1Department of Chemistry; 2Center for Nanohybrid Functional Materials; 3Department of Biological Systems Engineering; 4Biomedical Engineering Program; 5Department of Chemical and Biomolecular Engineering; 6Department of Electrical Engineering; University of Nebraska-Lincoln, U.S.A.

Characterization of biological thin films with combinatorial spectroscopic ellipsometry and piezoelectric nanogravimetry

Characterization of biological thin films with combinatorial spectroscopic ellipsometry and piezoelectric nanogravimetry

Biofunctionalizing Sculptured Thin Films

Abstract

J. Gerasimov1,2* K.B. Rodenhausen2,5, T. Kasputis3,4, D. Schmidt2,6, H. Wang2,6, A.K. Pannier3, R. Lai1,2, and M. Schubert2,6

J. Gerasimov1,2* K.B. Rodenhausen2,5, T. Kasputis3,4, D. Schmidt2,6, H. Wang2,6, A.K. Pannier3, R. Lai1,2, and M. Schubert2,6

Sequence-specific DNA Detection

Experimental Design

Future Work

Acknowledgements: The Procter & Gamble Co., J.A. Woollam Co., Inc., NSF EPS-1004094, NSF CAREER CHE-0955439 Nebraska EPSCoR RII External Reviewer Panel Visit, University of Nebraska-Lincoln, August 2011

The formation and function of biotechnologically relevant functional thin films was studied in real-time using tandem spectroscopic ellipsometry (SE) and quartz crystal microbalance (QCM). Simultaneously monitoring changes in film thickness given by each technique allows us to derive the porosity of the bioactive layer under investigation and gives clues about the morphology of the surface-confined biomolecules. A correlation of structural morphology with film function makes possible the directed optimization of bioactive films based on measurable parameters. We present the results of two investigations involving the formation of bioactive hybrid materials. In the study of DNA-incorporating monolayers for sequence-specific DNA detection, two DNA probes were analyzed for their ability to specifically recognize their complement in solution. The formation of protein layers on smooth and nanostructured surfaces was studied for its utility as a scaffold for whole-cell immobilization and future work in gene transfer studies.

S S S S SS SS S

S S S S SSS S S

Probe Adsorption

lightsource

polarizer

optical window

liquidinlet

liquidoutlet

optical window

detector

QCM-Dcontrol

gasket surface

analyzers

polarized light

polarized light

affected by sample

Instrument

S S S S SSS S S

Target Detection

0 500 1000 1500

-0.2

0.0

0.2

0.4

0.6

Thi

ckne

ss (nm

)

Time (min)

0 100 200 300 400 500-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

Thi

ckne

ss (

nm)

Time (min)

-0.1

0.0

0.1

0.2

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0.4

0.5

aligned

buffercomplement

targetnon-

complement DNA

buffer buffer

0 100 200 300 4000

1

2

3

4

Thi

ckne

ss (nm

)

Time (min)

0.2

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0.6

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1.0

f0v

0 100 200 300 4000

1

2

3

4

Time (min)

Thi

ckne

ss (nm

)

0.2

0.4

0.6

0.8

f0v

S S S S SSS S S

Electrochemical DNA Sensor

Flush Probe

ElevatedProbe

eT

eT

Substrate

Structured Thin Film

Titanium Nanocolumns on Gold

• extracellular matrix protein• facilitates cell adhesion,

spreading, and proliferation • diverse applications in

biotechnology and engineering of hybrid materials

Fibronectin

• Electron transfer (eT) to the labeled, surface-bound stem-loop DNA probe is greatly impeded upon hybridization with complementary target.

• Decrease in eT efficiency is monitored using alternating current voltammetry

• SE/QCM provides structural information about DNA layer formation

0 100 200 300 400-0.1

0.0

0.1

0.2

0.3

0.4

0.5

dSE dQCM

dSEdQCM

-0.1 -0.2 -0.3 -0.4 -0.5-202468

1012141618

Cur

rent

(nA

)

Potential (vs. Ag/AgCl)

-0.1 -0.2 -0.3 -0.4 -0.5

0

1

2

3

Cur

rent

(nA

)

Potential (vs. Ag/AgCl)

unbound

bound

0 100 200 300 400-0.1

0.0

0.1

0.2

0.3

0.4

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dSE dQCM

dSEdQCM

12

10

8

6

4

2

00 100 200 300 4000

1

2

3

4

Time (min)

Thi

ckne

ss (nm

)

0.2

0.4

0.6

0.8

f0v

0 100 200 300 4000

1

2

3

4

Time (min)

Thi

ckne

ss (

nm)

0.2

0.4

0.6

0.8

f0v

1.0

0.8

0.6

0.4

0.2

0120100806040200 0 100 200 300 400

-0.1

0.0

0.1

0.2

0.3

0.4

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dSE dQCM

dSEdQCMf0

v

Flat Surface

Nanostructured Surface

140 160 180 200 2200

2

4

6

8

10

12

14

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18

20 X

QCM

XQ

CM (

mg/

m2 )

Time (min)

0

2

4

6

8

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14

Protein Fraction

Pro

tein

Fra

ctio

n (%

)

140 160 180 200 2200

2

4

6

8

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14

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18

20 X

QCM

XQ

CM (

mg/

m2 )

Time (min)

0

2

4

6

8

10

12

14

Protein Fraction

Pro

tein

Fra

ctio

n (%

)

• SE thickness (dSE) gives the thickness of adsorbed organic layer, excluding solvent.

• QCM thickness is derived from total adsorbed mass

• Assuming equivalence of solvent and adsorbate densities, porosity () may be expressed as a ratio of the thickness measured in SE to the thickness measured in QCM

Theory

𝑑𝑄𝐶𝑀= 1𝐴

(𝜌𝑜

𝑚𝑜

+𝜌𝑎

𝑚𝑎

)𝜌𝑜 ,𝑚𝑜=𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑎𝑛𝑑𝑚𝑎𝑠𝑠𝑜𝑓 h𝑡 𝑒𝑎𝑑𝑠𝑜𝑟𝑏𝑎𝑡𝑒

𝑑𝑆𝐸

𝑓 𝑜𝑉=𝑑𝑆𝐸

𝑑𝑄𝐶𝑀

buffer complementary target

non- complementary

target

Study surface functionalization for cell adhesion and gene

transfer

º Helmholtz Association of German Research Centres (2009, April 7). Fitting Pieces For Biosensors. ScienceDaily. Retrieved August 7, 2011, from http://www.sciencedaily.com /releases/2009/04/090407105149.htmººadapted from http://webs.wichita.edu/mschneegurt/biol103/lecture16/normal_rogue.gif

Monitor conformational change in surface-confined proteinsºº

In the near future, we will add an electrochemistry module to the SE/QCM to study the relationship between electrochemical trends and film morphology. We will direct further efforts toward the following goals:

Characterize peptide-based sensors for antibody detection

S S S S SSS S S S S S S SSS S S

Aptamer-based small molecule detectionº

*jennifer.gerasimov@huskers.unl.edu http://ellipsometry.unl.edu

𝑓 𝑜𝑉

𝑓 𝑜𝑉

𝑓 𝑜𝑉