

c) d)

e) f)

Figure 5.2 AFM height images of 130-133 PS-b-PMMA films a) after spin coating, b) annealed at 24 °C, pattern typical for 73 % of samples, c) annealed at 24 °C, pattern typical for 6 % of the samples, d) annealed at 24 °C,

pattern typical for 21 % of the samples, e) annealed at 2 °C, pattern typical for 38 % of the samples, and f) annealed at 2 °C, pattern typical for 62 % of samples. Image sizes: 1 μm x 1 μm. Z range: 30 nm. In the inset in all parts is a

Fourier transform of the corresponding AFM image.

81

In the case of solvent annealing at a low temperature, 2 °C, Figure 5.1E shows cylinders

perpendicular to the substrate. The brighter areas in the image correspond to the PS matrix,

while the darker areas correspond to the PMMA cylindrical domains. The Fourier transform of

the AFM image in the inset of Figure 5.1E shows a six-point pattern corresponding to ordered

cylindrical domains. The films are brought back to room temperature for imaging; thus, the

solvent evaporation that influences the defect removal is still in effect. However, the density of

the cylinders is different at this lower temperature as will be explained in detail later on. The

advancing water contact angle stayed the same at 84 ± 3°. There were also samples present that

self-assembled into cylinders orientated both parallel and perpendicular to the substrate as shown

in Figure 5.1F. The brighter areas in the image correspond to the PS matrix, while the darker

areas correspond to the PMMA domains. The Fourier transform of the AFM image in the inset

shows no ordered cylindrical pattern.

The lower temperature annealing yielded more samples that have the well-ordered cylindrical

pattern than in the case of samples prepared by room temperature annealing as shown in Figure

5.1B and D respectively. In the case of low-temperature annealing, 38 % of 30 samples had

cylinders perpendicular to the substrate, while 62 % had a mixture of cylinders parallel and

perpendicular to the substrate as shown in Figure 5.2. In contrast, the room temperature

annealing yielded only 6 % of 50 samples patterned with cylinders perpendicular to the substrate,

73 % samples with a mixture of cylinders parallel and perpendicular to the substrate, and 21 %

samples with lamella parallel to the surface. There is no well-ordered structure at the surface

that contains both blocks. This finding is similar to that observed by Peng et al., who solvent

annealed PS-b-PMMA films in a large and small chamber partially filled with acetone.12 Their

lower solvent swelled films dewetted less than the higher solvent swelled films; however, the

lowered solvent swelled films showed hills with a lamellar pattern and valleys with a cylindrical

pattern.

82

Figure 5.3 Type of structures present after solvent annealing at 24 °C and 2 °C as a percent of samples.

The 130-133 PS-b-PMMA films with an ordered cylindrical pattern were floated off the silicon

substrate and flipped over in order to image the bottom of the film. Figure 5.3A shows the

presence of cylinders at the bottom of the film; thus, the cylindrical pattern extends through the

film all the way to the substrate. In addition, the 130-133 PS-b-PMMA films were oxygen

plasma treated to selectively remove the PMMA block. Plasma etching produces films that have

a lower surface roughness on the PS sidewall than the alternative method of UV degradation and

acetic acid wash.14-15 Removing the PMMA domains results in a good contrast for imaging the

film’s cross section by SEM. The interior cross section allows access to the length of the

cylinders and thus the quality of the pattern. Figure 5.3B shows an AFM height image of the

film after PMMA removal. The PMMA cylinders are replaced by circular holes, and the overall

pattern is preserved. The films were fractured and the cross-sectional view of the interior

structure as imaged by SEM is shown in Figure 5.3C and D. The circular holes are present

throughout the film all the way to the silicon substrate. The film appears to be about 25 nm

thick. These two methods of probing the copolymer-substrate interface lead to the destruction of

the film; however, alternative nondestructive methods are also starting to emerge such as

Grazing Incidence Small-Angle Neutron Scattering.16

83

a) b)

c) d)

Figure 5.4 Images of 130-133 PS-b-PMMA films a) AFM height image showing the bottom of the polymer film. Image size: 1 μm x 1 μm. Z range: 10 nm, b) AFM height image showing the top of the film after oxygen plasma treatment. Image size: 1 μm x 1 μm. Z range: 30 nm, c) SEM image showing a cross-sectional view of the film interior at a 20º angle after oxygen plasma treatment and d) SEM image showing a zoom in area of cross-sectional view of the film

interior at a 20º angle after oxygen plasma treatment.

During solvent annealing, the annealing solvent swells the copolymer and confers enough

mobility for the copolymer to reorganize. The polymer-solvent interaction parameter (χP-S,

where P is polymer and S is solvent) determines the miscibility between a polymer and solvent.17

When χP-S is lower than 0.5, the polymer and solvent are completely miscible. The diblock

copolymer was solvent annealed in acetone, a polar solvent. For this case the solubility

parameter is separated into a polar and dispersion component for the polymer and solvent. The

final version of χP-S is

χP-S = Vs[(δdS - δdP)2 + (δpS - δpP)2] / RT Equation 5.1

where VS is the molar volume of the solvent, δd is the dispersion solubility parameter, δp is the

polar solubility parameter, R is the gas constant and T is the temperature.18-19 The experimental

values for acetone are VS 73.3 cm3, δdS 15.5 MPa-1, and δpS 10.4 MPa-1, while for PS the values

are δdP 17.6 MPa-1 and δpP 6.1 MPa-1, and finally for PMMA the values are δdP 18.8 MPa-1 and

84

δpP 10.2 MPa-1.20 As is shown in Table 5.2, acetone is a good solvent for PMMA, but not for PS

at the two temperatures. The value of χPMMA-acetone does not change with temperature, while the

value for χPS-acetone slightly increases with a decrease in temperature, an increase in the degree of

immiscibility of the polymer with the solvent. However, this difference is not large enough to

explain on its own the increase in patterned samples at the low temperature. According to Peng

et al., when acetone is used for solvent annealing at room temperature, the vapor molecules

attract the PMMA toward the surface of the film.12 The solvent vapor maximizes contacts with

PMMA, which results in a strong upward driving force that draws the PMMA through the PS-

rich layer to the surface faster than the PS aggregation, resulting in cylinders perpendicular to the

surface. On the other hand, Kim et al. propose that during solvent evaporation a gradient in

solvent concentration develops with a lower concentration at the surface; pattern formation starts

at the air-film interface and propagates toward the film interior.21 However, Kim et al. worked

with PS-b-PEO, which formed cylindrical domains even before solvent annealing, while in the

case of Peng et al. PS-b-PMMA was used, which had a wormlike morphology before solvent

annealing. The mechanisms of solvent annealing are not well understood.

Table 5.2 Polymer-solvent interaction parameters (χP-S) calculated for different temperatures and polymer-solvent pairs.

Acetone 24°C Acetone 2°C

PS 0.68 0.73

PMMA 0.32 0.35

High-resolution carbon 1s (C 1s) XPS spectra were obtained for cylindrical patterned films and

films with no pattern after 2 h of solvent annealing as shown in Figure 5.4A and B. The main

hydrocarbon peak at binding energy of 285.0 eV is a typical peak for both the PS and PMMA

blocks and is in good agreement with the literature.22 The beta-shifted carbon at 286.0 eV, the

methoxy group carbon at 287.2 eV, and the carbon in the ester group at 289.5 eV are attributed

to the PMMA block. The spectra of the cylindrical patterned films and films with no surface

structure after 22 h of solvent annealing are very similar to those of the 2 h annealed films; the

same four peaks are present as shown in Figure 5.4C and D. In all four cases, patterned films

85

and films with no surface structure at 2 h and 22 h, the PMMA block is present at the surface.

Thus, for the case when the film has no surface structure, some of the PMMA block is

segregated to the surface.

a) b)

c) d)

Figure 5.5 High-resolution C 1s spectra with fitted curves for a) films with a cylindrical pattern after 2 h annealing, b) films with no surface structure after 2 h annealing, c) films with a cylindrical pattern after 22 h annealing, and d) films

with no surface structure after 22 h annealing.

On the other hand, the relative chemical composition of the films varies as shown in Table 5.3.

The atomic % of the hydrocarbon peak decreases while the atomic % of the beta-shifted carbon

and the carbon in the ester group increases as the annealing time increases for the cylindrically

patterned film. The concentration of PS at the surface decreases as the concentration of PMMA

increases over time. The atomic % of the hydrocarbon peak is lower while the atomic % of the

beta-shifted carbon, the methoxy group carbon, and the carbon in the ester group is higher for the

film with no surface structure compared to the cylindrically patterned film. The film with no

surface structure present has a higher concentration of PMMA at the surface than the patterned

film. When the film with no surface structure is left in the solvent annealing chamber for 22 h,

86

the atomic % of the hydrocarbon peak slightly decreases while the atomic % of the beta-shifted

carbon and methoxy group carbon increases. The concentration of PMMA at the surface

increases over time. The exact concentration of PS present at the surface cannot be determined,

since there is no peak associated with PS that can be distinguished from the PMMA peaks. In

turn, the exact concentration of PMMA cannot be determined; however, since PMMA has

distinguishable peaks, the presence or absence of PMMA can be seen as well as the relative

composition. Angle-resolved XPS cannot give an exact value either, since the surface

contamination with carbon and oxygen will affect the peak intensity and the film might not have

a homogeneous composition throughout. Thus, there might be a very thin layer of PMMA at the

surface of the film or more likely at the bottom of the film.

Table 5.3 XPS analysis of samples with cylindrical and no pattern morphologies over time.

Peak Binding

energy (eV)

Cylindrical

pattern 2h

annealing

Atomic %

No surface

structure 2h

annealing

Atomic %

Cylindrical

pattern 22h

annealing

Atomic %

No surface

structure 22h

annealing

Atomic %

C-C/C-H 285.0 59.5 46.2 47.6 44.5

Beta-shifted C 286.0 14.6 23.9 23.2 26.5

C-O 287.2 14.9 16.5 14.9 17.0

O-C=O 289.5 11.0 13.4 14.3 12.0

The segment-segment (Flory-Huggins) interaction parameter (χ) is inversely proportional to

temperature. As reported in the literature,23 for nondeuterated PS-b-PMMA χ can be

approximated by

χ = (0.0282 ± 0.002) + (4.46 ± 0.6) / T Equation 5.2

The value of χN for the 130-133 copolymer is above the order-disorder transition for both

temperatures as is shown in Table 5.4. The expected pattern would be lamellar. There is no

87

apparent difference between the values at room temperature and low temperature. However,

these values are approximations only, and in the case of thin films other factors come into play as

discussed in the Introduction. The interaction parameter does affect the formation of the pattern,

and it is expected to increase as the temperature is decreased; however, the extent to which this

effect takes place is not large for this temperature range.

Table 5.4 Characteristics of the PS-b-PMMA diblock copolymers. ƒPS is the volume fraction of the PS block calculated from ƒPS = (wPS/ρPS) / ( (wPS/ρPS) + (1 - wPS)/ρPMMA) by using the following densities for the PS and PMMA block: ρPS = 1.05 g cm-3 and ρPMMA = 1.18 g cm-3.17, 24 χ is determined from χ = 0.0282 + 4.46/T as reported in literature for PS-b-

PMMA.23

Label N ƒPS χN at 24 °C χN at 2 °C

160-160 3140 0.50 136 139

130-133 2580 0.49 111 115

105-106 2070 0.50 89 92

66-63.5 1270 0.51 55 56

52-52 1020 0.50 44 45

The difference in pattern probability for the low and room temperature cases could be explained

by a kinetic effect. There is an increase in the mixture of cylinders parallel and perpendicular to

the substrate, at 24 °C than at 2 °C after 2 h of acetone vapor annealing, indicating that the

mobility of the polymer is higher at 24 °C. This increased mobility can be explained by the

thermal motion of the polymer and the saturated vapor pressure of acetone. The thermal motion

of the polymer at 24 °C is greater than at 2 °C, leading to higher chain mobility. The saturated

vapor pressure of acetone at 24 °C is nearly 3 times higher than that at 2 °C; thus, more acetone

is expected to dissolve in the PMMA domains at 24 °C, also leading to higher mobility. The

volume concentration of a gas, c, in a polymer can be approximated by Henry’s law

c = Sp Equation 5.3

88

where S is the Bunsen solubility constant and p is the partial pressure.20,25 The solubility does

not vary greatly for different polymers for a specific gas. The solubility of the gas can be

approximated for amorphous polymers using the boiling point of the gas, Tb, by

log S = -2.1 + 0.0123Tb Equation 5.4

The boiling point of acetone is 330 K; thus, the expected maximum concentration for PS and

PMMA decreases from 27 cm3 of gas/cm3 of polymer at room temperature to 9.4 cm3 of gas/cm3

of polymer at the lower temperature. The degree of copolymer swelling will be lower at the

lower temperature. In this study, the polymer films were taken out of the solvent annealing

chamber after 2 h in order to freeze the surface morphology at a stage where the cylinders are

perpendicular to the substrate.

Kinetic effects explain the formation of more cylinders parallel to the substrate at 24 °C than at 2

°C; however, thermodynamics may explain the difference in the fine structural difference at

these two temperatures. In particular, the difference of samples whose major composition is

cylinders perpendicular to the substrate were examined in detailed since it is much easier to

characterize these cylindrical domains quantitatively. The surface coverage of PMMA in the PS

matrix for the 130-133 PS-b-PMMA was investigated. In the AFM height images the taller

features are PS domains and the shorter domains are PMMA; however, depending on the choice

of the threshold height, the surface coverage changes considerably. In addition, the absolute

height values differ from scan to scan, such that a single value cannot be chosen as the threshold.

Thus, the images were first flattened by applying plane fits in both x and y directions with a

mask that includes only the PS matrix, allowing for the use of a single threshold value for the

entire image. Then a surface height histogram was created as seen in Figure 5.5A, which shows

a distribution composed of more than one component. The peak at higher x value corresponds to

the topographically higher PS matrix, while the lower portion is related to the PMMA cylinders.

A Gaussian fit to the higher peak to each scan was made, and each scan was offset such that the

Gaussians align; thus, the PS matrix was at exactly the same height for the different images. In

order to see the compositional difference between samples, the normalized accumulative

histogram of the height topography was used as shown in Figure 5.5B, which gives the

percentage of PMMA coverage at an arbitrary threshold value, the x-axis.

89

a)

b)

Figure 5.6 130-133 PS-b-PMMA films a) Surface height histogram, and b) Normalized accumulative histogram of the height topography. T – Temperature, R – Room.

In the case of the 130-133 PS-b-PMMA films, there is a 6 - 10 % more surface coverage by

PMMA domains at 24 °C than at 2 °C across a broad range of threshold values, showing that the

difference in surface coverage is systematic and is relatively independent of the objective choice

of the threshold value. The number of cylinders per μm2 at 24 °C was also higher than at 2 °C by

14 % on average. The apparent PMMA surface coverage falls between 40 and 52 %. For

example, if the threshold dividing PS and PMMA was arbitrarily defined as the height that yields

50 % PMMA coverage at 24 °C, then the surface coverage of PMMA at 2 °C is 40 % as shown

in Table 5.5. The size of the cylinder domains does not vary; however, the PS-b-PMMA

interfacial length per μm2 at 24 °C is 25 % higher than at 2 °C. The significant increase of

PMMA coverage and interfacial length per μm2 was a mixed result of swelling due to more

acetone absorption in the PMMA domains and the lowered PS-b-PMMA interfacial tension at

the higher temperature. A linear fit to the PS-b-PMMA interfacial tension as a function of

temperature shows that the interfacial tensions at 2 and 24 °C are roughly 3.57 and 3.29 mN/m,

90

respectively.26 This change is small; however, it is not insignificant in determining the surface

morphology of the polymer thin film.

Table 5.5 Example of characteristics of the 130-133 PS-b-PMMA diblock copolymers when the height threshold value yields 50 % PMMA surface coverage at 24 °C.

Temperature Surface

Coverage

Number of

cylinders

per µm2

Cylinder

diameter (nm)

PS-PMMA interface

length per unit area

(1/µm)

24°C 50% 100 ± 2 80.0 ± 0.5 25.0 ± 0.2

2°C 40% 86 ± 3 77.2 ± 0.9 20.7 ± 0.4

The slower polymer mobility due to lower temperature and less dissolved acetone in the polymer

film causes the morphology at 2 °C to be predominantly cylinders perpendicular to the substrate,

in comparison to 24 °C. Therefore, eventually the morphology evolves to the equilibrium

lamellar structure. In the case of room temperature annealing, with time the morphology

changes from cylinders perpendicular to the substrate to lamellae perpendicular to the substrate

and finally to lamellae parallel to the substrate after 10.5 days as shown in Figure 5.6. For the

low-temperature annealing case, with time the morphology changes from cylinders perpendicular

to the substrate to lamellae parallel to the substrate after 13 days as shown in Figure 5.7. The

films dewet with time for both cases; however, the dewetting is slower at the low temperature

than at room temperature. Even though the evolution of the overall surface morphology is

ultimately driven by thermodynamics from cylinders to lamellae, the local structure around each

cylinder is likely to reside in a local free energy minimum. The time scale of the local

restructuring around single cylinders should be considerably faster than the global restructuring

rate, as the global restructuring that involves joining of separated cylinder to form lamellar

structure requires significant energy barrier crossing. Thus, it is reasonable to explain the size of

individual cylinders by thermodynamic considerations as they are in local equilibrium, while

explaining the global morphology by considering kinetics, since the system has not reached a

global energy minimum, the rate as well as the time allowed for the system to evolve will

determine its morphological outcome.

91

a) b)

c) d)

e)

Figure 5.7 AFM height images of PS-b-PMMA film solvent annealed at room temperature, 24ºC, a) Cylinders perpendicular to the substrate after 2 h to 5 days of annealing, b) Cylinders both perpendicular and parallel to the

substrate after 6 days to 8 days of annealing, c) Lamellae perpendicular to the substrate after 8.5 days of annealing, d) Lamellae both perpendicular and parallel to the substrate after 9.5 days of annealing, and e) Lamellae parallel to

the substrate after 10.5 days of annealing. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

92

a) b)

c) d)

e) f)

Figure 5.8 AFM height image of PS-b-PMMA film solvent annealed at low temperature, 2ºC, a) Cylinders perpendicular to the substrate after 2 h to 5 days of annealing, b) Spheres after 6.5 days to 8.5 days of annealing, c)

Spheres and lamellae perpendicular to the substrate after 9.5 days of annealing, d) Lamella perpendicular to the substrate after 10 days of annealing, e) Lamellae both perpendicular and parallel to the substrate after 11 days to

12.5 days of annealing, and f) Lamella parallel to the substrate after 13 days of annealing. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

93

5.4.2 Pattern Spacing and Dimensions

Low-temperature solvent annealing was used to fabricate patterned films from PS-b-PMMA

diblock copolymers of different molecular weights. Four additional diblock copolymers were

used, and they all produced cylindrical domains perpendicular to the substrate as shown in

Figure 5.8. As before, the brighter areas in the image correspond to the PS matrix, while the

darker areas correspond to the PMMA cylindrical domains. Figure 5.9A shows the percent of

samples that self-assembled into a particular pattern for the different copolymers at the two

annealing temperatures. The low-temperature annealing had a large effect on the yield of this

pattern with cylinders perpendicular to the substrate for the lower molecular weight copolymers.

For example, the 52-52 PS-b-PMMA copolymer assembled into this pattern only at the low

annealing temperature. The product χN decreased as the molecular weight of the copolymer

decreased, as is shown in Table 5.4; however, the values for χN are still above the value for the

order-disorder transition, 10. Statistically, the advancing water contact angle did not change as

the molecular weight increased, remaining constant with a value of 83 ± 3°.

The center-to-center cylinder spacing D and the cylinder diameter d were measured from the

height images and plotted as a function of PS block molecular weight as shown in Figure 5.9B.

As expected, both the spacing and the cylinder diameter increased as the molecular weight of the

PS-b-PMMA diblock copolymer increased. Helfand et al. developed a theory for equilibrium

patterns to calculate the domain size and spacing using the narrow-interface-approximation block

copolymer theory for spheres,27 lamellae,28 and cylinders.29 The domain size and spacing

increase with an increase in molecular weight. For the samples studied here Helfand’s equation

cannot be used to get an exact value since there is a trapped morphology; however, the overall

trend is still present. As can be seen from both Figures 5.9 and 5.10, low-temperature annealing

combined with different block copolymer molecular weights can provide samples for

experiments in which different domain spacing and size are desired.

94

a) b)

c) d)

Figure 5.9 AFM height images of PS-b-PMMA films annealed at 2°C a) 160-160, b) 105-106, c) 66-63.5, and d) 52-52. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

95

a)

b)

Figure 5.10 a) Type of structures present after solvent annealing at 24 °C and 2 °C for different PS-b-PMMA copolymer as a function of PS Mn and as a percent of samples; b) D, center-to-center cylinder spacing, and d,

cylinder diameter, as a function of PS Mn for different PS-b-PMMA copolymers.

5.5 Conclusions PS-b-PMMA diblock copolymers were used to fabricate patterned surfaces with cylinders

perpendicular to the substrate. In this work, a method of solvent annealing using a low-

temperature environment was demonstrated as well as investigated the effectiveness of various

approaches to explain the phase behavior of the diblock copolymer films produced by this

method. The lower temperature annealing improved the yield of patterned surfaces without

affecting the pattern type and properties. The increase in patterning by surface perpendicular

96

cylinders is due to slower polymer mobility caused by a lower temperature and less dissolved

acetone in the polymer film. As a further demonstration, this procedure was applied successfully

and increased the yield of surface perpendicular cylinders for other PS-b-PMMA diblock

copolymer molecular weights. The cylinder dimensions and spacing were found to increase as

the molecular weight of the copolymer increased.

5.6 References (1) Bates, F. S.; Fredrickson, G. H. Annu. Rev. Phys. Chem. 1990, 41, 525–557.




(5) Hashimoto, T.; Tanaka, H.; Hasegawa, H. Macromolecules 1990, 23, 4378-4386.

(6) Orso, K. A.; Green, P. F. Macromolecules 1999, 32, 1087-1092.

(7) Jeong, U.; Kim, H. C.; Rodriguez, R. L.; Tsai, I. Y.; Stafford, C.M.; Kim, J. K.; Hawker, C. J.; Russell, T. P. Adv. Mater. 2002, 14, 274-276.

(8) Stuen, K. O.; Thomas, C. S.; Liu, G.; Ferrier, N.; Nealey, P.F. Macromolecules 2009, 42, 5139-5145.

(9) Yaws, C. L.; Narasimhan, P. K.; Gabbula, C. Yaws’ Handbook of Antoine Coefficients for Vapor Pressure; Knovel: Norwich, 2005.

(10) Green, P. F.; Christensen, T. M.; Russell, T. P.; Jerome, R. J. Chem. Phys. 1990, 92, 1478-1482.

(11) Peng, J.; Wei, Y.; Wang, H.; Li, B.; Han, Y. Macromol. Rapid Commun. 2005, 26, 738-743.


(13) Mueller, K.; Yang, X.; Paulite, M.; Fakhraai, Z.; Gunari, N.; Walker, G. C. Langmuir 2008, 24, 6946-6951.

(14) Ting, Y.-H.; Park, S.-M.; Liu, C.-C.; Liu, X.; Himpsel, F. J.; Nealey, P. F.; Wendt, A. E. J. Vac. Sci. Technol. B 2008, 26, 1684-1689.

(15) Thurn-Albrecht, T.; Steiner, R.; DeRouchey, J.; Stafford, C. M.; Huang, E.; Bal, M.; Tuominen, M.; Hawker, C. J.; Russell, T. P. Adv. Mater. 2000, 12, 787-791.

(16) Müller-Buschbaum, P; Schulz, L.; Metwalli, E.; Moulin, J.-F.; Cubitt, R. Langmuir 2008, 24, 7639-7644.

(17) Brandrup, J.; Immergut, E. H.; Grulke, E. A.; Abe, A.; Bloch, D. R. Polymer Handbook, 4th ed.; John Wiley & Sons: New York, 1999.

97

(18) Chen, S.-A. J. Appl. Polym. Sci. 1971, 15, 1247-1266.

(19) Chen, Y.; Huang, H.; Hu, Z.; He, T. Langmuir 2004, 20, 3805-3808.

(20) Van Krevelen, D. W. Properties of Polymers; Elsevier Scientific Publishing Company: Amsterdam, 1976.

(21) Kim, S. H.; Misner, M. J.; Xu, T.; Kimura, M.; Russell, T. P. Adv. Mater. 2004, 16, 226-231.

(22) Ton-That, C.; Shard, A. G.; Teare. D. O. H.; Bradley, R. H. Polymer 2001, 42, 1121-1129.

(23) Yue, Z.; Sivaniah, E.; Hashimoto, T. Macromolecules 2008, 41, 9948-9951.

(24) Guo, R.; Huang, H.; Du, B.; He, T. J. Phys. Chem. B 2009, 113, 2712-2724.

(25) Haward, R. N. The Physics of Glassy Polymers, John Wiley & Sons: New York, 1973.

(26) Carriere, C. J.; Biresaw, G.; Sammler, R. L. Rheol. Acta 2000, 39, 476-482.

(27) Helfand, E.; Wasserman, Z. R. Macromolecules 1978, 11, 960-966.



98

6 Polystyrene-block-poly(2-vinyl pyridine) and Polystyrene-block-poly(methyl methacrylate) Surfaces with a Range of Nanopatterns for Inhibition of Algae Zoospores and Diatoms

6.1 Overview

Polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) and UV treated PS-b-P2VP mixed with

benzophenone diblock copolymers nanopatterned surfaces were fabricated on silicon substrates,

whereas polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymer

nanopatterned surfaces were fabricated on silicon and nylon coated silicon or glass substrates.

The diblock copolymers showed self-assembled cylindrical structures orientated perpendicular to

the substrate varying in size from 30 nm to 80 nm. In addition, patterns such as lamellar and

spherical were also studied for the PS-b-PMMA case. The films were annealed using

appropriate solvent gradients. In particular, the PS-b-PMMA films were annealed by a new low

temperature solvent annealing method. PS-b-P2VP and UV PS-b-P2VP thin films showed

reduced settlement of Ulva linza zoospores for all sizes expect the PS-b-P2VP 172-42 case.

There was a slight decrease in settlement as the pattern dimension increased. Overall, the

nanopatterning effect showed a moderate influence on zoospore settlement. In the case of PS-b-

PMMA copolymers, all films showed reduced zoospore settlement except PS on silicon

substrates and nylon coated silicon. The PS-b-PMMA 160-160 showed an intermediate

settlement on the silicon substrates. No apparent trend was observed with a change in pattern

dimensions. PMMA and PS-b-PMMA 130-133 on nylon coated glass slides showed the lowest

settlement in Navicula perminuta diatoms. Overall, for the PS-b-PMMA case, the

hydrophobicity effect was predominant in determining the final organism attachment, meanwhile

the nanopatterning showed a weak or non-existent influence. The surface structure of the

polymers films were investigated using Atomic Force Microscopy (AFM).

99

6.2 Introduction Untreated artificial surfaces such as ships’ hulls or aquaculture nets immersed in seawater

accumulate biofoulers such as algae zoospores, diatoms or barnacle cyprids soon after

immersion.1-3 Algae zoospore can settle on both pristine surfaces and surfaces already covered

with a biofilm.4 Zoospores are motile organisms using their four flagella to actively approach

and explore the surface before becoming permanently attached.5 Another type of algae, the

diatom, is an early unicellular surface colonizer which are a main part of the slime layers on

marine biofouled surfaces.4 On a ship’s hull, these slime layers lead to an increase in fuel cost

up to 16 % due to an increase in hydrodynamic drag.6 Diatoms are made of a silica cell wall or

frustule around the cell protoplast.7 The frustule is made of overlapping valves and a number of

girdle bands. A common diatom biofouler is the Navicula species characterized by bilateral cell

symmetry with an elongated slit in each valve known as the raphe. Extracellular polymeric

substances are secreted from the raphe and play a role in adhesion and motility through gliding.

Gliding is achieved using an adhesion complex which connects the extracellular adhesive strands

adhered to the substrates and the actin filaments - myosin molecules complex besides the raphe

opening. Diatoms, unlike zoospores, can probe the surface by gliding along it in order to find a

suitable location for settlement. However, diatoms are not motile in seawater like zoospores;

they reach a surface with the help of gravity or water currents not by active movement. 8-9

Diblock copolymers can self-assemble in a variety of ordered structures such as regular spaced

cylinders in a hexagonal pattern at the nanoscale.10-13 These nanopatterns permit the probing of

biological responses on specific length scales. PS-b-P2VP and PS-b-PMMA diblock copolymer

patterns with a range of pattern dimensions were fabricated on a number of substrates and

assessed for zoospore antifouling properties. In addition, PS-b-PMMA patterns of different

dimensions and types were evaluated using diatoms. Various pattern dimensions were obtained

by varying the molecular weight of the constituting blocks of the copolymers. These

experiments are aimed at further exploring the properties of diblock copolymers in order to

discover optimal pattern dimensions to organism settlement inhibition and the efficacy across

marine species, as well as the extent of nanoscale patterning effect on settlement as opposed to

hydrophobicity. A major challenge in antifouling coating design is that a surface that is optimal

for inhibiting settlement of one type of biofoulant may not work well for another. Thus, the

100

diatom settlement assay presented in this chapter offers a new viewpoint on the efficacy of the

coatings exposed to a different marine organism.


6.3.1 Materials

Various PS-b-P2VP diblock copolymers (Polymer Source) and the photoinitiator benzophenone

(BP; Sigma Aldrich) were used as received. The properties of the PS-b-P2VP copolymers can be

seen in Table 6.1. Thin polymer films were prepared by spin coating 0.3 wt% toluene solutions

of the diblock copolymers or the diblock copolymers mixed with BP (1:1 w/w) on silicon

substrates at 2000 rpm for 45 s. The silicon substrates were prepared by cleaning in piranha

solution (3:1 v/v concentrated H2SO4 : 30 % H2O2) for 10 min. Caution: Piranha is a very strong

oxidant. The thin films were solvent vapor annealed using toluene and chloroform (1/1 v/v) for

2 h. The PS-b-P2VP with BP films were UV irradiated using a Mercury Arc Lamp (Pen-Ray,

90-0012-01) with an intensity of 15 mW/cm2 for 5 min in air.

Table 6.1 PS-b-P2VP diblock copolymers properties. Mn is the number average molecular weight and the unit of measure is g/mol. PDI is the polydispersity index.

Label for PS-b-P2VP PS Mn P2VP Mn PDI

75-21 75000 21000 1.06

172-42 172000 42000 1.08

325-92 325000 92000 1.06

a) b)

Figure 6.1 a) Diblock copolymer PS-b-P2VP, and b) Photoinitiator BP.

101

PS-b-PMMA diblock copolymers (Polymer Source) were used as received. The properties of the

diblock copolymers are shown in Table 6.2. Thin polymer films were prepared by spin coating 1

wt% toluene solutions on various substrates at 2000 rpm for 45 s. The copolymers were coated

on piranha cleaned silicon substrates and nylon coated glass slides. In addition, the PS-b-PMMA

105-106, 66-63.5 and 52-52 were coated on nylon coated silicon substrates. Nylon substrates

were prepared by dissolving a nylon 6, 6 piece (Thyssenkrupp Materials) and recasting it. Thin

nylon films were prepared by spin coating 0.3 wt% formic acid solutions on piranha cleaned

silicon or glass slides at 2000 rpm for 45 s. The polymer thin films were solvent vapor-annealed

using acetone for 2 h at 2 °C.


Label for PS-b-PMMA PS Mn PMMA Mn PDI

160-160 160000 160000 1.09

130-133 130000 133000 1.10

105-106 105000 106000 1.13

66-63.5 66000 63500 1.08

52-52 52000 52000 1.09

a)

b)

Figure 6.2 a) Diblock copolymer PS-b-PMMA, and b) Nylon 6, 6.

102

Homopolymers PS (Polymer Source), P2VP (Polymer Source) and PMMA (Polymer Source)

were used as received. Random copolymers polystyrene-co-2-vinyl pyridine (P(S-r-VP);

Polymer Source) and polystyrene-co-methyl methacrylate random copolymer (P(S-r-MMA);

Polymer Source) were also used. The properties of the polymers can be seen in Table 6.3. Thin

films were prepared by spin coating 1 wt% toluene solutions on piranha clean silicon substrates

at 2000 rpm for 45 s. In addition, PS, PMMA and P(S-r-MMA) were also spin coated on nylon

coated silicon substrates and glass slides.

Table 6.3 Properties of homopolymers and random copolymers. Mn is the number average molecular weight and Mw is the weight average molecular weight. The unit of measure is kg/mol. PDI is the polydispersity index.

Label Mn Mw PDI

PS 131 138 1.05

P2VP 22 24 1.09

PMMA 106 114.5 1.08

P(S-r-2VP) 75 128 1.7

P(S-r-MMA) 102 173 1.7

a) b)

c)

Figure 6.3 a) PS, b) P2VP, and c) PMMA.

103


All polymer films were imaged using AFM to examine the surface topography. The AFM



frequencies in the range 280-320 kHz and a spring constant of 40 N/m were used.







zoospore suspension in the dark at ~ 20°C. Each dish contained one polymer substrate on silicon

or nylon coated silicon. After 45 min the substrates were washed in seawater to remove

unsettled zoospores. Substrates were fixed using 2.5% glutaraldehyde in seawater. The density

of zoospores attached to the surface was counted on each of the replicate substrates using an

image analysis system (Imaging Associates Ltd.) attached to an epifluorescence microscope

(Zeiss, Aksioskop 2). Spores were visualized by autofluorescence of chlorophyll. Counts were

made for 30 fields of view (each 0.17 mm2) on each polymer.

6.3.4 Navicula Diatom Settlement Assay

Navicula perminuta cells were cultured in F/2 medium contained in 250 ml conical flasks. After

3 days the cells were in log phase growth. Cells were washed 3 times in fresh medium before

harvesting and diluted to give a suspension with a chlorophyll a content of approximately 0.25

μg ml-1. Samples were equilibrated in 0.22 µm filtered artificial seawater for one hour before

testing. Cells were settled in individual dishes containing 10 ml of suspension at ~20oC on the

laboratory bench. Each dish contained one polymer substrate on nylon coated glass slides. After

2 h the slides were exposed to a submerged wash in seawater to remove cells which had not

104

attached. The immersion process avoided passing the samples through the air-water interface.

Samples were fixed in 2.5% glutaraldehyde, air dried and the density of cells attached to the

surface was counted on each slide using an image analysis system attached to a fluorescence

microscope. Counts were made for 30 fields of view (each 0.15 mm2) on each slide.


6.4.1 PS-b-P2VP Diblock Copolymers on Si Substrates

The morphology of PS-b-P2VP diblock copolymers thin films on silicon substrates is shown in

Figure 6.4. Figure 6.4A shows the AFM height image of PS-b-P2VP 75-21, whereas part B

shows the same diblock copolymer mixed with BP, UV irradiated and immersed 2 hours in

water. Figure 6.4C shows the PS-b-P2VP 172-42, while part D shows the equivalent diblock

copolymer mixed with BP, UV irradiated and immersed 2 hours in water. Finally, Figure 6.4E

shows the PS-b-P2VP 325-92, whereas part F shows the same diblock copolymer mixed with

BP, UV irradiated and immersed 2 hours in water. The morphology of the films is nanoscale

sized cylinders orientated perpendicular to the substrate. The brighter areas in the image

correspond with the PS matrix, while the darker areas correspond to the P2VP cylindrical

domains. The volume fraction of PS, ƒPS, is keep around 0.78 as the molecular weight of the PS

and P2VP blocks increases in order to maintain the cylindrical pattern of the system. The center-

to-center cylinder spacing and the cylinder diameter were measured from the height images and

can be seen in Table 6.4. The cylinder spacing and diameter increase as the molecular weight of

the blocks increase, 46 nm to 66 nm for the cylinder spacing and 30 nm to 48 nm for the

diameter. This increase in pattern dimensions is much smaller than for PS-b-PMMA diblock

copolymers.

Helfand et al. developed a theory for equilibrium patterns to calculate the pattern spacing and

size for cylinders and applied it to experimental data.20 The calculated values were sometimes

higher than the experimental values in particular for larger molecules. The discrepancies in

values are due to uncertainty in the copolymer physical constants and closeness to final

equilibrium dimensions. Similar copolymers in molecular weight to PS-b-P2VP includes a

polystyrene-block-polybutadiene (PS-b-PB) copolymer with a molecular weight of 25 kg/mol for

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PS and 76 kg/mol for PB with PS cylinders, a polyisoprene-block-polystyrene (PI-b-PS) with a

molecular weight of 48 kg/mol for PI and 173 kg/mol for PS with PI cylinders and a

polybutadiene-block-polystyrene PB-b-PS copolymer with a molecular weight of 115 kg/mol for

PB and 306 kg/mol for PS with PB cylinders. The pattern dimensions for PS-b-PB were 44.0 nm

for the spacing and 25.2 nm for the diameter experimental and 56.2 nm and 28.1 nm respectively

calculated. The pattern dimensions for PI-b-PS were 23 nm for the diameter experimental and

44.5 nm calculated. The pattern dimensions for PB-b-PS were 55 nm for the spacing and 33 nm

for the diameter experimental and 158 nm and 92.1 nm respectively calculated. The

experimental pattern dimensions do not increase much with an increase in molecular weight

similar to the PS-b-P2VP case. The higher increase observed in the PS-b-PMMA case is due to a

trapped morphology as opposed to a morphology close to or located at the equilibrium.

The advancing water contact angles slightly increase as the molecular weight increases for PS-b-

P2VP as seen in Table 6.5. The surfaces are hydrophobic similar to PS films. The difference in

contact angles between the diblock copolymers can be attributed to variations between samples.

In the case of PS-b-P2VP mixed with BP and UV irradiated films, the advancing water contact

angles are lower than for untreated PS-b-P2VP films. These surfaces are the most hydrophilic in

this study. The increase in hydrophilic character is due to oxygen incorporated groups during the

UV irradiation process.

106

a) b)

c) d)

e) f)

Figure 6.4 AFM height images of PS-b-P2VP films on silicon a) 75-21, b) 75-21 and BP after UV irradiation and 2 hours water immersion, c) 172-42, d) 172-42 and BP after UV irradiation and 2 hours water immersion, e) 325-92,

and f) 325-92 and BP after UV irradiation and 2 hours water immersion. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

107

Table 6.4 PS-b-P2VP nanopattern dimensions.

Label for PS-b-P2VP Center-to-center spacing (nm) Cylinder diameter (nm)

75-21 46 ± 3 30 ± 3

172-42 57 ± 4 38 ± 3

325-92 66 ± 4 48 ± 3

Table 6.5 Advancing water contact angles of polymers on silicon.

Label Contact Angle

PS 90 ± 2°

P2VP 61 ± 2°

P(S-r-VP) 87 ± 2°

PS-b-P2VP 75-21 86 ± 2°

PS-b-P2VP 172-42 91 ± 3°

PS-b-P2VP 325-92 91 ± 2°

PS-b-P2VP 75-21 + BP UV 39 ± 2°

PS-b-P2VP 172-42 + BP UV 25 ± 2°

PS-b-P2VP 325-92 + BP UV 34 ± 2°

108

In Figure 6.5 the height images of the respective homopolymers and random copolymer control

surfaces can be seen. Figure 6.5A shows a PS film, while part B shows a P2VP film. These

films show no surface structure as expected from pure homopolymers. The advancing water

contact angles are 90 ± 2° and 61 ± 2° respectively. Figure 6.5C shows a P(S-r-2VP) random

copolymer film. The random copolymer does not shows nanoscale structures too. The

advancing water contact angle of P(S-r-VP) is 87 ± 2°.

a) b)

c)

Figure 6.5 AFM height images of polymer films on silicon a) PS, b) P2VP, and c) P(S-r-2VP). Image sizes: 1 μm x 1 μm. Z range: 30 nm.

109

6.4.2 PS-b-PMMA Diblock Copolymers on Si Substrates

Figure 6.6 shows the height images of solvent annealed PS-b-PMMA diblock copolymers films

on silicon with various molecular weights. Figure 6.6A shows the morphology of PS-b-PMMA

52-52, part B shows PS-b-PMMA 66-63.5, part C shows PS-b-PMMA 105-106, part D shows

PS-b-PMMA 130-133 and finally part E shows PS-b-PMMA 160-160. The copolymer films

show nanoscale sized patterns of cylinders orientated perpendicular to the substrate. The

brighter areas in the image correspond with the PS matrix, while the darker areas correspond to

the PMMA cylindrical domains. The volume fraction of PS, ƒPS, is constant at 0.5 as the

molecular weight of the PS and P2VP blocks increases in order to maintain the trapped

cylindrical pattern of the system. The copolymer films are solvent vapor annealed in acetone at a

low temperature of 2 °C. This method is used to increase the yield of patterned samples for all

copolymer sizes in order to evaluate for antifouling properties using algae zoospores. A lower

temperature during annealing leads to less acetone dissolving and swelling the polymer films and

eventually to a lower mobility of the copolymer chains. The center-to-center cylinder spacing

and the cylinder diameter were measured from the height images and can be seen in Table 6.6.

The cylinder spacing and diameter increase as the molecular weight of the blocks increase from

55 nm to 113 nm for the cylinder spacing and from 34 nm to 82 nm for the diameter. The pattern

range of dimensions probes is larger than for PS-b-P2VP. The advancing water contact angles

are around 83 ± 2° as seen in Table 6.7. The surfaces are less hydrophobic than the PS films;

they are similar to PMMA and P(S-r-MMA).

110

a) b)

c) d)

e)

Figure 6.6 AFM height images of solvent annealed PS-b-PMMA films on silicon a) 52-52, b) 66-63.5, c) 105-106, d) 130-133, and e) 160-160. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

111



52-52 55 ± 4 34 ± 4

66-63.5 72 ± 4 45 ± 4

105-106 90 ± 4 62 ± 4

130-133 109 ± 4 74 ± 4

160-160 113 ± 4 82 ± 4

Table 6.7 Advancing water contact angles of polymers on silicon.

Label Contact Angle

PS 95 ± 2°

PMMA 81 ± 2°

P(S-r-MMA) 84 ± 2°

PS-b-PMMA 52-52 80 ± 2°

PS-b-PMMA 66-63.5 85 ± 2°

PS-b-PMMA 105-106 83 ± 2°

PS-b-PMMA 130-133 83 ± 2°

PS-b-PMMA 160-160 81 ± 2°

112

Figure 6.7 shows the height images of the appropriate homopolymers and random copolymer

control surfaces. Figure 6.7A shows a PS film, while part B shows a PMMA film. These films

show no surface structure since they consist of pure homopolymers. The advancing water

contact angles are 95 ± 2° and 81 ± 2° respectively. Figure 6.7C shows a P(S-r-MMA) random

copolymer film. In this case too there are no nanoscale structures at the surface. The advancing

water contact angle of P(S-r-MMA) is 84 ± 2°.

a) b)

c)

Figure 6.7 AFM height images of polymer films on silicon a) PS, b) PMMA, and c) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm.

113

6.4.3 PS-b-PMMA Diblock Copolymers on Nylon Coated on Si Substrates

The lower molecular weight PS-b-PMMA diblock copolymers were also spin-coated and solvent

annealed on nylon coated silicon as shown in Figure 6.8. Figure 6.8A shows the morphology of

PS-b-PMMA 52-52, while part B shows PS-b-PMMA 66-63.5 and part C shows the PS-b-

PMMA 105-106. The brighter areas in the images correspond to the PS matrix, while the darker

areas correspond to the PMMA domains made of cylinders orientated perpendicular to the

surface. The pattern dimensions increases as the molecular weight increases as seen in Table

6.7. The low temperature solvent annealing method was successfully applied to sample

patterning arrangement on new nylon substrates. The contact angles of these copolymer films

shown in Table 6.8 are lower than on silicon, 75 °C as opposed to 83°C, as observed in a

previous chapter due to a reduced attraction of the PMMA to the substrate.

a) b)

c)

Figure 6.8 AFM height images of PS-b-PMMA films on nylon coated silicon a) 52-52, b) 66-63.5, and c) 105-106. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

114

Table 6.8 Advancing water contact angles of polymers on nylon coated silicon.

Label Contact Angle

Nylon 49 ± 2°

PS 87 ± 2°

PMMA 72 ± 2°

P(S-r-MMA) 73 ± 2°

PS-b-PMMA 52-52 75 ± 2°

PS-b-PMMA 66-63.5 75 ± 2°

PS-b-PMMA 105-106 74 ± 2°

The control surfaces in this case too include homopolymers thin films and a random copolymer

surface as seen in Figure 6.9. In addition, the effect of the nylon on silicon substrate is also

evaluated. Figure 6.9A shows a nylon coating on a silicon substrate, while part B shows a PS

film, part C shows a PMMA film and part D shows a P(S-r-2VP) random copolymer film. The

nylon surface shows randomly distributed irregular nanosized globular structures. This surface

is the most hydrophilic in this study with an advancing water contact angle of 49 ± 2°. The PS,

PMMA and P(S-r-2VP) films show no surface structures. The advancing water contact angles

for these surfaces are 87 ± 2°, 72 ± 2° and 81 ± 2° respectively, lower than on pure silicon

surfaces.

115

a) b)

c) d)

Figure 6.9 AFM height images of polymer films on nylon coated silicon a) Nylon, b) PS, c) PMMA, and d) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm.

6.4.4 PS-b-PMMA Diblock Copolymers on Nylon Coated Glass Substrates

All five PS-b-PMMA diblock copolymers were spin coated on nylon coated glass slides and

solvent annealed as shown in Figure 6.10 in order to investigate their efficacy across marine

species using another organism, the diatom. Figure 6.10A shows the morphology of PS-b-

PMMA 52-52 films, part B of PS-b-PMMA 66-63.5 films, part C of PS-b-PMMA 105-106

films, part D of PS-b-PMMA 130-133 films and part E of PS-b-PMMA 160-160 films. These

films also display the cylindrical pattern orientated perpendicular to the substrate pattern similar

to the one observed for previous substrates. The brighter areas in the images correspond to the

PS matrix, while the darker areas correspond to the PMMA domains made of cylinders

orientated perpendicular to the surface. This trapped morphology is achieved in sufficient

quality and quantity using low temperature acetone solvent annealing. The advancing water

contact angle of these thin films is around 74 ± 2° similar to previous copolymers on nylon

116

coated silicon substrates but lower than for copolymers on silicon substrate caused by a reduced

attraction of the PMMA block to the substrate.

a) b)

c) d)

e)

Figure 6.10 AFM height images of PS-b-PMMA films on nylon coated glass slides a) 52-52, b) 66-63.5, c) 105-106, d) 130-133, and e) 160-160. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

117

Table 6.9 Advancing water contact angles of polymers on nylon coated glass slides.

Label Contact Angle

Nylon 58 ± 2°

PS 93 ± 2°

PMMA 73 ± 2°

P(S-r-MMA) 76 ± 2°

PS-b-PMMA 52-52 71 ± 2°

PS-b-PMMA 66-63.5 74 ± 3°

PS-b-PMMA 105-106 76 ± 2°

PS-b-PMMA 130-133 74 ± 2°

PS-b-PMMA 160-160 72 ± 2°

PS-b-PMMA 130-133 Before annealing 89 ± 2°

PS-b-PMMA 130-133 Cyl + Lam 74 ± 2°

PS-b-PMMA 130-133 Spherical 77 ± 2°

PS-b-PMMA 130-133 Lamellae 77 ± 2°

Additionally, the type of PS-b-PMMA 130-133 patterns is also varied in this study as can be

seen in Figure 6.11. The PS-b-PMMA films before solvent annealing is shown in Figure 6.11A

displaying a wormlike surface morphology. This surface is more hydrophobic than solvent

annealed films, advancing water contact angle of 89 ± 2°. The fast solvent evaporation during

spin coating decreases the time for chain rearrangement leading to a different morphology than

118

the equilibrium morphology. The PS block is attracted to the air interface due to a lower surface

energy, while the more polar PMMA block is attracted to the substrate.21 In the case of 130-133

PS-b-PMMA copolymer, 62 % of films show a mixture of cylinders parallel and perpendicular to

the substrate, which can also be called cylinders and lamellae patterns, when solvent annealed at

a low temperature. This morphology is shown in Figure 6.11B.

Figure 6.11C shows a spherical morphology and part D shows lamellae morphology. These

structures can be achieved by solvent annealing at a low temperature for a long time as shown in

the previous chapter, days instead of hours; however, the films dewet with time and a uniform

polymer layer is necessary for the settlement assay. Thus, different methods were used to

prepare these samples still using the low temperature annealing. In the case of the spherical

pattern, the amount of solvent in the annealing chamber was reduced by half similar to the

strategy used by Peng et al to obtain this pattern.22-23 The lamellae pattern was obtained by

increasing the amount of polymer placed on the substrate during spin coating leading to a

slightly thicker film similar to the study by Knoll et al showing a change in the polymer pattern

with increasing the film thickness.24

The advancing water contact angles for the solvent annealed diblock copolymers in Figure

6.11B, C and D are 74 ± 2° for part B and 77 ± 2° for parts C and D. The copolymer chains

rearrange during solvent annealing resulting in an increase in the presence of the PMMA block at

the surface.

119

a) b)

c) d)

Figure 6.11 AFM height images of PS-b-PMMA 130-133 films on nylon coated glass slides a) Before solvent annealing, b) Cylinders orientated parallel and perpendicular to the substrate (Cyl + Lam surface), c) Spheres, and d)

Lamellae. Image sizes: 1 μm x 1 μm. Z range: 30 nm.

Control surfaces include nylon coated glass slides and polymer spin coated on these nylon coated

glass slides, PS, PMMA and P(S-r-MMA). Figure 6.12A is the height image of a nylon coated

glass slides showing nanosized surface structures. These surfaces are the most hydrophilic of

this study, advancing water contact angle of 58 ± 2°. Figure 6.12B shows the height image of a

PS film, while part C shows a PMMA film and part D shows a P(S-r-MMA) film. These

polymer thin films show no surface structure. The advancing water contact angles are 93 ± 2°,

73 ± 2° and 76 ± 2° respectively.

120

a) b)

c) d)

Figure 6.12 AFM height images of polymer films on nylon coated glass slides a) Nylon, b) PS, c) PMMA, and d) P(S-r-MMA). Image sizes: 1 μm x 1 μm. Z range: 30 nm.

6.4.5 Settlement of Algae Zoospores on PS-b-P2VP Diblock Copolymers

Silicon substrates are flat surfaces minimizing the influence on the overall polymer film

roughness coated on top. In addition, they are typically used for diblock copolymer self-

assembly resulting in a vast body of knowledge on theory and experiments explaining the factors

affecting phase separation. PS-b-P2VP block copolymers with different pattern dimensions and

same pattern type were fabricated on silicon. The settlement density of zoospores attached to

these copolymer thin films and polymer controls on silicon substrates is shown in Figure 6.13A.

The settlement density was greatest on the hydrophobic PS surface and lower on the more

hydrophilic P2VP. These surfaces displayed no surface structure. This trend corresponds with

previous literature where settlement density on smooth surfaces correlated with increasing

hydrophobicity.14-15 The random copolymer P(S-r-VP) and PS-b-P2VP 172-42 polymer films

showed and intermediate settlement density. The number of attached spores was similar on the

121

PS-b-P2VP 75-21 and 325-92 and the three PS-b-P2VP UV irradiated films. The settlement

density was slightly lower on the higher molecular weights copolymers.

The large difference in contact angle between the nanopatterned non-UV treated and UV treated

diblock copolymers did not affect the settlement as expected from previous studies. An

exception to this is the PS-b-P2VP 172-42 coating that had almost double the settled zoospores.

All of the non-UV treated samples were hydrophobic with a similar contact angle to PS and P(S-

r-2VP), whereas the UV-treated samples were the most hydrophilic in this case as shown in

Figure 6.13B. The UV-treated samples were expected to have a low settlement density based on

hydrophobicity as observed, while the non-UV treated samples were expected to have a higher

settlement density. Overall, the change in nanopattern dimensions did not affect zoospore

settlement which could be due to the small range in pattern dimensions being probed; once more

the PS-b-P2VP 172-42 was an exception. The nanopatterns in general showed the lowest density

in zoospore attachment suggesting a stronger effect on the settlement than the hydrophobicity.

The assay results imply that there is something special with the PS-b-P2VP 172-42 coating or

that the other non-UV treated samples, PS-b-P2VP 75-21 and 325-92, were abnormal. There

was nothing irregular observed in sample preparation, characterization and testing; however, the

possibility of something wrong with the samples cannot be discarded entirely. If hydrophobicity

alone was responsible for the settlement density trend, than the PS-b-P2VP 75-21 and 325-92

films should have shown the same behavior as the 172-42 film and also all of them would have

had a high zoospore settlement. If nanopatterning alone was responsible for the settlement

density trend, than the PS-b-P2VP 172-42 film could have shown this special behavior, however,

the UV treated counterpart should also have shown this behavior. Interplay between

nanopatterning dimensions and surface hydrophobicity could explain the behavior for the 172-42

case with the hydrophobicity element having a stronger influence on the settlement at this

particular dimension than for the other samples where the nanopatterning effect seems to

predominate.

122

a)

b)

Figure 6.13 a) The density of attached Ulva spores on polymers on silicon. Each point is the mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95% confidence limits. and b) advancing water contact angle for

polymers on silicon.

123

6.4.6 Settlement of Algae Zoospores on PS-b-PMMA Diblock Copolymers

The settlement density of zoospores attached to PS-b-PMMA diblock copolymers thin films of

various pattern dimensions and respective controls coated on silicon substrates is shown in

Figure 6.14A. The hydrophobic PS surface had the highest settlement density analogous to the

hydrophobicity trend from literature on smooth and uncharged surfaces.14-15 The PS-b-PMMA

160-160 showed a lower settlement than the PS surface. All of the other surfaces, PMMA, P(S-

r-MMA) and the rest of the PS-b-PMMA diblock copolymers, showed a low number of attached

zoospores. In particular, the PS-b-PMMA 66-63.5 showed a slightly lower settlement value.

The surfaces have similar hydrophobic property except for PS which is more hydrophobic as can

be seen in Figure 6.14B. In this case, the hydrophobicity effect not the nanopattern appears to

play an important role in settlement. If there is an effect from the nanopatterning, it might be too

small to be evident in the results. An exception to this hydrophobicity trend is the PS-b-PMMA

160-160 where the nanopattern effect dominates and leads to an increase in zoospore attachment

which has not been observed before. There were large differences in zoospore attachment

between replicate samples for this copolymer size leading to large error bars divergent from the

other samples. There is a stronger possibility for pattern abnormality in this case.

124

a)

b)



125

The PS-b-PMMA 52-52, 66-63.5 and 105-106 diblock copolymers and control polymers were

also spin coated on nylon coated on silicon substrates in order to mimic nylon aquaculture nets.

The larger diblock copolymers PS-b-PMMA 130-133 and 160-160 were not included due to time

constrains in sample preparation corresponding to the end of the algae zoospore collection

season; however, the PS-b-PMMA 105-105, 130-133 and 160-160 were previously tested for

antifouling properties as shown in Chapter 4. The settlement density of tested samples is shown

in Figure 6.15A. Once again, the hydrophobic PS surface had the highest settlement density

corresponding to the hydrophobicity trend from literature.14-15 The other surfaces, nylon,

PMMA, P(S-r-MMA) and the PS-b-PMMA diblock copolymers, showed a low number of

attached zoospores. In particular, the PS-b-PMMA 105-106 and nylon surfaces showed a

slightly lower settlement value. The PMMA films showed a lower zoospore settlement than

observed previously on nylon coated silicon substrates.

The hydrophobic behaviors of these polymer surfaces are similar except for PS which is more

hydrophobic and nylon which is the most hydrophilic as can be seen in Figure 6.15B. The

influence in zoospore settlement is dominated by the hydrophobic effect instead of the

nanopatterning. Once more, the effect from the nanopatterning is not strongly evident with these

surfaces. The chemical composition of the diblocks, in particular the PMMA block as a

substitute for the P2VP block could lead the zoospores to interact differently with the PS-b-

PMMA surfaces as well as the PMMA based controls.

126

a)

b)

Figure 6.15 a) The density of attached Ulva spores on polymers on nylon coated silicon. Each point is the mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95% confidence limits. and b) advancing water

contact angle for polymers on nylon coated silicon.

127

6.4.7 Settlement of Diatoms on PS-b-PMMA Diblock Copolymers

The efficacy of the PS-b-PMMA diblock copolymers were tested for antifouling properties

across marine species by evaluating the number of attached organism using another popular

marine biofouler, the diatom. In particular, Navicula perminuta diatoms were used in this

experiment. The density of attached Navicula cells to various pattern dimensions and types of

diblock copolymers spin coated on nylon coated glass slides can be seen in Figure 6.16A. Nylon

surfaces were used as substrates to imitate actual aquaculture net material. The thin nylon film

was supported using glass slides. Glass slides were used to minimize the area available for

attachment around the samples and to optimize the fabrication parameters and comparison of this

study with future experiments on the strength of cell attachment in a water channel and water jet

apparatus that is designed to work only with glass slides.

The number of diatoms unlike zoospores will be similar on all the coatings at the end of the

laboratory assay. In the water column, the diatoms are not motile and they will reach a surface

with the aid of gravity or water currents not due to active movement.8-9 In the laboratory assay

instance, the diatoms will sink to the polymer films with the help of gravity leading to an even

covering of the entire polymer test surfaces. The polymer samples are washed at the end of the

assay to remove cells unattached or weakly attached before fixing the cells and counting them to

obtain the attached cell density. Thus, this measurement evaluates the capacity of cells to attach

strongly to the surface and resist removal during the washing process.

Previous studies have shown that diatoms are also influenced by the hydrophobicity of the

surface analogous to zoospores.15, 25-26 As the water contact angle of samples increases

corresponding to an increase in hydrophobicity, the number of attached diatoms increases and

the adhesion strength also increases. The strength of attached diatoms was also shown to

increase with an increase in hydrophobicity even in cases where the initial number of attached

diatoms was analogous.27

The highest cell attachment was observed for the nylon coated on glass slides. This was unusual

since this is the most hydrophilic coating as can be seen in Figure 6.16B. This response is also

opposite to the previous low settlement observed with these films on the zoospores. The

presence of the irregular sized nanofeatures on the surface appears to enhance the attachment or

the presence of these ill-defined nanofeatures with this particular hydrophobicity could lead to

128

this observed high diatom presence. The PS and PS-b-PMMA 130-133 before solvent annealing

had a lower number of settled cells than the nylon coated glass films. This higher attachment on

the most hydrophobic polymer films corresponds to the literature when compared with the

appropriate PMMA based polymer films. A large difference between the different pattern

dimensions and type and random copolymer was not observed; these coatings had a similar

attachment density, lower than for the PS films. An exception are the PMMA and PS-b-PMMA

130-133 films which showed the lowest cell density; however, these films were not much better

at inhibiting attachment. All of these polymers thin films have a similar intermediate advancing

water contact angle in the mid seventy degrees range. The hydrophobicity effect has a strong

influence on the diatoms attachment for these coatings, whereas the nanopatterning effect has a

weak or non-existent influence. In this aspect, the diatoms did not behave much differently than

the zoospores.

129

a)

b)

Figure 6.16 a) The density of attached Navicula diatoms on polymers on nylon coated glass slides. Each point is the mean from 90 counts on 3 replicate slides (30 on each substrate). Bars show 95% confidence limits. and b)

advancing water contact angle for polymers on nylon coated glass slides.

130

6.5 Conclusions PS-b-P2VP and UV treated PS-b-P2VP mixed with benzophenone and PS-b-PMMA diblock

copolymers nanopatterned surfaces showed generally no major influence on the attachment

density of zoospores or diatoms with a change in pattern dimensions in the region probed or

pattern type. The PS-b-P2VP and UV PS-b-P2VP thin films showed reduced zoospore

settlement for the nanopatterned films as opposed to the control unpatterned polymer films

suggesting an intermediate effect of this parameter. The change in hydrophobicity between the

diblock copolymers did not affect the settlement. The PS-b-P2VP 172-42 pattern showed a

larger settlement than the rest of the diblock copolymer which might be attributed to a special

case due to the interplay between the nanopattering and hydrophobicity or some undetected

abnormality with some of the samples. In the case of PS-b-PMMA copolymers, the

nanopatterning did not appear to influence either the zoospore settlement or diatom attachment,

while the hydrophobicity effect dominated the final organism attachment. There was a concern

with the PS-b-PMMA 160-160 pattern normality due to a higher zoospore settlement than the

rest of the PMMA based samples. The two diblock copolymers showed a different method of

influencing zoospore attachment.


(2) Rascio, V. J. D. Corros. Rev. 2000, 18, 133–154.

(3) Champ, M. A. Sci. Total Environ. 2000, 258, 21–71.

(4) Callow, J. A.; Callow, M. E. in Antifouling Compounds. Progress in Molecular and Subcellular Biology, Sub-series Marine Molecular Biotechnology, ed. Fusetani, N.; Clare, A. S. Springer-Verlag, Berlin, Heidelberg, 2006, pp. 141–170.

(5) Callow, M. E.; Callow, J. A.; Pickett-Heaps, J. D.; Wetherbee, R. J. Phycol. 1997, 33, 938-947.

(6) Schultz, M. P. Biofouling 2007, 23, 331-341.

(7) Molino, P. J.; Wetherbee, R. Biofouling 2008, 24, 365-379.

(8) Kiorboe, T. Adv. Mar. Biol. 1993, 29, 1-72.

(9) Krishnan, S.; Wang, N.; Ober, C. K.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Hexemer, A.; Sohn, K. E.; Kramer. E. J.; Fischer, D. A. Biomacromolecules 2006, 7, 1449–1462.


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(16) Ederth, T.; Nygren, P.; Pettitt, M. E.; Östblom, M.; Du, C.-X.; Broo, K.; Callow, M. E.; Callow, J. A,; Liedberg, B. Biofouling 2008, 24, 303-312.





(21) Green, P. F.; Christensen, T. M.; Russell, T. P.; Jerome, R. J. Chem. Phys. 1990, 92, 1478-1482.


(23) Peng, J.; Wei, Y.; Wang, H.; Li, B.; Han, Y. Macromol. Rapid Commun. 2005, 26, 738-743.


(25) Holland, R.; Dugdale, T. M.; Wetherbee, R.; Brennan, A. B.; Finlay, J. A.; Callow, J. A.; Callow, M. E. Biofouling 2004, 20, 323-329.

(26) Finlay, J. A.; Callow, M .E.; Ista, L. K.; Lopez, G. P.; Callow, J. A. Integr. Comp. Biol. 2002, 42, 1116-1122.

(27) Finaly, J. A.; Bennett, S. M.; Brewer, L. H.; Sokolova, A.; Clay, G.; Gunari, N.; Meyer, A. E.; Walker, G. C.; Wendt, D. E.; Callow, M. E.; Callow, J. A.; Detty, M. R. Biofouling 2010, 26, 657-666.

132

7 Thermal Nanoimprint Lithography Nanopatterned Polystyrene and Surface-Initiated Polymerized Poly(2-vinyl pyridine) Surfaces for Algae Zoospores Assays

7.1 Overview Nanopatterned polymer surfaces of polystyrene (PS) poly(2-vinyl pyridine) (P2VP) were

fabricated using Thermal Nanoimprint Lithography (TNIL). PS polymer films were prepared

using spin coating from solution, whereas P2VP polymer films were grown as brushes using

surface-initiated atom transfer radical polymerization (ATRP). The polymer films were

patterned using silicon templates with lines ranging in size from 200 nm to 900 nm and pattern

depths from 15 nm to 100 nm. PS based nanopatterns showed a higher settlement of zoospores

compared to P2VP due to a higher surface hydrophobicity. The zoospores showed a high

response to the nanopatterning in particular the surface roughness. Zoospores attached the most

on the patterns with the lowest pattern depth, the 15 nm deep samples, in both polymer cases.

Nonetheless, the depth is not the only parameter varied, the line and groove width also changes,

and thus, the effect of these parameters cannot be completely discarded. Atomic Force

Microscopy (AFM) was used to investigate the polymer surface topography and ellipsometry

was used to investigate the polymer film thickness.

7.2 Introduction Surfaces immersed in seawater, in particular man-made artificial structures including such as

ship hulls and aquaculture nets, are prone to marine biofouling if left untreated. 1 A common

marine biofouler, the zoospores of the Ulva linza algae, was shown to respond to a variety of

surface properties for example wettability2-3, surface chemistry4-5 and topography6-7. The

response of zoospores to diblock copolymer patterned samples displaying both nanoscale

topographic features and variation in local chemistry were shown to reduce the number of

attached zoospores. These differences in hydrophobicity and surface structures may have

enhanced efficacy by working either independently or together. A more in depth study with

133

patterns of various dimensions demonstrated that for PS-b-P2VP films the nanopattering reduces

the settlement of zoospores as opposed to surface hydrophobicity; however, for the range of

cylinder diameter studied 30 nm to 48 nm, nanopatterning in general did not have an effect. The

PS-b-PMMA diblock copolymer on the other hand showed a strong response to surface

hydrophobicity as opposed to nanopattern dimensions in the range of cylinder diameters of 34

nm to 82 nm. These diblock copolymer surfaces are very complex, a simpler case where only

the size of nanopatterns is varied or only the chemistry of the surface is varied would lead to a

better understanding of factors governing zoospore settlement at the nanoscale.

Nanopattern fabrication using diblock copolymers relies on the copolymer’s ability to self

assemble in ordered nanosized features producing complex surfaces leading to a method

operating from the bottom-up.9-11 Alternative technologies for pattern fabrication at the nano and

micron scale exist that operate from the top-down based on lithographic techniques such as

photolithography, e-beam lithography and nanoimprint lithography (NIL).12-13 These approaches

are not exclusionary; they have at times been combined to obtain desired patterning in surfaces

such as using a photolithography top-down approach to direct the self assembly of block

copolymers, a bottom-up approach.12, 14 NIL is an emerging method with a low cost, high

resolution, feature smaller in size than 10 nm, and high throughput.15-17 The NIL method

involves the use of a patterned template/stamp to transfer a pattern by mechanically contact and

3D material displacement into a material coated on a hard substrate.17 NIL techniques include a

thermal based method (TNIL, historically know as hot embossing lithography) and an UV curing

based method.

In TNIL, a thin thermoplastic material is compressed between a hard template and hard substrate

and the material is displaced by squeeze flow using pressure. The resolution of the final pattern

depends on the features present in the template. Both the template and the film coated substrate

can be damaged during the imprinting due to the high pressure. The templates can become

contaminated with particles from the atmosphere and polymer residues. The templates are

treated with a release layer to minimize the contamination; however, over time this layer wears

out and must be reapplied. Template cleaning techniques are still not fully developed yet. NIL

can also be used to replicate templates, thus, extending the life of the original pattern and

minimizing the effects of template damage and contamination. During imprinting, air inclusions

134

trapped in the template and uneven template filling will also lead to defects in the final pattern.

The advantages of NIL far outweigh its shortcomings leading to a powerful pattering technique.

The thin thermoplastic materials used for TNIL are usually thin polymer single layers.17 A

widespread method of synthesizing polymers is based on radical polymerization.18 A large

variety of polymers have been synthesized with this method. It requires the absence of oxygen,

water and impurities are easily accepted, occurs at easily accessible temperatures; however,

reaction control is difficult leading to polymers with high molecular weights and

polydispersities.19 Controlled versions based on this method were developed, in particular Atom

Transfer Radical Polymerization (ATRP) has been very successful.19-20 Synthesized polymers

have well controlled molecular weights, low polydispersities, well defined end groups and

functionality can be easily incorporated. In ATRP, a transition metal forms a complex with an

appropriate ligand. The catalyst complex abstracts a halogen from an initiator alkyl halide,

which in turn can add to the double bond of the alkene, the polymer monomer, and propagate.

This new radical can continue to grow by adding to more alkenes or can abstract the halogen

back from the catalyst complex to form an alkyl-polymer chain-halide product. The catalyst

complex controls the reaction through a reversible equilibrium between the growing polymer

chain radical and the dormant polymer chain. Surface-initiated ATRP has been used to grow

polymer chains attached at one end to a surface, also called polymer brushes, for a variety of

applications such as specific protein immobilization,21-22 pH responsive surfaces23 and protein

and cell antifouling surfaces.24-25 Tethering the polymer to the surface eliminates polymer

detachment during measurements in solution.

In this study, PS and P2VP homopolymer surfaces were nanopatterned using templates with lines

of various dimensions under 1 µm by the TNIL method. PS polymer films were fabricated using

spin coating from solution, while P2VP polymer films were grown as brushes using surface-

initiated ATRP. The polymer film is usually spin coated onto a substrate and then the TNIL

method is used for patterning; however, if a more complex pattern is required, part of the TNIL

polymer pattern is etched to the substrate and polymers grafted to the substrate.26 In this

procedure, P2VP polymer brushes are used instead of spin coated films for the TNIL process.

This eliminates additional etching steps and backfilling of the pattern and leads to a robust film

in solution. These PS and P2VP nanopattern samples allow for the separation between the

physical and chemical effect of patterning in order to better understanding the factors governing

135

zoospore settlement. In addition, the optimal pattern size for zoospore inhibition is also

explored.


7.3.1 Materials

PS (Sigma Aldrich) was used as received. The number average molecular weight is 113125

g/mol and the weight average molecular weight is 269477 g/mol leading to a polydispersity

index of 2.38. Thin polymer films were prepared by spin coating 2 wt% toluene solutions of PS

on silicon substrates at 2000 rpm for 45 s. The silicon substrates were prepared by cleaning in

piranha solution (3:1 v/v concentrated H2SO4 : 30 % H2O2) for 15 min. Caution: Piranha is a

very strong oxidant.

Figure 7.1 PS.

2-Vinylpyridine (97 %, contains 0.1 wt% p-tert-butylcatechol as inhibitor, Sigma Aldrich) was

vacuum distilled using calcium hydride to remove the inhibitor. The monomer was stored in the

refrigerator immediately following distillation. Tris[2-(dimethylamino)ethyl]amine (Me6TREN,

Sigma Aldrich) was used as received. Copper (I) chloride (CuCl, Sigma Aldrich) was previously

purified washing in glacial acetic acid, filtrated and rinsed with ethanol and diethyl ether.

Copper (II) bromide (CuBr2, Sigma Aldrich) was used as received. 2-Propanol (Sigma Aldrich)

was used as received. 3-(2-Bromoisobutyramido)propyl(trimethoxy)silane in toluene was

previously synthesized and stored under argon gas in the refrigerator.21

Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA, Sigma Aldrich) was used as

received.

136

7.3.2 Surface-Initiated ATRP of P2VP

Figure 7.2 shows an outline of the procedure used for the surface-initiated ATRP of P2VP from

the ATRP initiator immobilization onto silicon substrates, part A, to the synthesis of P2VP

brushes, part B, and the removal of the copper catalyst afterwards, part C.

a)

b)

c)

Figure 7.2 Surface-initiated ATRP polymerization of P2VP procedure a) immobilization of initiator, b) brush synthesis, and c) catalyst removal.

Silicon substrates were cleaned in piranha solution for 15 min prior to use. The silicon substrates

were exposed to the ATRP initiator by immersion in a 3-(2-Bromoisobutyramido)propyl(tri-

methoxy)silane toluene solution for 3 min. The substrates were removed from the reaction

solution and rinsed extensively with toluene, dried under a flow of nitrogen and placed in

Schlenk flasks for surface polymerization. The substrates were degassed by three argon-vacuum

cycles. The substrates were left in an argon atmosphere.

137

CuCl (158 mg, 1.6 mmol) and CuBr2 (7 mg, 0.032 mmol) were added to a Schlenk flask and

degassed by three argon-vacuum cycles. 2-Vinylpyridine (17.3 mL, 160 mmol), Me6TREN

(0.428 mL, 1.6 mmol), 11.14 mL of 2-propanol and 11.14 mL of ultrapure water (Mili-Q 18

MΩ) were placed in a Schlenk flask and degassed by three freeze-thaw-pump cycles. This

solution was transferred to the CuCl and CuBr2 flask using a degassed syringe. The mixture was

stirred at room temperature for 30 min until the formation of homogenous light green Cu

complex solution. The solution was transferred using a degassed syringe into the flaks

containing the initiator functionalized silicon substrates. The polymerization was left to react for

4 h at room temperature under argon gas. The substrates were removed from the Schlenk flasks

and washed with 2-propanol to removed untethered materials and dried under a flow of nitrogen.

The samples were placed in a deep-well plate and an EDTA (1.826 g, 5 mmol) solution was

added to remove the Cu catalyst. The well plate was placed for 18 h on a plate shaker. The

samples were rinsed with ethanol and dried under a nitrogen flow.

7.3.3 Thermal Nanoimprint Lithography

The PS and P2VP polymer samples on silicon were patterned using TNIL as show in Figure 7.3.

Silicon templates with linear patterns with various nanosized dimensions were used as seen in

Table 7.1. The size of the templates is approximately 1 cm x 1cm. Preceding the imprinting, the

templates were treated with a release layer of tridecafluoro-1,1,2,2-tetrahydrooctyl)trichloro-

silane (ABCR GmbH & Co. KG) to prevent adhesion of the template to the imprint material.

The templates were modified using vapor phase deposition by placing the templates beside a

glass vial containing 30 µL of the silane solution at the bottom of a desiccator and leaving the

system under vacuum for 17 h. The templates were rinsed with ethanol and dried under a flow

of nitrogen.

Figure 7.3A shows the treated silicon template and the polymer coated silicon substrate. The

template was brought into contact with the polymer film on silicon and the resulting system was

wrapped in aluminum foil and apiece of heat conductive rubber was placed on top to equilibrate

pressure and heat. Everything was again wrapped in aluminum foil in order to keep the machine

clean and the system was placed between the plates of a nanoimprinting press. The polymer

nanoimprinting was performed using a commercial hydraulic press (Specac, 15 tons manual

138

press with electrical heating plates and a temperature controller). The PS samples were heated to

the imprint temperature of 150 °C, whereas the P2VP samples were heated to the imprint

temperature of 170 °C. At the imprint temperature, an imprint pressure of 60 bar was applied to

transfer the pattern into the polymer. The imprint temperature and pressure were kept constant

for 10 min while the polymer moved to fill the template as shown in Figure 7.3B. Afterwards,

the system was cooled to 80 °C meanwhile maintain the imprint pressure. The pressure was then

released and the polymer system was taken out and unfolded. The template was lifted from the

polymer film as shown in Figure 7.3C.

a)

b)

c)

Figure 7.3 Polymer Thermal Nanoimprint Lithography a) release layer coated silicon template and the polymer film coated on a silicon substrate, b) imprinting at a temperature above the Tg and high pressure, c) template demolding

from imprinted polymer film.

Table 7.1 Silicon Templates Parameters.

Line width

(nm)

Groove width

(nm)

Depth

(nm)

Pattern size

(cm)

200 200 100 1.1 x 1.1

300 700 30 1.0 x 0.7

500 900 200 1.0 x 0.5

139


Topography images of the silicon templates and polymer samples in air were obtained in

Tapping Mode using a Molecular Force Probe AFM (Asylum Research, MFP-3D). Rectangular

shaped silicon probes (NanoWorld, NCH) with resonance frequencies in the range 280-320 kHz

and a spring constant of 40 N/m were used. Measurements in solution were obtained in Contact

Mode. V-shaped, silicon nitride cantilevers (Veeco, DNP) exhibiting a nominal spring constant

of 0.12 N/nm were used.

Polymer film thickness was measured using a rotating compensator ellipsometer (J. A. Woollam

Co., Inc., M-2000X) operating in the UV-visible range at multiple incidence angles, 65°, 70° and

75°. The results were modeled using the commercial software package (Complete EASE v.4.41)

part of the system.

A contact angle meter (dataphysics, OCA) was used to measure the advancing contact angle of

the films using ultrapure water (Mili-Q 18 MΩ).





zoospore suspension in the dark at ~ 20°C. Each dish contained one polymer substrate on

silicon. After 45 min the substrates were washed in seawater to remove unsettled zoospores.

Substrates were fixed using 2.5% glutaraldehyde in seawater. The density of zoospores attached

to the surface was counted on each of the replicate substrates using an image analysis system

(Imaging Associates Ltd.) attached to an epifluorescence microscope (Zeiss, Aksioskop 2).

Spores were visualized by autofluorescence of chlorophyll. Counts were made for 30 fields of

view (each 0.17 mm2) on each polymer.

140


7.4.1 PS and P2VP Polymer Films Nanoimprinting

Polymer thin films were nanopatterned using TNIL. This method uses a pattern template for

imprinting the polymer films. The AFM height images of the patterned silicon templates used in

this experiment are shown in Figure 7.4. Figure 7.4A shows the smallest pattern used, 200 nm

wide line x 200 nm wide groove x 100 nm deep, while part B show a larger pattern with 300 nm

wide line x 700 nm wide groove x 30 nm deep, and part C shows the largest pattern used, 500

nm wide line x 900 nm wide groove x 200 nm deep. The templates were treated with a release

layer using vapor phase deposition. This antiadhesive layer promotes the release of the template

from the polymer after imprinting and reduces the amount of polymer residue on the template

leading to a longer template lifetime. A schematic of the TNIL process is shown in Figure 7.3.

The template is placed on the polymer film and they are pressed together. The system is heated

to a temperature above the polymer glass transition temperature, Tg, and high pressure is applied,

60 bar in this case. In the case of PS, the system was heated to 150 °C, Tg of 97 °C for 290 nm

and thinner films,27 while for P2VP the system was heated to 170 °C, Tg of 130 °C for 160 nm

thin film.28 The polymer system is kept at these conditions for 10 min to allow for the polymer

to fill the grooves in the template. Then the polymer is cooled below the Tg, 80 °C, maintain the

high pressure leading to the hardening of the polymer inside the template. The polymer is

demolded from the template and a reverse pattern is obtained, lines in the template become

grooves in the polymer and the grooves in the template become raised lines in the polymer.

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a) b)

c)

Figure 7.4 AFM height images of silicon templates with dimensions a) 200 nm wide line x 200 nm wide groove x 100 nm deep, b) 300 nm wide line x 700 nm wide groove x 30 nm deep, and c) 500 nm wide line x 900 nm wide groove x

200 nm deep. Image sizes: 5 μm x 5 μm. Z range: 100 nm.

The morphology of the PS thin films was characterized using AFM. Figure 7.5A shows the

height image of a spin coated PS films. The surface shows no surface structures as observed

previously. The thickness of these films is 131 ± 1 nm as measured by ellipsometry. The films

are hydrophobic with an advancing water contact angle of 88 ± 2°. The PS films were imprinted

by TNIL using the three different silicon templates leading to an inverse nanopattern. The

surface topography of alternating lines and grooves is shown in Figure 7.5B, C and D

respectively. The lines are half as deep as the template due to incomplete template filling during

imprinting, however, the width of the lines and grooves are as expected. The water contact angle

of the samples is higher than for unpatterned PS similar to behavior observed for PS surfaces

patterned with 300 nm nanorods.29

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a) b)

c) d)

Figure 7.5 AFM height images of PS films a) flat unmodified surface, b) 200 nm wide line x 200 nm wide groove x 50 nm deep nanopatterned surface, c) 700 nm wide line x 300 nm wide groove x 15 nm deep nanopatterned surface,

and d) 900 nm wide line x 500 nm wide groove x 100 nm deep nanopatterned surface. Image sizes: 5 μm x 5 μm. Z range: 50 nm.

Table 7.2 Advancing water contact angles of PS polymers on silicon.

Label Contact Angle

PS 88 ± 2°

PS 200 x 200 x 50 94 ± 2°

PS 700 x 300 x 15 94 ± 2°

PS 900 x 500 x 100 103 ± 2°

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P2VP was synthesized by surface-initiated ATRP using a modified procedure of

Matyjaszewski’s work on poly(4-vinyl pyridine).30 P2VP was synthesized as polymer brushes

from a silicon substrate. The outline of the synthesis procedure is shown in Figure 7.2. The 3-

(2-Bromoisobutyramido)-propyl(trimethoxy)silane initiator was attached to silicon substrates by

the condensation reaction between the hydroxyl groups on the silicon substrate and the

trimethoxysilane ends of the initiator. The P2VP brushes were grown from this modified silicon

substrates. A CuCl/CuBr2/Me6TREN catalyst system in 2-propanol-water 1:1 water mixture at

room temperature was used. The polymerization time was 4 h. CuCl was used as catalyst due to

its strong bond to carbon leading to a stability of the radical activation and deactivation by the

catalyst system and also due to its poor performance as a leaving group leading to reduction in

nucleophilic substitution reactions with pyridine.30 CuBr2 was also added to the reaction to

increase the concentration of deactivating species to make sure the reaction is properly

controlled. The 2-vinylpyridine monomer as well as the P2VP polymer are strong coordination

ligands and will compete for the binding to the Cu catalyst with the ligand. There is a great

chance of forming this complex due to an excess presence of monomer over the ligand in the

synthesis. This complex formation is not desired due to its poor efficacy as a catalyst. Thus, a

very strong coordination ligand is employed, Me6TREN. Polar solvents are required for the

reaction to solubilize the polymer, 2-propanol, as well as decrease the catalyst contamination by

hydrogen bonding to the monomer and polymer, 2-propanol and water. At a high water

concentration, the monomer starts to precipitate out of solution, thus, a solvent mixture is

required for the synthesis.

The morphology of the P2VP thin films can be seen in Figure 7.6. Figure 7.6A shows the height

image of a P2VP films synthesized using surface-initiated ATRP. The surface shows no surface

structures typical of homopolymers; however, there are some particles present that were not

completely removed during polymer rinsing. The average thickness of these films is 142 ± 27

nm as measured by ellipsometry. The films are hydrophilic with an advancing water contact

angle of 41 ± 2°. These films were also imprinted using TNIL with the three different silicon

templates. The surface topography shows an inverse template nanopattern of alternating lines

and grooves as shown in Figure 7.6B, C and D respectively. The lines are half as deep as the

template in this case too due to incomplete template filling during imprinting, however, the width

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of the lines and grooves are as expected. The water contact angle of the samples is similar to

unpatterned PS.

a) b)

c) d)

Figure 7.6 AFM height images of P2VP films a) flat unmodified surface, b) 200 nm wide line x 200 nm wide groove x 50 nm deep nanopatterned surface, c) 700 nm wide line x 300 nm wide groove x 15 nm deep nanopatterned surface, and d) 900 nm wide line x 500 nm wide groove x 100 nm deep nanopatterned surface. Image sizes: 5 μm x 5 μm. Z

range: 50 nm.

Table 7.3 Advancing water contact angles of P2VP polymers on silicon.

Label Contact Angle

P2VP 41 ± 2°

P2VP 200 x 200 x 50 38 ± 2°

P2VP 700 x 300 x 15 39 ± 2°

P2VP 900 x 500 x 100 38 ± 2°

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7.4.2 PS and P2VP Polymer Films Behavior Underwater

The PS and P2VP nanopatterned films with pattern dimensions of 900 nm wide lines x 500 nm

wide grooves x 100 nm deep were immersed in water and AFM height images were obtained

after 10 min and 2 hours with the measurement performed in water. After 10 min water

immersion the pattern is present for the PS and P2VP films shown in Figure 7.7A and B

respectively. After 2 hours of water immersion, the pattern in still present for both polymers as

seen in Figure 7.7C for PS and part D for P2VP. During the zoospore settlement assay the

pattern will be present with no major polymer restructuring.

a) b)

c) d)

Figure 7.7 AFM height images of nanoimprinted films of PS left side and P2VP right side with dimensions 900 nm wide line x 500 nm wide groove x 100 nm deep in water a) and b) 10 min immersion, c) and d) 2 h immersion. Image

sizes: 5 μm x 5 μm. Z range: 50 nm.

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7.4.3 Settlement of Algae Zoospores on PS and P2VP Nanopatterned Polymer Films

The settlement density of zoospores attached to PS and P2VP polymers thin films with various

pattern dimensions is shown in Figure 7.8A. The PS surfaces with and without a nanopattern

had a higher settlement density than the P2VP samples. All of the PS surface are hydrophobic as

can be seen in Figure 7.8B and this observed settlement trend is consistent with an increase in

zoospore attachment trend as the hydrophobicity of the sample increases seen previously and in

the literature.2-3 In both sample groups, PS and P2VP, the highest settlement was on the 700 nm

wide line x 300 nm wide groove x 15 nm deep nanopatterned surface similar to the flat polymer

surfaces. The zoospore density was lower on the 200 nm wide line x 200 nm wide groove x 50

nm deep nanopatterned surface, while the 900 nm wide line x 500 nm wide groove x 100 nm

deep showed the lowest settlement density. The number of attached zoospores can be seen

visually from epifluorescence microscopy images in Figure 7.9. The PS samples show more

zoospores attached in large patches typical of surface prone to biofouling.

The hydrophobicity effect influences the settlement; nevertheless, the sample nanopatterning has

a strong effect in reducing zoospore attachment. The 700 nm wide line x 300 nm wide groove x

15 nm deep nanopatterned surface showed a high settlement. This surface had a much shallower

pattern than the rest of the tested surfaces. The zoospores appear to react to nanometer

roughness and are able to discriminate between patterns preferring to settle on shallow patterns

closer in morphology to a flat surface. Unfortunately, templates were not available where only

one parameter was varied, the line and groove width as well as the depth varied, thus, the width

of the pattern cannot be completely ignored. Nonetheless, this study shows that the antifouling

properties of a surface can be improved by incorporating nanometer features into the surface.

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a)

b)



148

a) b)

c) d)

e) f)

g) h)

Figure 7.9 Typical epifluorescence microscopy images of zoospores on polymers on silicon of PS films left side and P2VP right side of a) and b) unmodified films, c) and d) 200 nm wide line x 200 nm wide groove x 50 nm deep

nanopattern, e) and f) 700 nm wide line x 300 nm wide groove x 15 nm deep nanopattern, g) and h) 900 nm wide line x 500 nm wide groove x 100 nm deep nanopattern Images width: 450 μm.

149

7.5 Conclusions Nanopatterned PS and P2VP surfaces with alternating lines and grooves of dimensions under 1

µm were fabricated using TNIL. PS polymer films were spin coated from solution and P2VP

polymer films were successfully grown as brushes by surface-initiated ATRP. The TNIL

process was successfully combined with polymer brush synthesis to fabricate samples that are

robust in water and do not require additional fabrication steps such as polymer etching and

backfilling of the pattern with the desired polymer. The PS samples showed a higher settlement

of zoospores compared to P2VP samples due to a higher surface hydrophobicity. However, the

zoospores showed a high response to the patterning in particular the surface nanometer size

roughness. In both polymer cases, zoospores settlement was higher on the shallowest patterns,

the 15 nm deep samples. The line and groove width of the pattern also changes and these effects

cannot be completely overlooked. However, the zoospores appear to be able to discriminate

between different pattern roughnesses at the nanometer. Further experiments where only the

depth is varied will help to validate this observation. The antifouling properties of a surface can

be greatly improved by incorporating appropriate nanometer sized features into the surface

design.









150




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8 Conclusions and Future Directions

Marine biofouling is an interesting problem which has frustrated people for a long time,

thousands of years, ever since the first man made surfaces has been placed in seawater.1

Unwanted marine species have settled and grown over time on these surfaces leading to adverse

effects. Strategies to combat these species have focused on adding a protective coating to the

surface; however, some of these coatings have proven to have toxic side effects. Nowadays,

there is a shift in research towards new environmentally friendly coatings. In this thesis, various

polymer based coatings were investigated for antifouling properties against algae leading to a

better understanding of the factors that affect settlement and sensing at the nanometer length-

scale. Ordered nanopatterned coatings are optimal surfaces for probing biological responses on

specific length scales. Diblock copolymer coatings based on polystyrene-block-poly(2-vinyl

pyridine) (PS-b-P2VP) and polystyrene-block-poly(methyl methacrylte) (PS-b-PMMA) and

nanoimprinted homopolymer coatings based on polystyrene (PS) and poly(-2-vinylpyridine)

(P2VP) were studied in this thesis.

8.1 Conclusions Diblock copolymer surfaces can self-assemble into a variety of patterns with nanometer sized

features. These surfaces provide both physical and chemical nanopatterning due to the nature of

the diblock copolymer architecture made of two independent polymer blocks with different

properties covalently attached. PS-b-P2VP and PS-b-PMMA diblock copolymer thin films were

prepared by spin coating from solution and vapor solvent annealed. Thin films of PS-b-P2VP

block copolymer mixed with the photoinitiator benzophenone (BP), spin coated from solution,

vapor solvent annealed and UV irradiated were also prepared. All of the diblock self-assemble

into cylindrical domains perpendicular to the substrate, in this case silicon. UV irradiation

induces crosslinking in the polymer films; however, chain scissioning also occurs which

introduces oxygen groups into the polymer chain causing the hydrophobicity of the films to

decrease. The Ps-b-P2VP and BP UV irradiated films showed cylindrical patterns when the

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sample was dried after five weeks in water and three weeks in artificial seawater. This simple

route can be used to obtain surfaces that retain their nanoscale patterns in water. The PS-b-P2VP

and BP UV irradiated films and PS-b-PMMA films reduced the settlement of Ulva linza algae

zoospores. The hydrophobicity effect which shows that zoospores prefer to settle on

hydrophobic surface observed previously in literature for uniform surfaces does not fully explain

the observed phenomena. The unique features of the diblock surfaces, chemical and

topographical heterogeneity incorporated into a nanometer sized pattern, inhibit the settlement of

zoospores. The difference in hydrophobicity between the two blocks, the P2VP block or the

PMMA block respectively forming the cylinders and the PS block as the matrix, and/or the

nanoscale roughness may discourage the zoospores in attaching to these surfaces. Then again

the zoospores might be receiving conflicting signals from these textured surfaces. The zoospores

are able to respond to features at the nanometer scale, a dimension not fully explored yet.

Thin polymer films of PS-b-P2VP and PS-b-PMMA were also fabricated on nylon coated

surfaces instead of model silicon substrates in order to mimic real world applications,

aquaculture cage nets. In this case too, the PS-b-P2VP and BP UV irradiated films and PS-b-

PMMA films, both surfaces nanopatterned with cylindrical domains, show a decrease in

zoospore settlement. PS-b-PMMA films were also fabricated with different pattern dimensions

which increased as the molecular weight of the PS-b-PMMA copolymers increased. There was a

slight reduction in settlement density as the pattern dimensions decreased. Zoospores were left

to grow into sporelings for two types of PS-b-PMMA films and the settlement density was

evaluated. The PS-b-PMMA diblock copolymers as well as PMMA and P(S-r-MMA) showed

reduced settlement in zoospore and sporelings. The nanoscale polymer patterning continues to

influence the zoospore settlement; however, the hydrophobicity effect appears to play a

dominant role in determining zoospore attachment in PS-b-PMMA films. These surfaces have a

complex morphology and it is hard to quantify the exact nature of the forces operating in

inhibiting the attachment of zoospores. All these effects could be working together,

independently and or against each other. Additionally, the nylon thin films also showed

antifouling properties against algae zoospores as well as for sporelings. The thin nylon films

show nanoscale sized features as well as hydrophilic behavior. However, unpublished data on

long term seawater submersion show much better antifouling properties against marine species

for triblock copolymer coated nylon nets than untreated nylon nets.

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Next, the fabrication method for PS-b-PMMA diblock copolymers was reevaluated due to a low

number of successful samples assembled into the desired pattern. The yield of good quality

cylindrical patterns orientated perpendicular to the substrate for the PS-b-PMMA diblock

copolymers was highly improved, from 23 % to as high as 86 %, by modifying the vapor solvent

annealing. The solvent annealing was performed in a low-temperature environment, 2 °C, as

opposed to the typical room temperature methods used throughout the literature. At a lower

temperature leads to slower polymer mobility and a lower amount of dissolved acetone in the

polymer film. The system self-assembles at a lower rate and it allows for stopping further

rearrangement when the desired morphology is reached.

PS-b-PMMA diblock copolymers with various pattern dimensions, from 34 nm cylinder

diameter to 82 nm cylinder diameter, were fabricated using the low temperature vapor solvent

method for zoospores and diatoms, a unicellular type of algae, settlement assays. In addition,

PS-b-P2VP and UV treated PS-b-P2VP mixed with BP copolymer films were fabricated with

cylinder diameters ranging from 30 nm to 48 nm for the zoospores settlement assay. The change

in pattern dimensions for the two types of diblock copolymers showed little influence on the

number of attached zoospores or diatoms in the pattern range probed. However, the type of

diblock copolymer utilized revealed a different mechanism of zoospore inhibition. In the case of

PS-b-P2VP based copolymers the hydrophobicity difference between the UV irradiated and non-

UV irradiated samples did not affect the settlement, except in the case of the PS-b-P2VP 172-42

pattern which showed a larger settlement that might be attributed to a special case due to the

interplay between the nanopattering and hydrophobicity or some undetected abnormality with

this sample type or the non-UV irradiated samples. The nanopatterned PS-b-P2VP and UV PS-

b-P2VP thin films had a reduced zoospore settlement as opposed to the control unpatterned

polymer films suggesting a dominant effect of the nanopatterning parameter. The PS-b-PMMA

copolymers case, showed no influence of the nanopatterning in the settlement of the zoospores or

diatoms. However, the effect of hydrophobicity dominated the final organism attachment with

all of the samples showing similar attachment with a similar hydrophobicity. Once again, there

was a concern with one of the samples, the PS-b-PMMA 160-160, due to a higher zoospore

settlement than the rest of the PMMA based samples.

PS and P2VP homopolymer surfaces were patterned with alternating lines and grooves from 200

nm to 900 nm in length and 15 nm to 100 nm deep using Thermal Nanoimprint Lithography

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(TNIL) to separate the physical and chemical effect of patterning as well as optimizing pattern

dimension for zoospore inhibition leading to a better understanding of the factors affecting

zoospore settlement. The PS polymer films were spin coated from solution. The TNIL process

was successfully combined with surface-initiated ATRP polymer brush synthesis to fabricate

P2VP samples that are robust in water and do not require additional fabrication steps. The

hydrophobic PS samples had a higher zoospores attachment compared to the hydrophilic P2VP

samples as seen previously in the literature for unpatterned surfaces. The zoospores, however,

showed a high response to the sample nanopatterning, in particular to the surface nanometer size

roughness. For both the PS and P2VP polymer samples, the zoospores settlement was higher on

the shallowest patterns, the 15 nm deep samples.

Chemical and topographical surface heterogeneity, combined effects in the diblock copolymer

systems and independent effects in the nanoimprinted PS and P2VP homopolymers, were shown

to affect the settlement of algae zoospores. The antifouling properties of a surface can be

enhanced by incorporating appropriate nanometer sized features into the surface design.

However, when the pure homopolymer, the PMMA case, already shows a good inhibition in

algae settlement, the incorporation of nanometer pattering did not have a strong effect on the

algae settlement in the short time used in laboratory assays. The chemical choice is of critical

importance in coating fabrication. Nonetheless, nanopatterning shows promise in the battle

against algae zoospores. Nanoscale features can directly target small marine species as well as

the sensing apparatus of larger marine species.

8.2 Future Directions

All the same, this dissertation is a work in progress. Further experiments are required to expand

the base built by this thesis in exploring nanometer sized patterned materials as potential

environmental friendly antifouling marine coatings. The TNIL method shows promise in

fabricating nanopatterns of various dimensions to test for effects of patterning in three

dimensions and is applicable to different polymers; however, availability and cost of the

templates could be an issue since nanosized patterns are made using e-beam lithography, an

expensive technique for large patterns necessary in marine species assays. This could be

circumvented by purchasing commercially available patterns, limited selection, or collaborating

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with various groups to build a database of patterns. Patterns should be made where only one

dimension is varied at a time such as only the depth of the pattern is changing. Patterns should

also be tested using different designs and polymers to test for hydrophobicity effects and

optimize the pattern.

The block copolymer method is a more straightforward alternative in making nanopatterns as in

this thesis. Different block copolymers should be tested to find the optimal block combinations

as well as the optimal pattern size. The cost of purchasing block copolymers can become quite

high, in particular if custom synthesis is required to test certain molecular weights and block

types. An alternative is to synthesize the required materials in-house or purchase block

copolymers produced in large quantities at low cost for other applications and test their

adaptability as marine antifouling paints. Short laboratory assays are an indication of potential

antifouling properties, however, more realistic experiments such as long term marine species

exposure and immersion in actual seawater with all the different marine species present at once

are necessary to ultimately decide a coatings antifouling properties. If a coating inhibits the

settlement of one type of marine species, it does not lead to general inhibition of all the other

species present in the water column.

The focus in this thesis has been on environmentally friendly coatings which were tested for

antifouling properties. However, marine testing should be further extended to explore the foul

release properties. Block copolymers are amphiphilic systems; they are made of distinct polymer

blocks with different properties such as hydrophobicity. Thus, a hydrophobic block can be

combined with a more hydrophilic block. The hydrophobic block will have foul release

properties while the hydrophilic block will have antifouling properties as shown in the

literature.2-3 This combination of properties increases the performance of the coating. Thus, it is

important to also test for foul release in order to determine the full potential of the coating.

Other strategies to improve marine coatings include the use of biocide boosters and natural

derived biocides.1 Biocide boosters such as zinc pyrithione are more environmentally friendly

and perform better than tributyl tin based coatings. However, if these biocides are added to

existing coatings to improve their performance, care must be taken to perform in depth studies on

their effect on the environment. Natural derived biocides can also be incorporated into a coating

to improve its performance. These natural biocides can dissolve the adhesive of the biofoulant,

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can inhibit settlement or can interfere with the metabolism of the biofoulant. These compounds

are harder to produce on a large scale and there is always the risk of a decrease in performance

once the compound is placed into a coating. Even if these compounds are found in nature, their

toxicity effects must also be fully studied. Future work can also include the incorporation of

these promising natural derived biocides to boost the performance of block copolymers.


(2) Krishnan, S.; Ayothi, R.; Hexemer, A.; Finlay, J. A.; Sohn, K. E.; Perry, R.; Ober, C. K.; Kramer, E. J.; Callow, M. E.; Callow, J. A.; Fischer, D. A. Langmuir 2006, 22, 5075–5086.

(3) Gudipati, C. S.; Finlay, J. A.; Callow, J. A.; Callow, M. E.; Wooley, K. L. Langmuir 2005, 21, 3044-3053.

Nanopatterned Polymer Coatings for Marine Antifouling Applications

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