A Gold Nanoparticle-Based Assay for the Measurement of

Nitrite and Nitrate Levels for Soil Macronutrient Measurement

By

Muhammad Shadman Zaman

A thesis submitted in conformity with the requirements

for the degree of Master of Applied Science

Department of Chemical Engineering and Applied Chemistry

University of Toronto

© Copyright by Muhammad Shadman Zaman 2017

ii

A Gold Nanoparticle-Based Assay for the Measurement of Nitrite and

Nitrate Levels for Soil Macronutrient Measurement

Master of Applied Science

Muhammad Shadman Zaman

Department of Chemical Engineering and Applied Chemistry

University of Toronto

2017

Abstract

Soil conditions are a key parameter that determines yield and quality of the crops harvested from

it. The levels of nitrates, phosphates and potassium are the essential nutrients regarding soil, and

most developing parts of the world do not have the luxury to perform any sort of soil analysis.

Commercially available soil test kits were tested for their quantitative ability, but were found to

be inadequate for regular soil testing in all parts of the world. This project takes a step towards

addressing this problem by developing a nitrite/nitrate sensor that is precise, accurate and cost

efficient using gold nanotechnology. While the platform is promising for widespread commercial

soil testing, particularly in developing nations, the reaction has to be performed at 95oC for 1 hour.

The alternatives for improving the kinetics of the reaction to allow its implementation at lower

temperatures and over shorter times are presented.

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Acknowledgements

I would like to begin by thanking Dr. Arun Ramchandran for making this project a success, and

for guiding me in times of struggle and failures.

I would like to express my sincerest gratitude to the Bender Lab and the Chan lab for all their

kindness in letting me use their equipment. I would also like to express Susie from biozone the

highest level of gratitude for assisting me in anything that was needed, from equipment to

consumable to recommendations.

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Table of Contents

Acknowledgements ...................................................................................................................................... iii

List of Tables ................................................................................................................................................ v

List of Figures .............................................................................................................................................. vi

List of Appendices ...................................................................................................................................... vii

1. Motivation ................................................................................................................................................. 1

2. Literature Review ...................................................................................................................................... 6

3. Materials and Methods ............................................................................................................................ 15

3.1 Soil Test Kit ...................................................................................................................................... 15

3.2 Gold Nanoparticles ........................................................................................................................... 16

3.2.1 Nanoparticle Synthesis ............................................................................................................... 16

3.2.2 Nanoparticle Purification and Characterization ......................................................................... 17

3.3 Organic Probes .................................................................................................................................. 17

3.3.1 Organic Synthesis ...................................................................................................................... 17

3.3.2 Purification and Characterization ............................................................................................... 18

3.4 Functionalization of Gold Nanoparticles .......................................................................................... 18

3.5 Nitrite Measurements ........................................................................................................................ 18

4. Results and Discussion ........................................................................................................................... 20

4.1 Soil Test Kit ...................................................................................................................................... 20

4.2 Gold Nanoparticles ........................................................................................................................... 23

4.2.1 Nanoparticle Synthesis ............................................................................................................... 23

4.2.2 DLS Measurements .................................................................................................................... 26

4.2.3 TEM Characterization ................................................................................................................ 28

4.3 Organic Synthesis and Characterization ........................................................................................... 30

4.4 Loading and Testing ......................................................................................................................... 33

5. Conclusions and Future Works ............................................................................................................... 48

References ................................................................................................................................................... 52

v

List of Tables

Table 1 - Nanoparticle Synthesis Parameters ............................................................................................. 16

Table 2 - Soil Test Kit Calibration Data ..................................................................................................... 21

Table 3 - Summarized and Condensed DLS data ....................................................................................... 27

Table 4 - Concentrations of NaCl for samples with salt added ................................................................... 40

vi

List of Figures

Figure 1 - indicates the different regimes of fertilizers and their effects [3] ................................................ 2

Figure 2 - Effect of curvature on distance between DNA strands .............................................................. 11

Figure 3 - A) shows the Griess reaction, B) shows the gold nanoparticles functionalized with the Griess

probes. Weston et al [17] ............................................................................................................................ 12

Figure 4 - On-Off behaviour of kinetic endpoint detection of nitrite with AuNP probes. Weston et al [17]

.................................................................................................................................................................... 13

Figure 5 - DPAA reaction ........................................................................................................................... 18

Figure 6 - DPAN reaction ........................................................................................................................... 18

Figure 7 - Calibration curve for potassium using Rapitest Soil test kit ...................................................... 20

Figure 8- Calibration Curve for nitrate assay of Rapitest Soil Test kit ....................................................... 21

Figure 9 - Absorbance spectrum for phosphorus measurement .................................................................. 22

Figure 10 - Absorbance curves for AuNPs ................................................................................................. 23

Figure 11 - Absorbance of 5x diluted samples after centrifugation at 600xg for 1.5 hours ........................ 24

Figure 12 - UV-vis spectra for the functionalized gold nanoparticles with DPAA probes and DPAN

probes for latest set of experiements. 1 and 2 refer to batch number but are identical in composition ...... 25

Figure 13- DLS number average size distribution of a) sample after centrifugation and b) a sample before

centrifugation ............................................................................................................................................. 26

Figure 14 - TEM image for sample E ......................................................................................................... 29

Figure 15 - TEM image for sample M ........................................................................................................ 29

Figure 16 - TLC for crude DPAN ................................................................................................................... 32

Figure 17 - TLC for crude DPAA ................................................................................................................... 33

Figure 18 - Kinetic measurement of different concentrations of nitrite (150-450 ppm) using functionalized

AuNPs and the effect of salt (0.16M NaCl) ................................................................................................ 34

Figure 19 - Calibration curve for nitrite detection with no additional salt .................................................. 35

Figure 20 - Calibration curve for nitrite detection with no additional salt and evaporation issues corrected

.................................................................................................................................................................... 36

Figure 21 - Calibration curve for nitrite detection with no additional salt added with different batch of

functionalized gold nanoparticles ............................................................................................................... 37

Figure 22 - Calibration curve for nitrite detection with the addition of different volumes of 2M NaCl; a)

20 μL, b) 30 μL, c) 60 μL, d) 80 μL, e) 160 μL .......................................................................................... 39

Figure 23 - Calibration curve for nitrite detection with various amounts of 2M NaCl added .................... 41

Figure 24 - H-NMR for DPAN after column purification .......................................................................... 58

Figure 25 - C-NMR for DPAN after column purification .......................................................................... 59

Figure 26 - H-NMR for DPAA after column purification .......................................................................... 60

Figure 27 - C-NMR for DPAA after column purification .......................................................................... 61

Figure 28 - The general steps to the Griess Reaction.................................................................................. 62

vii

List of Appendices

Appendix A – Procedures ........................................................................................................................... 55

Appendix B – NMR Spectra ....................................................................................................................... 58

Appendix C – Reaction Steps ..................................................................................................................... 62

1

1. Motivation

Food and nutrition have been two major fields of interest for centuries and still continues to be.

The shortage of foods in developing nations, in addition to the lack of nutrition in the food products

is among the leading problems associated with food. Furthermore, the storage and lifetime of food

is a major concern, often more than the yield of crops themselves. A large component to this

problem is soil quality. Soil quality directly affects the nutrition of the crops grown in it, and the

yield of the crops. In fact, there are several positions that this issue occupies in the 50

breakthroughs required for sustainable global development article [1]. This article addresses many

key issues in the developing world ranging from waste treatment to food security.

Breakthroughs 28 and 35 in the document outline issues regarding soil nutrients and their

measurements and/or treatment [1]. The first related engineering breakthrough is number 28 on

the list. This is in regards to low cost systems for precision applications of fertilizers and water.

The key point here is that in order to control the application of fertilizers, one must be able to

measure the fertilizer levels in a quick, accurate, and precise manner. Overuse of fertilizer is a

major issue and causes both economic waste for the farmers (who are generally very poor) and

severe environmental damage. Breakthrough 35 is the need for a portable toolkit for agricultural

extension workers and livestock veterinarians. In essence, there is dire need for farmers to be able

to measure soil quality and nutrient levels in order to optimize the yields and improve crop/produce

quality. These are two breakthroughs which are directly related to soil quality, however there are

several more breakthroughs which are indirectly related to soil quality.

Soil quality is one the most important, if not the most important factor regarding proper crop yields

and nutrition/health. Soils in developing nations are in very poor conditions with complete lack of

the required nutrients (all nations around the Saharan desert) due to high salinity and imbalance of

nutrients in the soil. Nitrate levels largely determine the yield of a crop and also the quality/nutrient

value of a crop as lack of nitrates will cause plants to be yellow in colour rather than green (due to

a lack of chlorophyll). At the same time, too much nitrate will negatively affect the quality and

yield of the plant and thus this must be controlled. Furthermore, a macronutrient such as potassium

plays an important role in regulating the nitrate levels in addition to helping the growth of crops.

The effects of soil nutrients are paramount to high yields and quality, but this is seldom

2

implemented. The main nutrients required are the macronutrients: nitrates, phosphates and

potassium. Micronutrients such as magnesium, calcium and many more also play an important and

subtle role, yet not as significant as the macronutrients. The amount of food produced is only part

of the solution, the other part comes in food security and preservation. Low quality crops will start

to perish in significantly shorter periods of time. Thus, the soil quality not only addresses the food

shortage issues, but also the storing of food. These two are two of the three main issues regarding

food security in developing nations.

A major factor that determines soil health is the proper use of fertilizers. Over fertilization can

harm the soil quality, but more importantly, it can have long lasting effects on the environment

and ecosystems nearby. Leaching of fertilizers cause nitrates and phosphates to be transported into

groundwater and lakes. For example, the excess leaching of fertilizers can lead to eutrophication

of lakes (case study, Lake Erie [2]), which is extremely harmful for the ecosystem as

eutrophication starves the affected body of oxygen, causing the ecosystem to collapse. The limiting

component for eutrophication is phosphorus which is leaching from poor agricultural practices.

Even 10% over usage of phosphorus can be a major concern due to the sensitivity of eutrophication

to phosphorus levels. Thus, it is important to be able to measure nutrient levels as accurately as

possible.

Figure 1 - indicates the different regimes of fertilizers and their effects [3]

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One of the challenges that remains to this day is the inability to monitor soil nutrient levels over

the course of the season. While the initial conditions for soil is paramount for good crop health

and yield, adjustments are critical over the course of the crop growth. An example of this is the

rice crop. The demands for the different nutrients vary with time. While there is a certain minimum

amount of nitrate required at the start, the amount required in the middle stages are not as

important. Instead, the phosphate demand in the intermediate term of the rice crop growth is

imperative for proper yields and health of the crop. Lab tests can provide accurate results; however,

they are expensive and have a lead time which may be too long to be able to make the adjustments

as they are time sensitive. Furthermore, the cost of doing multiple lab tests over the course of the

growth of the crop is prohibitive. Soil test kits lack the quantitative information required for the

application of the correct amount of fertilizer. Both under-fertilization and over-fertilization are

detrimental to the health of the crop which is indicated by Figure 1. Other devices meant for the

measurement of soil nutrient levels are simply too expensive for sustainable usage. There are many

instances of such devices in literature.

It seems that there have been many attempts to create a nitrate/nitrite sensor due to the importance

of its measurement. Currently there are major compromises for each of the current techniques and

devices for the inexpensive measurement of nitrites for soil nutrient detection. It should be noted

that the first step in the procedure for measuring nitrate is to first reduce the species into nitrite.

Thus, any measurement regarding nitrites automatically extends to nitrates.

From an engineering perspective, cost of devices is the limiting factor for whether or not a device

or product is viable. This is where all of the current techniques are extremely lacking. While soil

test kits are relatively cheap with respect to lab analysis, it is still far too expensive for rapid

multiple diagnostic applications. Developing nations and even most people in developed nations

cannot afford the use of these soil test kits. The use of these test kits is the cheapest option on the

market at the moment. The approximate cost of a nitrite analysis for ten samples is 6.3 CAD for

the Rapitest soil test kit, and 2.4 CAD for the LaMotte test kit. It is important to note that there

was an academic discount applied to the LaMotte test kit and thus the actual cost for a consumer

is approximately double that going off the price listed on Amazon.com. While this may seem like

a negligible cost to us, this cost is orders of magnitude too expensive for developing nations. With

just considering cost arguments, these kits are already insufficient. This problem is exacerbated if

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nutrient levels are monitored several times throughout the growth of the crop at several different

locations in a field, which is a practice that is not commonly followed but critical for proper growth

of crops. It is clear that an alternative kit is required, one that can provide precise, accurate, fast

results, and is cost effective.

A device which can satisfy all the conditions required for widespread use has been in high demand.

A sensor with these capabilities will change the way farming and gardening are done, on a global

scale. Not only will this sensor be cost-effective such that it can be used in developing nations, the

rapid diagnostic with high precision and accuracy will be valuable regardless of developing or

developed nations. Accuracy refers to how close a value is to the actual value is and precision

refers to the reproducibility of a value. Performing soil analysis is currently too expensive in Africa

and many parts of South Asia. Even the kits, which are the most cost-effective option currently, is

far too expensive for the average farmer. Furthermore, lab analysis is often impossible due to either

accessibility or cost. The ability for any farmer, anywhere to be able to measure soil nutrient levels

which are both accurate and precise while being fast and affordable would be revolutionary. The

ability to increase crop yields and crop health/nutrition would most certainly go a long way to

combat global hunger on a significant scale.

This project set out to develop a sensor that can determine levels of macronutrients in a manner

that is accurate and precise while being cost efficient and fast. In order to develop a sensor that

can meet all these criteria, nanotechnology is incorporated. Gold nanoparticles are an exceptional

platform for optical sensors with high accuracy and precision in addition to minimized costs due

to lower amounts of materials and chemicals required. A sensor using gold has the potential fill

the void of a device which can accurate and precisely determine concentrations of macronutrients

while being cost efficient such that it may be used in developing nations.

This project aims to engineer a sensor based on gold nanoparticle technology such that it has the

required accuracy and precision, in addition to keeping the cost efficiency as high as possible.

Gold nanoparticles are at the forefront of optical sensors due to their various advantages such as

extremely high molar absorptivity, chemical versatility and the decoupling of the reaction from

the colour. The decoupling of the reaction from the colour is of great importance because

traditional chemistry is extremely limiting as the reaction is required to produce both the specificity

and the colour. The use of gold nanoparticles will allow for a much wider variety of chemistry as

5

the chemistry is only required to maintain specificity while the gold nanoparticles themselves will

give the colorimetric response.

This thesis will first demonstrate that the existing soil test kits are not equipped for quantitative

analyses. Following this, the design and engineering of a gold nanoparticle based nitrite assay is

done in order to demonstrate the ability for an accurate and precise quantitative nitrite assay which

is both cost-efficient and reliable.

6

2. Literature Review

The importance of the measurement of the main nutrients attributed to soil health and quality is

significant. In this regard, there is very little work done to determine the accuracy of off-the-shelf

soil test kits. These commercial off-the-shelf kits measure nitrate, phosphate, potassium and pH of

the soil. A comparison of the accuracy of the three leading soil test kits to analytical tests conducted

by Faber et al [4] shows variations in the performance of different brands of soil test kit. The

results reported by the available test kits are in brackets of low, medium, and high and the

corresponding values for these brackets were determined by Faber et al. and these brackets depend

on the macronutrient. For example, low K level was found to be less than 50 ppm of potassium

whereas low N level corresponded to less than 25 ppm. The high bracket for N is above 60 ppm

and 80 ppm for K. For P, less than 6 ppm of phosphates fell into the low bracket and greater than

10 ppm the high bracket. The medium ranges are simply the range between the low brackets and

high brackets. While there is agreement between quantitative analytical results and the qualitative

soil kit brackets, the brackets of high-medium-low are very large [2]. As a result, the test kit

measurements cannot provide accurate and precise quantitative information to deal with the

problem of soil quality and health. For example, medium nitrate levels account for anything

between 25-60 ppm. The results from the work of Faber et al [4] show a 94% match between

analytical and test kit for the LaMotte brand and a 92% match for the Rapitest. The fact that they

do not match the analytical results even though the guidelines for this match are so lenient is cause

for concern with the current technologies. In terms of overall performance with cost factors

included, the LaMotte test kit seems to be superior to the Rapitest test kit. There are no other

published scientific works done regarding the accuracy and precision of these test kits.

A significant gap in knowledge and understanding regarding soil nutrient levels is the precision

and accuracy required for their measurement. In fact, different crops require different level of

precision and accuracy for the measurement of the soils they are grown in. A report published by

University of Florida [5] indicates crops such as tomatoes are extremely sensitive to macronutrient

levels, and thus, a more refined test kit is required to properly address the quality and soil

measured. The report simply accumulates several bodies’ worth of findings into a single report. It

is apparent that there is a “cutoff” mark after which the fertilizer no longer has a positive effect in

7

terms of yield and quality and a maximum yield mark. These numbers such as 240 lb./acre (120

ppm), are quite specific and thus requires more accuracy than a “range” that includes 120 ppm.

For example, if these test kits were used, there would be no information the moment the potassium

levels surpassed 80 ppm, which is extremely far off for the ideal 120 ppm that tomato crops require.

Even minor levels above or below this mark will be negatively affecting the yield and/or quality

of the tomato crop. Thus, it is best to have a method that is as accurate and precise as possible,

since the required specifications depend on the type of crop.

There is a significant amount of work done in literature to attempt to fill the void of a diagnostic

tool that has the capabilities for soil nutrient measurements (specifically N) with superior precision

and accuracy compared to commercially available options (test kits and lab analysis). All of the

colorimetric devices for the measurement of nitrite use the Griess reaction due to its high

specificity to nitrites and because it develops a colour upon reaction. This implies that most work

regarding diagnostic devices for the measurement of nitrites have the necessary accuracy and

precision. However, traditional Griess reaction does not have a limit of detection low enough for

proper analysis, which is typically reported to be around 10 μM. In addition, the limiting factor

regarding the viability for commercializing nitrite assays for mass use for diagnostic applications

is cost. There is much work regarding nitrite diagnostic devices in literature for the measurement

of nitrite such as the use microfluidic devices in order to control the reaction and mixing [6] [7]

[8]. The use of paper microfluidic channels is a promising endeavor due to the low cost of paper

in terms of material cost and handling costs (low weight, easier to transport). Regardless, the cost

due to the high amounts of Griess reagents required removes the viability of this device for multiple

assays as the cost will be too high for those in developing nations and perhaps even those in

developed nations.

Another technique for measurement of nitrite is electrochemical sensing. This has several

limitations such as the need for more advanced equipment, and thus significant costs. Furthermore,

the limit of detection is poor at around 5 μM nitrite concentration [8]. These electrochemical

methods include both voltammetric [9] detection and amperometric detection [10]. Due to poor

limit of detection in addition to poor cost-efficiency, these techniques cannot fill the need for a

cheap diagnostic device.

8

Development of chemical sensors for the detection of specific targets is currently a very active

field of research. Over the past few decades, the use of gold nanoparticles has taken to the forefront

for chemical sensor applications. Gold nanoparticles have several high desirable traits for sensor

applications which include but are not limited to, unparalleled optical properties, ease of tuning

and chemical functionalization, improved limit of detection, low cost (due to extremely small

amounts of materials required), and chemical versatility [11]. Gold nanoparticles can act as a

substrate for chemical probes, act as an activator to certain chemical species, and act as an inhibitor

to certain chemical species (fluorescent quencher). Gold nanoparticles are widely used for

colorimetric biosensors and fluorescent biosensors. Due to surface Plasmon resonance (SPR),

the optical intensity of gold nanoparticles is extremely high, and sensitive to surface changes to

the gold which include size and conformation. SPR is the oscillation of conduction electrons with

the electromagnetic field at its resonant frequency at an interface between a negative permittivity

material (nanoparticle) and a positive permittivity material (suspending medium, water). Changes

in the size of the particles have a strong effect in the case of nanoparticles due to quantum

confinement effects. The sensitivity allows for the differentiation of sizes on the order of one

nanometer. However, the instrumentation required to measure such a fine change in SPR is not

feasible for cost effective diagnostics. Thus, a larger shift in size of nanoparticles is required for

an observable change of colour. One way this is achieved is to induce aggregation of nanoparticles

through chemical reactions between a probe and its target. Furthermore, the high optical intensity

of gold nanoparticles allows for extremely small concentrations giving an intense colour, which

allows for the use of significantly less material for these optical sensors. This in turn reduces the

cost of the assays by an extremely large amount as the cost of the assays are almost always due to

the cost of the chemical species.

The synthesis of gold nanoparticles has evolved greatly, but has remained very simple. To this

day, the Turkevich method/Frens method remains as one of the best methods for synthesizing

nanoparticles within a certain size range. This method calls for the synthesis at 100°C of gold (III)

chloride salt with sodium citrate. The sizes of the nanoparticles can be controlled via many

different factors such as concentration of the reductant, concentration of the gold salt, temperature,

time of reaction, and salt concentration [12]. The ideal size of nanoparticles which exhibit high

monodispersity is between 10-20 nanometers using the Frens method. Frens method may be used

for the synthesis of larger nanoparticles, however it must sacrifice its monodispersity and spherical

9

morphology. Other methods for the synthesis of gold include the use of different reducing agents

(ascorbic acid, hydroquinone) and seed based synthesis procedures. Through these different

procedures, other size ranges of gold nanoparticles can be made.

The characterization of nanoparticles is necessary to determine its size, morphology and

polydispersity. There are several techniques used for the characterization of nanoparticles.

Dynamic light scattering (DLS) is a technique which is often used to determine the size

distribution of nanoparticle samples. DLS characterization has become standard when reporting

works related to nanoparticles. Khledtsov et al [13] describes the process as it relates to gold

nanoparticles, and cautions against the existence of false peaks and the difficulties of interpreting

the data due to the interferences with the environment. One of the benefits of DLS they state is the

ability to sample a large ensemble of particles in situ which cannot be done by other techniques,

primarily compared to transmission electron microscopy (TEM). DLS can be used to determine

the size distribution of a sample of gold nanoparticles. The distribution of the samples are given

by the polydispersity index (PDI) which is the standard deviation of the size distribution divided

by the mean of the standard deviation. In the case of gold nanoparticles, a sample is considered to

be of high quality if the PDI is less than 0.1.

TEM is the primary technique for the characterization of gold nanoparticles. That being said, TEM

is done in conjunction to UV-visible spectroscopy and DLS for a complete characterization. TEM

is a microscopy technique which goes beyond the diffraction limit of visible light by using

electrons as the light source. Thus, a sub-nanometer resolution image of a sample can be obtained.

Electron microscopy uses an electron beam, and thus requires a high-ultrahigh vacuum for proper

functioning of the instrument. There are generally two detectors, one for secondary electrons and

one for backscattered electrons. As opposed to scanning electron microscopy (SEM), TEM

requires a thin sample such that the electron beam can be “transmitted” through the sample.

Therefore, the sample preparation for TEM is more cumbersome and challenging; however, it also

provides better resolution than SEM. The technique of TEM will generate images of nanoparticles

deposited on a thin copper grid. The generated images are treated with image analysis and

statistical treatments to extract many pieces of necessary information including but not limited to

size distribution, morphology, and cleanliness of sample.

10

UV-visible spectroscopy is routinely performed after the synthesis of any gold nanoparticles. UV-

vis is a very efficient way to get a rough idea of the concentration and size of the nanoparticles.

The determination of approximate size and concentration can be done due to the work completed

by Liu et al [14]. For a certain size of nanoparticle, an approximate molar absorptivity can be used

in conjunction with Beer’s Law to get an approximate concentration. This study also presents an

empirical relationship between the absorbance data and particle size.

After synthesis of the gold nanoparticles, they must be functionalized with the desired probes for

the application. One of the major benefits to working with gold nanoparticles is self-assembly,

which is where the bonding and structuring of the molecule and a surface happens spontaneously

and without external stimuli, into a well-structured and defined pattern. This self-assembly occurs

as long as there is an open site on the gold surface and therefore forms a monolayer, also known

as self-assembled monolayers (SAM). The interaction between a thiol group and gold forms a

very strong attraction that is on the order of a covalent bond (on the order of 50-100 kJ/mol) [15].

The process of loading chemical groups onto gold is a facile process, however there are many

factors that determine the quality and success of the loading. The work done by Hurst et al [16]

explored the effects of multiple factors on the loading of nanoparticles including size, salt aging,

use of spacers, and sonication. In this particular case, the work done was with DNA loaded onto

gold nanoparticles. The findings show significant benefits from proper loading conditions

regarding all of the factors listed above. DNA loaded onto gold was maximized with salt aging of

0.7M NaCl, and through the use of polyethylene glycol (PEG) spacers. The use of spacers is

integral as they lead to a more “upright” configuration of the DNA, leading to more surface area

available for more DNA strands to be loaded onto. The presence of salt is also imperative as it

causes screening such that the negatively charged DNA strands do not experience as much

repulsion between each neighboring strand. Positive effects were observed until the approximately

0.7M NaCl at which point the effects stagnated. The increase in salt concentrations lead to a shorter

Debye length, hence reducing the electrostatic repulsion, which allows for the DNA to pack tighter.

Sonication also showed positive effects on the adenosine and thymine spacers as they tend to “lie”

down on the gold surface, and the sonication disrupts the interaction between the spacers and the

surface, causing a conformational change to a more upright position, thereby increasing loading.

For spacers like PEG that are already relatively “upright,” no effect was seen from sonication,

which is consistent with the previous explanation. Finally, as the size of nanoparticles increase, so

11

does the loading. This is expected as there is a larger surface area with larger nanoparticles.

However, the smaller nanoparticles have a higher loading density, which means that there are more

DNA strands per unit area. This is explained via curvature arguments: the smaller the

nanoparticles, the larger the degree of curvature at a local scale. A greater degree of curvature

means there is a greater lateral distance between the heads of the DNA, which reduces the

electrostatic repulsion. This phenomenon is shown in the diagram below (Figure 2).

Figure 2 - Effect of curvature on distance between DNA strands

An additional and equally important role of functionalization of gold nanoparticles is to stabilize

them. Gold nanoparticles are a colloidal system and can therefore be unstable via aggregation and

precipitation by gravity. The functionalization of gold nanoparticles also serves to improve the

stability. Typically, the particles are functionalized with stabilizing thiols immediately following

synthesis to increase their shelf life, and further functionalized later on for sensing purposes.

12

Figure 3 - A) shows the Griess reaction, B) shows the gold nanoparticles functionalized with the Griess

probes. Weston et al [17]

Work done by Weston et al [17] has set the foundations for this project. The work involved the

use of functionalized gold nanoparticles for the detection of nitrates using a kinetic endpoint. This

study shows the possibility to quantitatively probe for inorganic targets for sensor and diagnostic

applications. In this application, the Griess reaction is employed for the sensing of the nitrate via

nitrite. The reaction scheme is shown above in Figure 3.

The probes were synthesized such that they could functionalized onto gold nanoparticles (thiol

capped) while having the active components for the Griess reaction. The gold nanoparticles were

synthesized via Frens method (0.2g of AuCl3 dissolved in 490 mL of Milli-Q water and brought

to a boil at which point 10 mL of 0.0454 g/mL sodium citrate was added and refluxed for 10

minutes). The nanoparticles are reported to have been synthesized at a size of 13 nm ± 1 nm. The

organic probes were synthesized using standard organic chemistry procedures and are purified via

flash chromatography. This was followed by a full characterization with FTIR, NMR and mass

spectroscopy. Due to the chemical composition of the probes, a stabilizer probe was co-loaded

onto the gold so that the gold nanoparticles do not aggregate and precipitate out of water. The

stabilizer (11-mercapto-undecyl)-trimethyl-ammonium (MTA) is a probe with an ammonium

head group and a mixture of 15:1 of MTA to active probe ratio is used to properly stabilize the

loaded nanoparticles in water. After the loading process, the nanoparticle samples are cleaned by

centrifugation and are then tested with nitrite. In this study, a kinetic endpoint measurement was

done, and thus, the reactions were not taken to completion, but only for 25 minutes at 95°C. At

this point, the absorbance was measured using a microplate reader. Due to the nature of their study,

13

only a qualitative treatment of the presence of nitrites was possible, acting as an on off sensor for

a certain micromolar level of nitrite. Figure 4 below is taken from the paper which shows the on-

off behavior of the system under kinetic endpoint treatment. Although the results in the paper do

not show quantitative capabilities, as stated in the paper itself, given proper reaction time and

improved reactions and loading schemes, a fully quantitative treatment is possible. This is the basis

for the first major part of this project.

Figure 4 - On-Off behaviour of kinetic endpoint detection of nitrite with AuNP probes. Weston et al [17]

The nature of the Griess reaction (in this system) is such that it is a very slow reaction in terms of

the necessary point-of-care diagnostic specifications (Griess reagents are not in excess). For this

reason, a kinetic endpoint study is required as it was in the work summarized above. In order to

understand and engineer the correct system for such a device, the kinetics of the reaction itself

must be understood. While there are no studies for the kinetics of this reaction where the aniline

and naphthylamine groups are immobilized on surfaces, an understanding of the classical bulk

reaction provides some clues. For this reason, the kinetics of this reaction in a standard setting is

the lower bound of the rate of reaction involving the Griess reagents being functionalized onto

nanoparticles. A comprehensive study was conducted by Jay Fox Jr. [18] which studied the

kinetics and the mechanism for the Griess reaction. Firstly, the study demonstrates that the reaction

14

is first order with respect to the nitrite concentration. The order of the reaction with respect to the

aniline and naphthylamine group is not explicitly mentioned. This is justified because the standard

practice for this reaction is such that the concentration of the aniline and naphtylamine species are

100-fold of the nitrite for optimal colour production. Due to the relative concentrations of the two

reagents compared to the nitrite, the aniline and naphthylamine species concentrations can be

absorbed into the rate constants. The results also indicate that the formation of the intermediate

diazonium ion is the rate limiting step with the use of native aniline. The steps for the Griess

reaction involves the nitrosation of the aniline species followed by an internal diazonium ion

formation. The final step of the reaction involves the reaction of the diazonium ion with the

naphthylamine species which forms a double bonded nitrogen bridge between the aniline species

and the naphthylamine species. These reaction steps can be found in Appendix C (Figure 28). In

addition, this work identifies chemical group effects on the rate of reaction due to electron

withdrawing effects or electron donating effects. The species used in this thesis are simply aniline

and naphthylamine. The results from this kinetics work indicate that native aniline reaction rate is

significantly slower than other derivatives. Aniline species which involve an electron withdrawing

group show an exceptionally faster rate of reaction for the formation of the diazonium ion as it is

the electron withdrawing effect which initiates the reaction by weakening the N-O bond. This

information provides the means to increase the rate of reaction which is a limiting factor in the

Griess reaction.

15

3. Materials and Methods

3.1 Soil Test Kit

Two different soil test kits were procured for testing of their quantitative capabilities, the Rapitest

1601 soil test kit and the LaMotte soil test kit. Their capabilities were tested by making a

calibration curve using freshly made standards. Stock solutions of potassium nitrate (239 mg in 20

mL) and sodium phosphate (25 mg in 500 mL) were made fresh. From these stock solutions,

solutions of 20 ppm, 40 ppm, 60 ppm and 80 ppm concentration solutions were made for nitrate

and 40 ppm, 60 ppm, 80 ppm and 100 ppm concentration solutions were made for potassium. In

the case of phosphate, concentrations of 2.5 ppm, 5 ppm, 7.5 ppm, and 10 ppm were made. Each

of these concentrations were tested with the soil test kits according to the instructions for each

respective kit. Once the colour had developed, the absorbance of each sample was measured using

a 96-well plate reader for multisampling purposes. Note that the phosphates were measured

individually using a UV-vis spectrometer instead. In order for a method to have the capability for

quantitative analysis, a linear relationship between absorbance and concentration must be present.

This is due to Beer’s Law which states that absorbance is proportional to concentration.

The procedure for the measurement of the kit’s capabilities followed the steps outlined on the test

kit. The instructions state waiting for around 7-10 minutes after mixing in the reagent with the soil

sample. Thus, each of the samples were measured using the plate reader (or UV-vis in the case of

phosphate) exactly 10 minutes after they were mixed with the reagent provided in the kit. It should

be noted that 10 minutes was the point at which the measurement began, not when the sample was

loaded into the plate reader or spectrometer. The samples were mixed using a vortex mixer in order

to remove mixing as a factor that could cause variability. Each sample was mixed for 25 seconds

using the vortex mixer before being loaded onto the well plate. The volume of the “soil” sample

is controlled by containers provided by the manufacturer. This amount corresponded to 5.6 mL as

measured out using a 10-mL graduated cylinder. Thus, the reagents were added to 5.6 mL of the

samples in a 20-mL scintillation vial. This procedure was used and repeated for NPK tests for both

kits. It should be noted that pH is also measurable by the test kits, however this was not tested.

16

This is because pH measurements are already well established and are not the limiting factor of

the kit. Highly precise and accurate pH measurements using colorimetric techniques already exist.

3.2 Gold Nanoparticles

The detailed procedures of the synthesis and characterization of the gold nanoparticles, the

synthesis and characterization of the probe molecules, and the functionalization of the

nanoparticles with probes are provided in the Appendix. Here, we provide short descriptions of

these steps.

3.2.1 Nanoparticle Synthesis

Gold nanoparticles were synthesized using the advance Turkevich method (also known as Frens

method). This synthesis methods involved the reaction of gold (III) chloride trihydrate (50% w/w

of gold) with citrate. The gold salt is added to a certain volume of water and brought to a boil while

stirring. Once boiling, a certain amount of citrate (between 4:1 and 8:1 citrate:gold) is added. The

citrate acts as both the reducing agent and the stabilizing agent (electrostatic and steric

stabilization). It should be noted that the procedure was streamlined with time and latest procedures

involve the reaction of 0.073 g of gold (III) chloride trihydrate in 200 mL of MilliQ water being

reduced by 0.456 g of citrate salt. In order to ensure proper amount of gold was being added, gold

stock solution was made and a volume corresponding to the weight of 0.073 g was added to the

water. This was the only way to have an accurate amount of gold added as the salt was difficult to

measure out with the required level of accuracy and precision.

Table 1 - Nanoparticle Synthesis Parameters

Batch name Approximate

Volume (mL)

Gold (III)

Chloride (mg)

Citrate (mg) Citrate:Gold

Ratio

A 600 39.0 881 30

B 600 23.5 927 53

C 450 48.4 152 4

D 900 105 305 4

E 900 105 610 8

M 500 mL 197 570 4

17

Each of the samples in Table 2 were made in 500 mL Erlenmeyer flasks except for the Mirkin

sample which was done in a 1 L three-necked round bottom flask. The temperature for each

reaction was 100°C. All sample besides the Mirkin sample were done without reflux, and thus the

volumes stated are approximate volumes. The Mirkin sample was boiled under reflux and thus the

volume is accurate at 500 mL. These were the earlier batches of gold nanoparticles; however, the

results of the synthesis are very good regardless of sub-optimal conditions for synthesis

(citrate:gold ratio below 4 or beyond 8). It should be noted that these gold nanoparticles were not

used. The gold nanoparticles used in the measurements were all synthesized using the method

mentioned above with a volume of 200 mL.

3.2.2 Nanoparticle Purification and Characterization

There are several steps to the characterization of gold nanoparticles. Centrifuging was used to

purify the nanoparticles and DLS, UV-vis and TEM were used to characterize the AuNPs.

3.3 Organic Probes

Henceforth, the first probe (5-[1,2]dithiolan-3-yl-pentanoic acid [2-(4-amino-phenyl)ethyl]amide)

will be abbreviated as DPAA, and the second probe (5-[1,2]dithiolan-3-yl-pentanoic acid [2-

(naphthalene-1-ylamino)ethyl]amide) will be abbreviated as DPAN.

3.3.1 Organic Synthesis

The two organic probes, DPAA and DPAN, were synthesized at room temperature for 24 hours.

The reaction involved was a DCC coupled condensation reaction to form an amide link between

the spacer molecule (lipoic acid) and the aniline/naphthylamine species. The reactions were carried

out in dichloromethane and the reaction vessel was sealed using a silicone septum cap to prevent

evaporation of the solvent. A needle was pierced into the septum cap to prevent over

pressurization.

18

Figure 5 - DPAA reaction

Figure 6 - DPAN reaction

3.3.2 Purification and Characterization

The purification of the organic compounds proved to be very challenging. The purification scheme

for both DPAA and DPAN were developed in-house.

In reality, the separation of DPAN was extremely complicated and details of this are presented in

the following section. Following the purification of the DPAA and DPAN using flash

chromatography, they were both characterized using NMR. It should be noted that further

characterization such as the use of mass spectrometry was not required as the NMR data for the

purified products are presented in the work from Weston et al [17] and thus were simply compared

against those results.

3.4 Functionalization of Gold Nanoparticles

The functionalization of the gold nanoparticles with the DPAA and DPAN probes is

straightforward. The process is completed over the course of approximately one hour with no

external stimuli required due to the self-assembly of thiol on gold. Store wrapped in aluminum foil

and refrigerate.

3.5 Nitrite Measurements

The measurement of nitrite using the functionalized gold nanoparticles (F-AuNPs) was done using

a 96-well plate reader. Each sample was prepared in a 1.5 mL microcentrifuge tube.

+

+

19

For experiments that do not add any additional salt (no NaCl), the reaction rate is so slow at room

temperature that t0 or the time at which the reaction starts is when it is placed into the oven. For

experiments involving additional salt, the aggregation induced by salt occurs even at room

temperature and thus must be stopped from occurring before placed into the oven by placing it in

an ice bath. This way t0 is when the samples are placed into the oven at 95°C.

20

4. Results and Discussion

4.1 Soil Test Kit

As mentioned earlier in the report, soil test kits of the two leading brands (LaMotte and Rapitest)

were tested for their quantitative capabilities. The results below show poor correlation for the P

and K assays, but show good correlation for the N assay. The results are summarized in Table 2.

The following calibration curve for the Rapitest potassium assay shows a clear indication that the

test kits are unable to quantitatively measure for soil macronutrients. There should be a strong

linear correlation (R2 value close to 1) during a calibration experiment/measurement. All standards

were prepared on the day of the test. All glassware was thoroughly cleaned using water, IPA, and

acetone. Results from the Rapitest potassium test are shown below.

Figure 7 - Calibration curve for potassium using Rapitest Soil test kit

The nitrate assays for both the Rapitest and LaMotte test kits yielded very respectable calibration

curves. Figure 7 shows the calibration curve for the Rapitest test kit and Table 2 includes the

regression information for all the assays for these two kits. While the results for the nitrate/nitrite

y = 0.0024x + 0.621R² = 0.6523

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

0 20 40 60 80 100 120

Ab

sorb

ance

Concentration (ppm)

Calibration Curve for Potassium Detection using Rapitest Soil Test Kit

21

assays imply that the assay holds enough precision and accuracy required for soil nutrient

measurement applications, it is far too costly as mentioned earlier. However, this result highlights

the strength of the Griess reaction: a specific reaction to detect nitrate with high level of precision

and accuracy. It should be mentioned that the kit cannot perform a nitrite analysis, but only a nitrate

analysis.

Figure 8- Calibration Curve for nitrate assay of Rapitest Soil Test kit

Table 2 - Soil Test Kit Calibration Data

Test Regression Fit (R2)

L-N Test y = 0.0414x - 0.6478 0.8667

L-P Test y = 0.0387x - 0.0358 0.9690

R-K Test y = 0.0024x + 0.621 0.6523

R-N Test y = 0.0301x + 0.5163 0.9397

R-P Test - -

y = 0.0301x + 0.5163R² = 0.9397

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40 50 60 70 80 90

Ab

sorb

ance

Concentration (ppm)

Calibration Curve for Nitrate Detection using Rapitest Soil Test Kit

22

The calibration curve was made using the maximum absorbance wavelength determined from the

absorbance curves. The absence of the Rapitest phosphorus test is due to correlation being

completely non-existent. Calibration curves involve the measurements at a wavelength for which

the absorbance is a maximum. This does not exist for this kit’s phosphate assay within the visible

range. The absorbance curve for the Rapitest phosphorus test is shown below in Figure 9.

Figure 9 - Absorbance spectrum for phosphorus measurement

Just from the results in table 1, it is clear that these correlations are far from what is needed for

quantitative analysis capabilities (besides the nitrite assay). Furthermore, the time required to

develop a colour is variable and stability of the colour once developed is very poor for all three

assays. This was tested by measuring the absorbance only five minutes after the first reading and

the readings were already extremely different. This was verified once more as the absorbance

readings were again very different at the 25-minute mark. Overall, the test kits’ abilities to perform

these assays with precision and accuracy are not satisfactory, in addition to cost issues that have

been discussed.

-3

-2

-1

0

1

2

3

4

5

6

150 250 350 450 550 650 750

Ab

sorb

ance

Wavelength (nm)

Rapitest P Test Absorbance Data

2.5 ppm

5 ppm

7.5 ppm

10 ppm

23

4.2 Gold Nanoparticles

4.2.1 Nanoparticle Synthesis

Figure 10 - Absorbance curves for AuNPs

Each one of the datasets in Figure 10 represent a batch synthesized in the summer of 2016. It

should be noted that every profile is the ideal and expected absorbance profile for gold

nanoparticles synthesized with the Turkevich method. Samples 1 and 2 were the first batches

synthesized and had suboptimal citrate:gold ratio for nanoparticle synthesis and can be seen to

have a peak shifted from the 524 nm peak exhibited by the other samples from Table 2. The other

AuNP samples were synthesized using a proper citrate to gold ratio, and as a result, showed the

expected behavior on the UV-visible spectra.

0

0.5

1

1.5

2

2.5

3

425 475 525 575 625 675

Ab

sorb

ance

Wavelength (nm)

Absorbance Spectra for AuNPs

A

B

C

D

E

M

24

Figure 11 - Absorbance of 5x diluted samples after centrifugation at 600xg for 1.5 hours

Due to the extremely high concentrations used in the Mirkin procedure, the synthesis comes with

some amount of larger aggregates. In order to purify the sample to only contain the nanoparticles

of the size desire (approximately 13 nm), the samples were centrifuged at low speeds (600xg) to

force the larger aggregates and particles to pellet. The supernatant was extracted and characterized

to see if the samples were purified. This was done through DLS which is presented in the following

section. Figure 11 is the UV curve for samples 1.3 and 1.4 after centrifugation (notice that the

samples were diluted for proper absorbance readings). Once again, the difference in the peak height

is due to the small variance in concentration due to different rates of evaporation during the

synthesis of different batches.

25

Figure 12 - UV-vis spectra for the functionalized gold nanoparticles with DPAA probes and DPAN

probes for latest set of experiements. 1 and 2 refer to batch number but are identical in composition

Figure 12 shows the UV-vis spectra for the functionalized gold nanoparticles. UV-vis readings

must be taken for the F-AuNPs in order to determine the concentration of the nanoparticles after

the washing step. This concentration is essential such that the nitrite assay can only be done with

a known concentration of AuNPs. Note that the nanoparticle samples must be diluted between

tenfold and twentyfold such that the absorbance readings do not saturate. The absorbance values

at 524 nm are thus adjusted by multiplying them by the dilution factor. From these values in

conjunction with the molar absorptivity value for 15 nm AuNP at 524 nm (ε = 2.7 x 108 M-1cm-1),

the concentration of the nanoparticles are determined.

The reason that these F-AuNPs do not appear identical on the UV-vis spectrum is because the

amount of water they are re-suspended in is not controlled/measured out. The reason this was not

done is because it does not have any effect on the measurements or how they are done as they

simply have to be added to a final concentration of 2.5 nM. Thus, for a lower concentration stock

of F-AuNPs, slightly more volume needs to be added to each assay sample, but the total volume

of the stock F-AuNP is also higher. The difference in the volumes are simply accounted for by

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

300 400 500 600 700 800 900

Ab

sorb

ance

Wavelength (nm)

Functionalized Gold Nanoparticle Spectra

DPAA 1

DPAA 2

DPAN 1

DPAN 2

26

adding more or less water. However, this process can be standardized such that all of these

concentrations are nearly identical if the gold nanoparticles are re-suspended in a fixed volume of

water during the washing stage and if a set and measured volume of the supernatant is discarded

from each sample. This standardization is possible but is not necessary for the assay.

4.2.2 DLS Measurements

DLS measurements were done in Professor Chan’s lab on several AuNP samples. The following

data presented outline the findings of these measurements.

Figure 13- DLS number average size distribution of a) sample after centrifugation and b) a sample before centrifugation

Figure 13 b) shows a clear peak at very high sizes which correspond to aggregates forming. These

aggregates are thought to be formed due to the high concentrations of precursors used in the AuNP

synthesis procedure. In order to remove the aggregates, the samples were centrifuged at low spin

to pellet all aggregates above 100 nm. Upon doing so, a size distribution seen in Figure 13 a) was

27

achieved as planned. This was seen for every sample, and their results can be seen in Table 4. It is

clear that there are larger aggregates in each of the unpurified samples (1.0 – 1.4) which causes the

polydispersity to be poor. However, these samples after centrifugation show the absence of the

larger peak altogether and a much better polydispersity (especially sample 1.3). However, all

samples show a significant decrease in the PDI after centrifugation, but sample 1.3 seems to have

the higher quality factor from all the samples listed in Table 4. It should be noted that the removal

of such a small number of larger particles are important in case any of the future treatments or

processing of these particles are surface area dominated. In that case, even a small amount of these

larger aggregates will be an issue. By ensuring that the sample is clean and void of any larger

aggregates, it prevents this issue altogether.

Table 3 - Summarized and Condensed DLS data

Sample Name Z-Ave

(d.nm)

PdI Number

Mean

(d.nm)

Pk 1

Mean

Int

(d.nm)

Pk 2

Mean

Int

(d.nm)

Pk 1 Area

Int

(Percent)

Pk 2 Area

Int

(Percent)

1.0 17.76 0.149 13.02 18.52 0 100 0

1.1 18.92 0.31 11.79 17 245.6 84.2 15.8

1.2 17.81 0.208 12.78 17.5 291.1 94.8 5.2

1.3 16.88 0.259 13.39 15.13 0 100 0

1.4.1 16.51 0.15 13.28 16.56 4325 96.7 3.3

1.4.2 19.77 0.225 13.65 15.67 201 93.4 6.6

1.3 sonic 22.48 0.46 13.72 15.84 151.4 69.2 29

1.4 sonic 113.9 0.163 13.96 16.44 292.4 79.7 20.3

1.4 sonic2 95.22 0.162 13.74 15.07 122.4 87.6 12.4

1.4 centrifuge

(500xg)

16.04 0.105 12.88 16.38 0 100 0

1.0 centrifuge

(600xg)

17.36 0.103 14.38 18.29 0 100 0

1.2 centrifuge

(600xg)

17.71 0.155 14.07 17.76 3829 95.5 4.5

1.3 centrifuge

(600xg)

15.93 0.052 13.74 16.52 0 100 0

1.4 centrifuge

(600xg)

16.19 0.1 13.5 16.4 0 100 0

Another interesting piece of data is from the experiments that included the sonication step. The

DLS measurements clearly show that after sonicating a sample for a short duration, it induces

breakup of the reversible aggregation portion of the aggregates. All sonication data show the peaks

28

of larger aggregates appear as smaller aggregates when compared to non-sonicated DLS data. In

general, the DLS data obtained shows the need for purification of the AuNPs synthesized by the

Mirkin recipe and shows a huge improvement in the quality of the nanoparticles (PDI) once

centrifuged. For this reason, the centrifugation of gold nanoparticles after their synthesis was added

to the procedure.

4.2.3 TEM Characterization

For every gold nanoparticle batch synthesized, it is characterized and imaged using TEM. The

preparation of the TEM samples includes the deposition of a small drop of the gold nanoparticle

solution onto a carbon grid (purchased from Ted Pella) and letting the particles adsorb onto the

copper grid for approximately 10 minutes. At that point, a Kimwipe was used to remove the excess

liquid on the copper grid using a gentle dab onto the liquid film. The TEM used for all images

including the ones not included in the report were taken using the CTEM setup in the Centre for

Nanostructure Imaging in the Department of Chemistry (Lash Miller), at the University of Toronto.

The accelerating voltage used was 75 kV.

29

Figure 14 - TEM image for sample E

Figure 15 - TEM image for sample M

30

Image analysis using ImageJ shows an average size of 12.85 nm ± 0.11 nm for Figure 14 and 13.01

nm ± 0.15 nm for Figure 15. This is consistent with the DLS results shown above. These numbers

are obtained using a small sample sizes, however for both the above images, multiple other images

were used to verify these values. Furthermore, it should be noted that the centrifugation process

was not carried out for the two samples corresponding to the two images. This was noticeable on

the TEM as there were sections of very large aggregates on the carbon grid. It should also be noted

that the synthesis protocol for sample M and its results were used for all further experiments. This

is the recipe used for the nitrite measurements.

The images in Figure 14 and Figure 15 show the morphologies and the size distribution of the

nanoparticle sample. Both these samples have good morphology with spherical geometries and

there do not seem to be much size discrepancies between the different particles in the image.

4.3 Organic Synthesis and Characterization

The crude product was purified using column chromatography. This was done by setting up a

column for 1 grams of crude product at a ratio of approximately 100:1 silica:crude ratio.

During the purification of the second probe, DPAN, there were severe issues in determining a

proper system for the chromatography. After many iterations, Figure 16 shows a system which

shows the different components of the crude product. Unlike DPAA which had three distinct spots,

DPAN had many more, and the separation is still not ideal for the 15:1 DCM:Methanol (MeOH)

2% trimethylamine (Et3N) system. The two bottom sets are not easily resolvable. However, this

was the best iteration without the use of exotic systems which are very complicated and may cause

more problems than good. The top spot was determined to contain the product and was collected.

However, after much trial and error and upon further analysis, the system of 1:1 DCM:EA (ethyl

acetate) showed the spreading of that single spot into four more spots. The crude showed the same

four spots in addition to a concentrated broad band near the origin which are the impurities already

separated using the 15:1 DCM:MeOH 2% NEt3 system. Even though this system was significantly

better, the product (which is the second spot from the solvent line), was not a distinct spot and

would be impossible to properly separate. Upon even further trial and error to try and obtain an

even better system, a 70:30 DCM:EA mixture was determined to be most effective for the

separation and purification of DPAN. It should be noted that even this system was not ideal and

31

could not effectively separate DPAN from its impurities. However, through half a year of trial and

error, this was determined to be the best system from all the systems tested. During this rigorous

trial and error process, even exotic systems (three solvent systems) were investigated but were not

successful.

The mobile phase used to extract DPAA was 95% DCM 5% MeOH. Notice in Figure 17 that there

is significant broadening of a spot (right plate). This is due to NHx groups “sticking” to the silica

and thus the addition of a small amount of trimethylamine primes the silica to prevent the

broadening. This is seen on the left plate in Figure 17. Using the system of 20:1 DCM:MeOH, the

purified DPAA elutes first (top spot). DPAA was very easy to separate without any issues once a

proper system was identified. Note that the RF values for this system is far from ideal (which is

approximately 0.3), however, clear separation was achieved.

Once both products were supposedly purified, they were characterized via NMR to ensure that the

product has been isolated. It should be noted that the crude was also characterized in order to

ensure that the chemical species of interest was in fact synthesized.

32

Figure 16 - TLC for crude DPAN

33

Figure 17 - TLC for crude DPAA

4.4 Loading and Testing

The first experiment conducted for the nitrite assay with F-AuNPs was a room temperature kinetic

reaction which was monitored over the course of 8000 seconds. The results, seen in Figure 18,

indicate that the reaction did not occur at room temperature. In the no salt case, the change in

absorbance is negligible and shows no sensitivity to the nitrite concentrations. The samples with

added salt show aggregation over the course of the experiment, however there is no difference in

the absorbance readings for the different concentrations of nitrite. This is a clear indication that

the salt aggregation mechanism does not discriminate with respect to the nitrite concentrations.

Thus, the salt is simply enhancing the aggregation and any change in the absorbance readings

during the assays will be purely from the nitrite reaction induced aggregation. The aggregation

observed in Figure 18 is simply due to colloidal instability due to the Debye length reduction from

the added salt.

34

Figure 18 - Kinetic measurement of different concentrations of nitrite (150-450 ppm) using functionalized

AuNPs and the effect of salt (0.16M NaCl)

There were two main objectives and goals for the measurements of nitrite using F-AuNPs: to

obtain a calibration curve for the change in absorbance as a function of nitrite concentration, and

to study the effects of salt on the aggregation process. The results show a very desirable effect with

the addition of salt, in addition to an on/off feature on top of the quantitative ability. Most

importantly, all the results obtained suggest the ability for these F-AuNPs to be used as a nitrite

detection assay with superior capabilities compared to existing sensors whether it is in terms of

precision and accuracy and/or cost.

0.15

0.25

0.35

0.45

0.55

0.65

0.75

0 2000 4000 6000 8000

Ch

ange

in A

bso

rban

ce

Time (s)

Kinetic Curves for Different Nitrite and Salt Concentrations

150 ppm

300 ppm

450 ppm

150 ppm no salt

300 ppm no salt

450 ppm no salt

35

Figure 19 - Calibration curve for nitrite detection with no additional salt

Figure 19 shows the measurement of nitrite using the F-AuNPs in the case where there was no

additional salt added. The spread in this data is quite poor with an R2 value of 0.92. For accurate

and precise measurements, a much lower spread of data is required in order to be used in a

quantitative manner. However, the reason for this spread was identified and remedied as seen from

later plots involving the same measurement repeated with the same batch of F-AuNPs and another

batch of F-AuNPs. The reason for the spread is due to evaporation effects. Since these experiments

are done at 95°C, evaporation effects are very prominent since it is an aqueous system. Between

this measurement and another measurement, it was observed that even though the caps of these

tubes clicked, there was still an opening in the middle or the back of the tube cap. This was noticed

after this experiment was completed and is a major reason for the spread. The final volume

remaining in the tubes after the heating in the oven was very different across almost all the tubes.

This indicates that a) there was evaporation and b) the rate of evaporation was different across the

different tubes. This is simply because some tubes do close properly and some require an extra

push. Through another experiment and a few salt experiments shown below, it was apparent that

y = 0.0049x - 0.068R² = 0.9243

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 20 40 60 80 100

Ch

ange

in A

bso

rban

ce

Nitrite Concentration (µM)

No Salt Calibration Curve

36

even if the cap was closed as tightly as possible, there were still evaporative effects. In order to

help minimize these effects, Teflon tape was used for the final set of no salt experiment (Figure 20

and Figure 21). As a result, the spread on the data from those experiments are greatly minimized.

The possibility of the nitrite itself evaporating must be considered. In these acidic solutions, nitrite

ions will be in the form on nitrous acid which can be volatile. However, due to the concentrations

of nitrite introduced into the solutions (on the order of 10 μM), the evaporation of nitrous acid is

negligible compared to the evaporation of water. But regardless of the spread, there is a very

strong positive and linear correlation in Figure 19 between the change in absorbance of the sample

before and after the reaction versus the nitrite concentration.

Figure 20 - Calibration curve for nitrite detection with no additional salt and evaporation issues

corrected

Once the spread problem was identified as an evaporation problem, the experiment was repeated

by wrapping by the tubes with Teflon tape at the cap. This resulted in drastically improved results

as seen in Figure 20 with the spread in the data greatly reduced. The R2 value increased to 0.98

which is a substantial improvement over the previous no salt measurement. This correlation proves

y = 0.0072x - 0.0398R² = 0.9787

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30 40 50 60 70 80 90 100

Ch

ange

in A

bso

rban

ce

Concentration of Nitrite (μM)

No salt Concentration Repeat

37

the ability to provide a means for accurate and precise quantitative measurements using these F-

AuNPs. These results can still be improved by obtaining a purer DPAN product as the impurities

in the product can be causing interferences.

Figure 21 - Calibration curve for nitrite detection with no additional salt added with different batch of

functionalized gold nanoparticles

Figure 21 shows a strong linear correlation between absorbance and the concentration of nitrite

similar to Figure 20. This experiment was done with an alternate batch of F-AuNPs. The amount

of DPAN on those nanoparticles are slightly higher which is the reason for slightly different values.

Once again it shows a significant improvement over the experiment shown in Figure 19 as the

evaporative effects are reduced. This level of correlation is similar to that of the soil test kit but

requires significantly less material and is exceptionally cheaper. Furthermore, these results can be

improved greatly by tuning different parameters such as cleaner DPAN product as mentioned

above but also many other adjustments which will be discussed in depth later.

Although the experiments with no salt added are presented first in this report, the experiments with

the additional of salt was performed first. This is relevant as the evaporation effects were still not

observed. Therefore, the results are expected to be significantly improved with Teflon tape as was

the case with the no salt experiments.

y = 0.0095x - 0.0817R² = 0.967

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 10 20 30 40 50 60 70 80 90 100

Ch

ange

in A

bso

rban

ce

Concentration of Nitrite (μM)

No salt data batch 2

38

As mentioned earlier, the addition of salt allows for an on/off capability. This simply means the

point at which the sample completely loses colour, or in other words, when all the gold precipitates

out. This is seen by the discontinuous data points seen in Figure 22 c) d) and e). This allows for

the experiments with salt to act both quantitatively and qualitatively. The point at which all the

gold precipitates out depends on the concentration of salt and is able to be tuned. Figure 22 is a

collection of all the data obtained for the different experiments involving addition of salt.

39

Figure 22 - Calibration curve for nitrite detection with the addition of different volumes of 2M NaCl;

a) 20 μL, b) 30 μL, c) 60 μL, d) 80 μL, e) 160 μL

y = 0.024x - 0.0148R² = 0.972

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30

Ch

ange

in A

bso

rban

ce

Nitrite Concentration (µM)

20 µL Salt

y = 0.0308x - 0.0691R² = 0.9535

0

0.2

0.4

0.6

0.8

1

0 10 20 30

Ch

ange

in A

bso

rban

ce

Nitrite Concentration (µM)

30 µL Salt

y = 0.0249x - 0.02R² = 0.9568

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40Ch

ange

in A

bso

rban

ce

Nitrite Concentration (µM)

60 µL salt

y = 0.0209x - 0.0121R² = 0.9813

0

0.2

0.4

0.6

0.8

1

0 10 20 30Ch

ange

in A

bso

rban

ce

Nitrite Concentration (µM)

80 µL Salt

y = 0.0239x + 0.0195R² = 0.96960

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30Ch

ange

in A

bso

rban

ce

Nitrite Concentration (µM)

160 µL Salt

a) b)

c) d)

e)

40

Table 4 - Concentrations of NaCl for samples with salt added

Amount of 2M NaCl added

(μL)

Concentration of NaCl

(M)

Boiling Point Elevation

(°C)

20 0.029 0.028

30 0.043 0.042

60 0.086 0.084

80 0.114 0.111

160 0.228 0.222

Unlike the initial experiment with no salt, evaporation effects were not as noticeable in this

experiment. One may think that the reason for this is the phenomenon of boiling point elevation,

but this is not the case, and can be proved as follows. The boiling point elevation ∆TB is given by

∆𝑇𝐵 = 𝐾𝑏 ∙ 𝑏𝑠𝑜𝑙𝑢𝑡𝑒 ∙ 𝑖 Equation 1

where Kb is the Ebullioscopic constant, b is the molality of the solution and I is the van’t Hoff

factor. The van’t Hoff factor for NaCl is 1.9.

∆TB = 0.512 °C ∙ kg

mol∙ 0.228

mol

kg∙ 1.9

∆TB = 0.222 °C

These values are very small with the greatest increase in the boiling point being 0.2 °C. That being

said, there is clearly a noticeable effect in the relative lack of evaporative effects compared to the

no salt experiment. In fact, there is very little spread in any of the additional salt experiments,

irrespective of the fact that they were not wrapped with Teflon tape. This is not to say that there

are no evaporative losses, and the results may be improved by incorporating the Teflon tape for

future experiments with salt.

It is very clear that subplots a) and b) in Figure 22 (20 μL and 30 μL of 2M NaCl added) do not

have an “off” point in the range of 0 μM – 28 μM of nitrite. However, once the salt level is

increased to 60 μL, the off switch occurs somewhere between 20 μM and 24 μM of nitrite. In fact,

regardless of increasing the amount of salt, up until 160 μL does not show a change in the “off”

point. Further experiments with a wider concentration range of salts will allow for a more confident

41

statement of the effect of salt on the on/off concentration. But, there seems to be sufficient evidence

of this phenomenon which is clearly seen between 30 μL of salt and 60 μL of salt.

The most important effect the addition of salt has is the enhanced “resolution” it provides. The

term resolution here is referring to the increase in the absorbance reading per unit change in the

concentration, or equivalently, the magnitude of the slope of the absorbance and nitrite

concentration data. The magnitude of the slope for the case with no salt added is approximately

0.008, whereas the magnitude of the slopes for the cases with salt added range from 0.02 to 0.03.

Thus, the resolution when salt is added to the system is approximately 3 times higher than without

given the data collected. This is extremely valuable as it allows for more accurate measurements.

The resolution required for the detector is significantly lower when the change in the signal is

higher. This is a very important factor for determining the viability for sensors in general, but

especially field sensors where the detectors are far less capable due to both mobility restrictions

and cost restrictions.

Figure 23 - Calibration curve for nitrite detection with various amounts of 2M NaCl added

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 10 15 20 25 30 35

Ch

ange

in A

bso

rban

ce

Nitrite Concentration (uM)

Calibration Curve with Salt Effects

20 uL Salt

30 uL Salt

30 uL Salt 2

60 uL Salt

80 uL Salt

160 uL Salt

42

Figure 23 shows all the salt experiments on a single plot and provides for comparison in the

resolution effects and how they vary with different salt concentrations. As it turns out, it seems

that the slopes for these experiments are the same within a 95% confidence interval. Four of the

six experiments have near identical slopes and the other two experiments have a slope 0.05 higher

and lower respectively. While it is too early to make a definitive statement, it seems as though the

salt is simply increasing the resolution of the readings but does not scale with the salt

concentration. With the knowledge that there are errors in these measurements due to the

evaporation effects (although not nearly as prominent as the no salt experiment), this may be the

reason for the two curves to not share a similar slope with the others as there does not seem to be

any logical dependence on the concentration of the salt. The 20 μL, 30 μL, 60 μL and 80 μL

concentrations of salt share nearly the same magnitude for the slope with another 30 μL experiment

has a higher magnitude for the slope and 160 μL having a lower magnitude for the slope. There

are no apparent trends that can be attributed to this behaviour and thus, it is possible that these two

slopes being different may be due to error.

Another important capability for samples with salt added is the ability to perform these assays for

shorter periods of time or at lower temperatures. The “on/off” point is at around 24 μM of nitrite

at the one-hour mark at 95°C, thus, if the reaction is only run for 15 minutes, a larger range of

nitrite concentration measurements is feasible. Therefore, the use of salt also allows for the

extension of the ranges without sacrificing the resolution effect that is absent in the case of no salt.

A potential issue with these results is the validity of the absorbance measurements. Given that

these experiments are measured using a plate reader, the ability for the same resolution of the

absorbance measurements in the field may not be possible. The measurement of these assays must

be possible in a cost-efficient manner otherwise such an assay will fail to meet the requirements

for this technology. Photomultiplier tubes, or PMTs, are one of the most cost-efficient detectors

for detecting light. Plate readers, including the plate reader used for these assays, also use PMTs

as the detector. Thus, given that these results and measurements of absorbance were done using

PMTs which are among the lowest resolution detectors, there is no doubt that this quantitative

nitrite assay is not only possible but also has all the right qualities for a cost-efficient alternative

that has been sought after. Furthermore, the intention is to use smartphones for the detection but

this will require calibration.

43

It should be noted that the oven used for the heating is not ideal. This is because there is a

temperature drop when the samples are inserted and thus the oven is not initially at 95°C.

Furthermore, the way the oven achieves the temperature programmed is to adjust by overshooting,

then undershooting until it stabilizes at the desired temperature. Depending on the amount of heat

lost during the insertion of the sample, the temperature profile over the course of the reaction is

different. This is undoubtedly causing some variance between the different sets of data. A better

method of heating will lead to better, more reproducible data. A different heating method is

required and is something to be investigated.

It is worthwhile to calculate the relative abundance of the different species in order to understand

the general mechanism for the reaction. By understanding the general mechanism of the reaction

in this particular system, steps can be made to optimize this assay in terms of the reaction time,

which is currently an issue.

Np =πR2

a,

where Np is the number of functional groups (probes) per gold nanoparticle, a is the area occupied

by a single probe and R is the radius of the gold nanoparticle. At this stage, we do not know the

value of a, and will assume a small value of 10 Å2 for subsequent calculations. We will examine

the sensitivity of our results to this value later.

Np =π(6.5 ∙ 10−9 m)2

10 ∙ 10−20 m2

Np ≅ 1327probes

particle

Given the concentration of both the DPAA functionalized and DPAN functionalized AuNPs were

2.5 nm, this corresponds to 4.9 x 10-7 g

L.

mass per particle = ρgold ∙4

3πR3

mass per particle = 19.32 ∙ 106g

m3∙

4

3π(6.5 ∙ 10−9 m)3

mass per particle = 2.22 ∙ 10−17g

particle

44

particles per liter =4.9 ∙ 10−7

gL

2.22 ∙ 10−17 gparticle

particles per liter = 2.21 ∙ 1010 particles

L

probes per liter = 2.21 ∙ 1010 particles

L∙ 1327

probes

particle

probes per liter = 2.92 ∙ 1013 probes

L

Cp =2.92 ∙ 1013

probesL

6.022 ∙ 1023 mol−1

Cp = 4.85 ∙ 10−11MAs

As a comparison, the final concentrations of nitrite tested are on the order of 1-100 μM, 5 orders

of magnitude higher than that of either probes. Note that this was assuming the upper bound

concentration of the probes where they occupy only 10 Å2 of surface area. If we took the surface

area of the nanoparticle occupied by each probe to 1 Å2 , which is an order of magnitude smaller,

then nitrite concentrations would still be 4 orders of magnitude larger than the probe concentration.

Simply put, CHNO2 >> Cp.

With the relative amounts of the species in the system being known, the mechanism for the

reactions and other factors causing aggregation can be studied. Note that nitrite will take the form

of nitrous acid in aqueous solution due to phosphoric acid in solution.

The various steps involved in the reaction induced aggregation process are

1. Nitrous acid diffuses from the bulk to the aniline AuNP surface across the concentration

boundary layer.

2. The aniline (DPAA) probe reacts with nitrous acid to form the nitrosated intermediate

3. The nitrosated intermediate undergoes a rearrangement to form the diazonium ion.

4. The aniline AuNP diffuses in the aqueous phase to collide with a naphthalene AuNP.

5. A reaction occurs between the aniline AuNP and the naphthylamine (DPAN) AuNP.

45

Step 1 is controlled by the mass transfer rate. But even if we consider the mass transfer to be

governed purely by diffusion, which provides the lowest rate of mass transfer, this diffusive

process is extremely fast. For a 15 nm gold particle, the diffusion time 2 /R D , where R is the

radius of the nanoparticle, and D is the diffusivity of nitrous acid in solution. With R = 7.5 nm,

and D of the order of 10-9 m2/s, the diffusion time is on the order of 100 nanoseconds, which is

much shorter than the time scales over which the changes in absorbance are observed in the

experiment (60 min). The nitrous acid molecules are thus available at the bulk concentration at

the subsurface of the aniline AuNPs.

Step 2 is controlled by the rate of the nitrosation reaction, and presumably follows the rate law,

2HNO .s

s

dCkC C

dt Equation 2

Here, k is a 2nd order rate constant, sC is the surface concentration of the free (non-nitrosated)

aniline groups, 2HNOC is the subsurface concentration of nitrous acid, and k is the reaction rate

constant.

From above, it is shown that the species that is limited in this reaction is the aniline probe; the

change in the nitrite concentration relative to its initial concentration is weak. This allows us to

integrate the above equation and derive the following expression for sC :

0 2HNOexp .s sC C kC t Equation 3

The characteristic time scale of this reaction is, therefore, 2

1

HNOkC

.

Step 4 involves the Brownian diffusion of the Aniline AuNPs to locate naphthalene AuNPs. The

time for this step can be estimated as follows. The average separation d between the nanoparticles

in a dilute suspension is given by

1/3

1/31/3

3

3,

842

3

R Rd

nR n

Equation 4

46

where is the volume fraction of the nanoparticles, and n is the number of nanoparticles per unit

volume. The time required by the particle to traverse this separation is again set by a diffusion

time, but governed by the particle’s Brownian diffusivity, DB, which is given by the Stokes-

Einstein-Sutherland equation:

.6

B

kTD

R Equation 5

The time for one nanoparticle to locate another is, therefore,

2/32

2/3

6 3.

8B

Rd

D kT n

Equation 6

This time scale turns out to be a few seconds, which, again, is much smaller than the experimental

time scales for absorbance changes. Step 4 cannot be the rate determining step.

Step 5 involves the collision between the two AuNP particles followed by aggregation. According

to Smoluchowski’s theory, if the nanoparticles are initially monodisperse and the aniline AuNPs

are in their diazonium form, then the initial rate of aggregation is governed by the differential

equation

24,

3

dn kTn

dt

Equation 7

where is the collision efficiency. The characteristic time scale of the collision and aggregation

process is, thus, 0kTn

, where n0 is the initial number of AuNPs per unit volume. If this were the

rate determining step, then collision efficiency required to produce a time scale of 60 min is about

60%, which is certainly possible. This step cannot be discounted as a rate determining step at this

stage.

From the discussion above, there are potentially three slow steps in the entire aggregation process:

step 2, step 3 or step 5. To determine which of these steps is the rate limiting step, we consider

the case where any step other than step 2 is the slowest step and controls the absorbance changes,

i.e. we assume that step 2 is a fast step. In this case, all the aniline probes on the nanoparticle

would then be quickly and completely nitrosated due to the stoichiometric excess of nitrites in the

47

solution. This would mean that the subsequent steps, 3, 4 and 5, would be independent of any

changes in the bulk nitrite concentration, as any such change would not change the level of

nitrosation of the aniline nanoparticles. However, this is not observed experimentally; increasing

the nitrite levels affects the absorbance change (see Figures 19-23). Therefore, step 2 has to be a

slow step in the nanoparticle aggregation process, and the other two steps have to be either

comparable in time or faster than step 2.

Since step 2 is the rate limiting reaction between the aniline probes and nitrous acid, higher surface

coverages of the aniline probe will lead to an increase in the rate of reaction. Currently, we use a

ratio of 15:1 between the stabilizer (MTA) and the probes. An improvement in the surface probe

concentration is expected to strongly enhance the available number of nitrosated and therefore

diazonium groups per unit area on the nanoparticle surface, and hence the reaction rate by

improving the collision efficiency . Another implication is that the experimental time to achieve

a given absorbance change will be lower at higher nitrite concentrations. According to Equation

3, this dependence is exponential, which suggests that at sufficiently high nitrite concentrations,

Step 2 will no longer be the rate determining step, and then, the absorbance changes will become

independent of the nitrite concentration for a fixed experimental time.

Other possible mechanisms for aggregation include but are not limited to

A. Aniline AuNPs collide and aggregate with each other

B. Naphthylamine AuNPs collide and aggregate with each other

In the absence of salt, electrostatic repulsion and steric hindrance due to the presence of the

stabilizers (cationic head group) greatly hinders the efficiency of the collision of type A and type

B, and is much lower than the reaction cross collision. Increasing the salt levels reduces the

electrostatic repulsion (shorter Debye lengths) and thus increases the efficiency of a collision to

lead to aggregation.

48

5. Conclusions and Future Works

The results obtained from this work clearly show the potential for F-AuNPs to be used for

quantitative assays with high degrees of both accuracy and precision while limiting the materials

and concentrations of chemical species, allowing for such assays to be done at extremely low cost.

The ability to perform nitrite assays in a cost effective and quantitative manner is novel, and opens

the way for many more possibilities such as extending this work to the other macronutrients, which

currently lack a specific reaction for their detections, unlike nitrite.

The cost this assay must be discussed in more detail. A very basic cost analysis for this sort of

system on a lab scale is shown below:

Cost of gold nanoparticles for a single assay: 0.016 CAD

Cost of probes for a single assay: 0.024 CAD

Cost of all additional species required for the assay: 0.005 CAD

Total cost: 0.045 CAD per assay, or 4.5 cents per assay

It should be noted these numbers involve the material costs only. They exclude packaging costs

and labour costs. Even so, this simple cost analysis shows the power of nanotechnology and cost

efficiency. Therefore, this assay not only demonstrates its capabilities to provide accurate and

precise results, but can be done at a fraction of the material cost compared to the alternatives

mentioned earlier in the report. It is also worthwhile to mention that the reagent costs from Sigma-

Aldrich for this Griess reagent leads to a cost of 12 CAD per 10 assays. Thus, this gold nanoparticle

assay for nitrite is a colossal improvement in cost-efficiency from all existing methods.

Furthermore, as discussed earlier, the measurements were done using low resolution detectors

which are the most cost-efficient detectors available on the market. Ideally, once smartphone

diagnostic software is developed, they will be implemented for this system to also reduce the

detection cost to levels that are globally feasible. It should be mentioned that all the works

completed directly apply to the measurement of nitrates. The nitrates simply need to be reduced to

nitrite before being measured and back calculated to determine the concentrations. The efficiency

of reduction is very high as a nitrate specific reducing agent (called nitrate reductase) can be used

49

to convert the nitrates into nitrite with extremely high efficiency. This reaction completes within

10-15 minutes at room temperature, and thus is an efficient means to reduce the nitrates into

nitrites.

The next steps for this project involve the optimization of the nitrite detection. Firstly, experiments

with evaporative effects not considered will be done again in order to obtained proper calibration

data for those experiments. Next, common interferences will be tested to ensure that this system is

indeed specific to nitrite. The following step is to determine ways to speed up the reaction.

Currently, this is the main drawback, as the reaction is being conducted at 95°C, which corresponds

to approximately 128 times faster reaction rate compared to room temperature. In order for this

assay to be viable for the use in developing nations, heating requirements must be minimized.

There are several ways to go about this issue, all of which focus on increasing the rate of reaction.

If the rate of reaction is sufficiently increased, then the reaction will no longer need to be heated

at such a high temperature.

Another option to be explored is the use of PEG as the base of the probe instead of lipoic acid.

This is for two reasons; the first is that PEG has an extremely high packing factor, or in other

words, it stands “upright” effectively increasing the total number of binding sites available to

probes by reducing the area spacer is occupying. On that note, it should also be noted that the

current DPAN and DPAA probes bind to the gold nanoparticles using two binding sites, instead

of the one binding site that is the case with PEG. This will also effectively increase the number of

active sites on the surface. This was shown to be very important in the previous section with the

rate limiting step being the nitrosation of the DPAA probes. Thus, a higher concentration of DPAA

probes leads to a faster reaction. The second reason follows much of the first reasons motivation:

in order to increase the stability of the particles in water. Currently a 15:1 stabilizer to probe ratio

is used, and in increase in the solubility of the probe will allow for the use of a smaller ratio,

thereby increasing the concentration of active probes on the surface. As in the first reason, this will

help improve the rate of the reaction significantly as it targets the rate limiting reaction. The length

of the PEG spacer is a variable that can be tuned to control the rate of reaction significantly.

Overall, the use of PEG as the spacer molecule in place of the lipoic acid has three distinct means

to increase the rate of reaction, with two of the three means targeting the rate limiting step.

50

During this step, the organic probes will be attached to the HS-PEG-COOH via condensation

reaction with the amine on the probes with a coupling agent (such as DCC). It should be noted that

the functionalization and loading onto the nanoparticles also includes “backfilling” with shorter

PEG molecules [19]. This is the process by which “holes” on the surface of the gold must be filled

with smaller spacers in order to ensure the active components (the analyte, nitrate in this case) do

not occupy those spaces, making it inaccessible for reaction. Once this process has been fine tuned

for nitrite, a reaction scheme for other macronutrients such as potassium and phosphate will be

explored. While there is currently a lack of specific reactions for phosphate and potassium,

complexing agents such as crown ethers may be tuned to be specific to potassium [20], and

similarly chelating agents may be engineered to be specific to phosphates.

Lastly, testing of this assay will be done with soil samples in order to determine the types of noise

and effects it has on the assay. With the possibility of high salinity of soil, it may have a very

significant effect in the aggregation and thus needs to be accounted for. But due to the high

specificity of the Griess reaction, inferences due to other chemical species should not pose a

problem. Furthermore, the effects of salt that exists in the soil will have negligible effects as they

are significantly less than the amount that is added into the samples in the assay. Thus, even though

the levels of salt are variable depending on the soil sample, the effects of their salt levels will not

be affecting the assay in any way as the salt effects are dominated by what is artificially added.

An important finding from the works investing the kinetics of the Griess reaction [18] is the effect

of the functional groups on the aniline and naphtylamine. For the measurements, the base molecule

of aniline and naphthylamine species were used. However, the kinetics of the reaction are much

higher with the use of these molecules with certain functionalization. The rate limiting step for the

bulk process is actually different than the rate limiting step that is found in this nanoparticle system.

The internal rearrangement after nitrosation into the diazonium ion was the rate limiting factor in

the bulk experiments and if the aniline compounds had an electron withdrawing group at the ortho

or para position with respect to the amine, the rate of this rearrangement was greatly accelerated.

While the rearrangement of the nitrosated species into the diazonium ion was not the rate limiting

step in the system with the gold nanoparticles, there is no evidence that this step was fast. In fact,

as it was mentioned previously, the rearrangement process can be on the same time scale of the

nitrosation or faster. Given the data presented in the kinetic works, there is no reason to assume

51

this step would be a fast step, and it is safe to assume that this is a slow step also. Therefore, by

using different probes, such as a p-nitroaniline based probe, the overall rate of reaction should be

significantly increased. It should be noted that the species would need to be synthesized and not

commercially available as it requires both the nitro- group in the para position, but also the

ethylamine group such that it can be linked onto a spacer molecule. Due to the time restrictions of

the project, this was not possible but is a very significant adjustment that can be made to speed up

the reaction. It should be noted that the functional groups of the naphthylamine species also played

a role, however it was not nearly as significant as the effects of the functional groups on the aniline

species.

During the process of developing the chemistry for the measurement of these macronutrients, the

platform for this will be developed using microfluidics. There are several possibilities in the

development of the platform, ranging from paper microfluidic devices to more complex polymer

based ones. For the purpose of low-cost soil diagnostic devices, paper microfluidics are ideal, but

also suffer from relatively limited capabilities as a result. However, given that the cost of the device

is the most important factor, paper based microfluidics is ideal for this application. Flow of the

samples inserted will be transported via capillary action and mixing can happen via wicking in the

paper device. Since transport of sample to the right place on the sensor and mixing of the sample

with the gold are the only two requirements for this assay, paper based microfluidic devices will

be evaluated in our initial trials.

52

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55

Appendix A – Procedures

Gold Nanoparticle Purification

1. DLS measurement to determine purity (PDI < 0.1 is the target for purity)

2. Centrifuge at 600 x g to remove particles over 100 nm in size

3. DLS measurement to determine purity (PDI < 0.1)

4. UV-vis measurement to verify purity and measure concentration (absorbance profile

measured from 400 nm to 600 nm with peak absorbance occurring at approximately 524

nm)

5. TEM to verify purity with regards to consistency in morphology and PDI

Organic Synthesis

1. Add 1.6g of (±)-1,2-dithiolane-3-pentanoic acid and 1.3 g of 4-(2-aminoethyl)benzenamine

in 100 mL of anhydrous CH2Cl2 (can use DCM with molecular sieves: add up to 25 mL of

the volume but do not surpass this as lower yields may result) in the presence of 3

equivalents of dicyclohexylcarbodiimide (DCC) to synthesize DPAA. Add 1.6 g of (±)-

1,2-dithiolane-3-pentanoic acid and 2.0 g 2-(1-naphthylamino)ethylamine dihydrochloride

in 100 mL of anhydrous CH2Cl2 (can use DCM with molecular sieves and follow previous

instruction on amount) in the presence of 3 equivalents of DCC to synthesize DPAN. Note

add DCC last to the mixture.

2. Once all reactants have been added to the reaction vessels, stopper the round bottom flasks

(are preferable for optimal mixing)/reaction vessels and pierce stoppers with syringe needle

(leave needle in) to relieve pressure. Allow mixing at sufficient speed at room temperature

for 24 hours (can use stir plate but make sure to stabilize reaction vessels).

Organic Purification of DPAA/DPAN

1. Suction filtration to extract the liquid phase which contains the product

2. Overnight cooling in the freezer

3. Suction filtration to remove particulates that precipitated out overnight

4. Liquid-Liquid extraction to remove water soluble impurities using equal parts product

in DCM and water

56

5. Acid-base treatment followed by liquid-liquid extraction for DPAN (was not required

for DPAA)

6. Removal of solvent using rotary evaporator with low-medium rotation speed

7. Thin-layer chromatography (TLC) in order to determine the mobile phase required for

column chromatography

8. Run a column with the chosen system determined by the TLC (must have distinct and

significant separation between different compounds)

Functionalization of AuNPs

1. Prepare a fresh solution of 0.45 mM MTA in water and adjust the pH to 9

2. In a separate vial/container, mix 6 μL of the 5 mM DPAA solution per 1 mL of the MTA

solution (15:1 MTA to probe ratio).

3. Add 100 μL of this MTA/DPAA mix per 1 mL of gold nanoparticles. Typically, 30 mL

of AuNPs, thus 3 mL of MTA/DPAA mix was added into the centrifuge tube containing

the 30 mL of AuNPs.

4. Repeat for DPAN with MTA.

5. Vortex and let self-assembly occur for 30-45 minutes.

6. Prepare 1M HCl and add 100 μL per 1 mL of AuNP solution into the centrifuge tubes

containing the functionalized particles.

7. Vortex and wash the particles by centrifuging at 16000g for 1.5 hours five times. Each

time after centrifuging, decant the supernatant and fill back up to approximately 30 mL.

8. Characterize by using UV-vis, DLS and TEM.

Nitrite Measurements

1. Prepare stock solution of 500 μM sodium nitrite solution in a volumetric flask. Stored by

wrapping in aluminum foil to prevent light based degradation.

2. Prepare 20 mL of 2M NaCl and 20 mL of 1.8M phosphoric acid. These were prepared

and used over the duration of the 3 weeks. They were stored in a dry and dark place

wrapped in aluminum foil to prevent any form of degradation.

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3. Prepare excel spreadsheet with all the volume amounts required for different nitrite

concentrations

4. Label and set up the microcentrifuge tubes

5. Ensure to always run a blank or control. This is simply the same composition as the

samples without any nitrite. Each different experiment requires a control sample

6. Add the required amount of MilliQ water into each tube. The amount of water varies for

different concentrations of nitrite

7. Add the fixed amount of phosphoric acid into each tube

8. Add required (and fixed) amount of the DPAA and DPAN functionalized gold

nanoparticles such that the final concentration of each F-AuNP is 2.5 nM in 1.4 mL of

sample

9. Add required amount of nitrite using the 500 μM stock solution

10. For experiments with additional salt, add the fixed amount of the 2M NaCl to each tube.

11. Close the cap and vortex each tube. For experiments with salt, place in ice bath after

vortex mixing

12. Transfer 3 aliquots of 200 μL of each sample onto the 96-well plate

13. Take a measurement using the plate reader at a wavelength of 524 nm

14. Close the caps tightly and wrap the caps with Teflon tape

15. Insert into the oven at 95°C for 1 hour

16. Take samples out and transfer the tubes into ice water to effectively stop the reaction

17. Transfer 3 aliquots of 200 μL of each sample onto the 96-well plate

18. Take a measurement using the plate reader at a wavelength of 524 nm

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Appendix B – NMR Spectra

Figure 24 - H-NMR for DPAN after column purification

59

Figure 25 - C-NMR for DPAN after column purification

60

Figure 26 - H-NMR for DPAA after column purification

61

Figure 27 - C-NMR for DPAA after column purification

62

Appendix C – Reaction Steps

Figure 28 - The general steps to the Griess Reaction