Understanding the Effects of Plasma Assisted Nanoparticle ...

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FIU Electronic Theses and Dissertations University Graduate School

6-12-2020

Understanding the Effects of Plasma Assisted Nanoparticle Understanding the Effects of Plasma Assisted Nanoparticle

Deposition for the Enhancement of Optical and Electrochemical Deposition for the Enhancement of Optical and Electrochemical

Response Response

Apurva Sonawane [email protected]

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FLORIDA INTERNATIONAL UNIVERSITY

Miami, Florida

UNDERSTANDING THE EFFECTS OF PLASMA ASSISTED NANOPARTICLE

DEPOSITION FOR THE ENHANCEMENT OF OPTICAL AND

ELECTROCHEMICAL RESPONSE

A dissertation submitted in partial fulfillment of

the requirements for the degree of

DOCTOR OF PHILOSOPHY

in

ELECTRICAL AND COMPUTER ENGINEERING

by

Apurva Sonawane

2020

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To: Dean John L. Volakis

College of Engineering and Computing

This dissertation, written by Apurva Sonawane, and entitled Understanding the Effects of

Plasma Assisted Nanoparticle Deposition for The Enhancement of Optical and

Electrochemical Response, having been approved in respect to style and intellectual

content, is referred to you for judgment.

We have read this dissertation and recommend that it be approved.

_______________________________________

Jean Andrian

_______________________________________

Nezih Pala

_______________________________________

Bruce McCord

_______________________________________

Mubarak Mujawar

_______________________________________

Shekhar Bhansali, Major Professor

Date of Defense: June 12, 2020

The dissertation of Apurva Sonawane is approved.

_______________________________________

Dean John L. Volakis

College of Engineering and Computing

_______________________________________

Andrés G. Gil

Vice President for Research and Economic Development

and Dean of the University Graduate School

Florida International University, 2020

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© Copyright 2020 by Apurva Sonawane

All rights reserved.

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DEDICATION

To H. H. Shri Mataji Nirmala Devi

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DEDICATION

To my husband, Mr. Shailendra Chivate

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ACKNOWLEDGMENTS

I am grateful to my major advisor, Dr. Shekhar Bhansali, for the opportunity to

perform this study towards my doctoral dissertation. I want to express gratitude from the

bottom of my heart for his continuous efforts in guiding me throughout this journey, which

helped me develop my skills as a researcher.

I am thankful to my committee members, Dr. Jean Andrian, Dr. Nezih Pala, Dr.

Bruce McCord, and Dr. Mubarak Mujawar, for their valuable input. Their comments

helped me understand the topic profoundly. I would like to thank Dr. Pala for his advice

on biosensing measurements. I am thankful to Dr. Bruce McCord for his insightful

comments on the validation of the hypothesis with cortisol detection. I thank Dr. Andrian

for his guidance in signal handling, processing, and analysis. I want to thank the Advanced

Materials and Engineering Research Institute (AMERI) at FIU, as Sensor fabrication and

Imaging could not have been carried out without its facilities and assistance. I am thankful

to the managers of AMERI labs for their services. My special thanks go to Dr. Mubarak

Mujawar for introducing me to the plasma field and his valuable comments on my work.

I am thankful to the Electrical and Computer Engineering department. I also thank

Ms. Pat Brammer, Ms. Layla El-Hilu, and Ms. Luisa Ruiz for their administrative support.

I am thankful to Mr. Gordon Osborne for encouraging me to pursue a Ph.D. program and

for mentoring me during my early days in the U.S. I would like to thank Dr. Pandiaraj and

Dr. Yogeswaran for helping me understand and clearing my doubts about electrochemical

measurements. I am thankful to Dr. Khalid Pasha and Krystine for training me on

microfabrication tools. I would like to thank my former and present fellow graduate

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students, Michelle, Pulak, and Lamar, for accompanying me and for their moral support

during this journey.

Special thanks to Mr. Anjan Upadhya and Mrs. Louisa Upadhya and Sahaja family

for believing in me and being supportive in my difficult times. I immensely thank my in-

laws, Mr. Suresh Chivate and Mrs. Supriya Chivate, for their love and motivation

throughout my research journey. I am grateful to my parents, Mr. Fakirrao Sonawane and

Mrs. Anita Sonawane, for the support they have provided me throughout my life and for

being the reason behind all my endeavors. Many thanks go to my husband, Mr. Shailendra,

who stood by me through thick and thin, and for all the efforts he has made to keep me

focused.

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ABSTRACT OF THE DISSERTATION

UNDERSTANDING THE EFFECTS OF PLASMA ASSISTED NANOPARTICLE

DEPOSITION FOR THE ENHANCEMENT OF OPTICAL AND

ELECTROCHEMICAL RESPONSE

by

Apurva Sonawane

Florida International University, 2020

Miami, Florida

Professor Shekhar Bhansali, Major Professor

In this work, the effects of atmospheric plasma treatment on morphology, optical,

and electrochemical properties of 10 ± 3nm spherical silver and gold nanoparticles (AgNPs

and AuNPs) functionalized substrates were studied. The nanoparticles (NPs) were

deposited on substrates by drop-casting, aerosol spray, and a low-temperature atmospheric

plasma-assisted aerosol jet. The reduction in nanoparticle size was observed, which was

explained by the redox reaction that occurs on the nanoparticle surface. This phenomenon

was evident by the presence of AgO, Ag2O, and AuOx Raman peaks in the treated sample.

The surface charge changed as a result of plasma treatment, as indicated by a significant

change in the zeta potential from +25.1 ± 4 mV for the untreated AgNPs to −25.9 ± 6 mV

after 15 minutes of plasma treatment and from -20.9 ± 4 mV to -43.9± 5 mV for AuNPs.

Surface-enhanced Raman spectroscopy of the plasma-treated films was carried out with

the fluorescent dye Rhodamine 6G, which showed a ~120-fold enhancement for AgNPs

and ~95 fold for AuNPs in the signal intensity relative to the untreated substrates. This

surface charge tuning during deposition led to the effective surface coverage with

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comparatively uniform NPs films as observed in Scanning Electron Microscopy, and

Transmission Electron Microscopy images. This technique can be applied to a wide range

of nanoparticle systems used in biosensing applications as substrates prepared by this

method can serve as effective SERS substrates due to the cumulative effect of surface

roughness, and size reduction.

The electrochemical performance of plasma-assisted NPs-modified

microelectrodes was studied. These microelectrodes were fabricated using standard

photolithography, Chrome/Gold evaporation, and lift-off techniques. These electrodes

showed more enhancement in the electroactive surface area and improved inter-electrode

variability than in other methods. Electrochemical Impedance spectroscopy results showed

the improvement in the conductivity of plasma-assisted NPs functionalized electrodes.

Cortisol was detected using self-assembled monolayer, and antibodies functionalized on

plasma-assisted NP-modified electrodes, which showed increased sensitivity.

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TABLE OF CONTENTS

CHAPTER ............................................................................................................ PAGE

1 INTRODUCTION ....................................................................................................1

1.1 Motivation ........................................................................................................1

1.2 Objectives .........................................................................................................5

1.3 Dissertation organization ...................................................................................5

2 BACKGROUND AND LITERATURE REVIEW ....................................................8

2.1 Biosensor ..........................................................................................................8

2.2 Properties of NPs and their applications ............................................................9

2.3 Plasmonic Nanoparticles ................................................................................. 10

2.4 Methods of Nanoparticle Deposition ............................................................... 12

2.5 Plasma ............................................................................................................ 15

2.5.1 Plasma parameters .................................................................................... 18

2.5.2 Cold Atmospheric Plasma ........................................................................ 20

2.5.3 Plasma-Induced Surface Modification ...................................................... 21

2.6 Nanoparticle-based Electrochemical Immunosensing ...................................... 21

3 EXPERIMENTAL PROCEDURE.......................................................................... 24

3.1 Aerosolized deposition and argon plasma assembly......................................... 24

3.1.1 Argon plasma ........................................................................................... 27

3.1.2 Plasma characterization ............................................................................ 28

3.1.3 Plasma treatment and thermal annealing of NPs........................................ 31

3.2 Surface Plasmon Resonance ............................................................................ 31

3.3 Surface charge measurements.......................................................................... 33

3.4 Surface-enhanced Raman spectroscopy ........................................................... 33

3.5 Imaging ........................................................................................................... 36

3.6 Microelectrodes Fabrication ............................................................................ 37

3.7 Electrochemical Immunosensing of cortisol .................................................... 42

3.8 Cyclic Voltammetry ........................................................................................ 43

3.9 Electrochemical Impedance Spectroscopy ....................................................... 44

4 EFFECTS OF COLD ATMOSPHERIC PLASMA TREATMENT ON

MORPHOLOGICAL, AND OPTICAL PROPERTIES OF PLASMONIC

NANOPARTICLES ............................................................................................... 47

4.1 Surface Plasmon Resonance (SPR).................................................................. 47

4.2 SEM Imaging .................................................................................................. 51

4.3 Surface charge measurements.......................................................................... 54

4.4 TEM and AFM imaging .................................................................................. 55

4.5 Mechanism of plasma-induced reduction in AgNP size ................................... 57

4.6 SERS characterization of the plasma-treated and thermally treated AgNP film 63

4.7 Stability of Plasma treated AgNPs film ........................................................... 66

4.8 Mechanism of Plasma Induced reduction in AuNPs size ................................. 67

4.9 SERS characterization of the plasma-treated and thermally treated AuNP film 69

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4.10 Summary of chapter 4 ..................................................................................... 70

5 PLASMA INDUCED ENHANCEMENT IN ELECTROCHEMICAL

PROPERTIES OF PLASMONIC NANOPARTICLES .......................................... 72 5.1 Fabrication of AgNPs and AuNPs modified electrodes .................................... 72

5.2 Surface functionalization of AgNPs and AuNPs based sensors for cortisol

measurement ................................................................................................... 74

5.3 Electrochemical Measurements ....................................................................... 75

5.4 Electrochemical Performance of AgNPs modified electrode ............................ 75

5.5 Electrochemical Performance of AuNPs modified electrode ............................ 78

5.6 Electrochemical detection of cortisol using modified SPCEs ........................... 80

5.7 Summary of chapter 5 ..................................................................................... 86

6 UNDERSTANDING THE RELATION BETWEEN NANOPARTICLE

DISTRIBUTION AND ELECTRODE RESPONSE ............................................... 87 6.1 Methods .......................................................................................................... 88

6.2 Electroactive surface area from the electrochemical response .......................... 89

6.3 Image quantification of AuNPs modified electroactive surface area ................ 90

6.4 TEM images ................................................................................................... 91

6.5 Nanoparticle deposition model ........................................................................ 92

6.6 Electrochemical characterization of AuNPs modified Au Electrode................. 94

6.6.1 CV characterization .................................................................................. 94

6.6.2 EIS characterization ................................................................................. 96

6.7 Inter-Electrode Variability of methods of deposition ....................................... 98

6.8 The relation between the Size of NPs and ESA ............................................. 101

6.9 Cortisol detection using AuNPs modified Au electrodes ................................ 103

6.10 Summary of chapter 6 ................................................................................... 106

7 SUMMARY AND FUTURE WORK ................................................................... 107

7.1 Summary....................................................................................................... 107

7.2 Future Work .................................................................................................. 109

REFERENCES ............................................................................................................ 111

APPENDICES ............................................................................................................. 122

VITA ........................................................................................................................... 128

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LIST OF FIGURES

FIGURE PAGE

Figure 1.1: Illustration of wearable sensors with remote health monitoring system

regenerated with permission[3] ................................................................2

Figure 2.1: Schematic representation of the sensing mechanism of biosensors ............8

Figure 2.2: Properties of NPs and their applications .................................................. 10

Figure 2.3: Schematic representation of various NP deposition techniques ............... 13

Figure 2.4: a) Inkjet-printed, b) drop-casted, c) electrodeposited NPs on SPCE, d)

hydrophobic coating, e)evaporation-induced AuNP film formation, and

f) ultra-thin AuNPs film using filtration technique, adapted from [45],

[46], [49]–[51] ........................................................................................ 14

Figure 2.5: Nanoparticle-based electrochemical immunosensing includes [a), and

b)] Competitive, c) sandwich, and d) NP as an underlying layer and

Ligand-based immunosensing. Adapted From [19] ................................. 22

Figure 3.1: a) Schematic illustration and b) photograph of the experimental set-up

for the deposition of Ag NPs on the substrate. c) Schematic illustration

and d) photograph of the experimental set-up of the cold plasma unit.

e) Ag NPs deposited on a flexible polyimide film. .................................. 26

Figure 3.2: Argon energy level diagram .................................................................... 27

Figure 3.3: Optical emission spectra for Ar plasma. Inset: relative intensity of

the Ar I and Ar II transitions with respect to the relative higher-energy

level. ...................................................................................................... 28

Figure 3.4: Schematic representation of SERS measurement technique, adapted

from[74]. ................................................................................................ 34

Figure 3.5: Layout of the electrode design ................................................................ 38

Figure 3.6: The schematic of a photoresist spin coater set-up, as regenerated from

[77]. ....................................................................................................... 39

Figure 3.7: Fabrication steps for microelectrodes ...................................................... 40

Figure 3.8: Picture of evaporator a) and b) planetary fixture ..................................... 41

Figure 3.9: Schematic of Antibody/DTSP SAM/NPs binding and stepwise

fabrication of cortisol immunosensor ...................................................... 42

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Figure 3.10: Equivalent circuit and Nyquist plot of the biosensor ............................... 45

Figure 4.1: SPR of untreated, 5 mins, 10 mins, and 15 mins a) plasma-treated; b)

thermally treated at 250̊ C and c) thermally treated at 400 ̊C and d)

Blueshift of a), b) and c) shows the blueshift in SPR spectra after the

thermal and plasma treatment. ................................................................ 48

Figure 4.2: a) SPR results of plasma-treated Au NPs and b) Blueshift in SPR after

the plasma treatment ............................................................................... 49

Figure 4.3: SPR and redshift of plasma-treated drop-casted a), c) AgNPs and b), d)

Au NPs ................................................................................................... 50

Figure 4.4: SEM images of AgNP films deposited on silicon substrates a) before

treatment, b) after 15 mins thermal annealing, and c) after 15 mins

plasma treatment. The SEM images processed in ImageJ to identify

nanoparticle boundaries [d), e) and f)]; and Feret’s diameter

distributions [g), h) and i)] corresponding to untreated, thermally

treated and plasma-treated AgNPs. The left shift in the AgNPs size

distribution was attributed to the size reduction after the treatments.

The plasma-treated AgNPs exhibited a more uniform film than other

samples. ................................................................................................. 52

Figure 4.5: SEM images of AuNP films deposited on silicon substrates (a) before

treatment, (b) after 15 mins of plasma treatment. The SEM images

processed in ImageJ to identify nanoparticle boundaries [(c) and (d)];

and Feret’s diameter distributions of (e) untreated and

(f) plasma-treated AuNPs film. ............................................................... 53

Figure 4.6: Zeta potential distribution of (a) untreated and (b) plasma-treated (15

minutes) Ag NPs. The surface charge changes from +25.1 to −25.9 mV

after treatment. ....................................................................................... 54

Figure 4.7: Zeta potential measurements for a) untreated and b) plasma-treated

AuNPs. The surface charge changed from -20.9 mV to -43.9mV. ........... 55

Figure 4.8: TEM images and diffraction patterns (insets) of (a), (c) untreated and

(b), (d) plasma-treated Ag NPs. AFM images of (e) untreated and (f)

plasma-treated Ag NP film; the roughness and texture are shown in the

insets. ..................................................................................................... 57

Figure 4.9: Representation of positively charged AgNPs using BPEI ........................ 58

Figure 4.10: a) Proposed mechanism by which plasma treatment modifies Ag NP

films. The bottom panel b) is the SERS spectra of the untreated and

plasma-treated NPs at 532-nm excitation, which validates the

hypothesis that removal of the BPEI coating and subsequent surface

oxide formation results in the observed size reduction. ........................... 59

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Figure 4.11: Schematic representation of AgNPs surface reaction within the plasma

sheath. .................................................................................................... 61

Figure 4.12: SERS response of R6G on as-deposited and plasma-treated Ag NP

films on a silicon substrate. The R6G response is enhanced until 15

minutes of plasma treatment. It is saturated because of the complete

surface coverage with the oxide layer after 15 mins of the treatment.

The inset in (b) shows the enhancement factor of R6G as a function of

treatment time......................................................................................... 64

Figure 4.13: SERS response of R6G molecules on thermally treated AgNPs at 400֯C . 66

Figure 4.14: SERS response for R6G on 15 mins plasma-treated AgNPs recorded at

a different time interval at room temperature. The samples were stored

at room temperature (25̊C) ...................................................................... 67

Figure 4.15: SERS of a) untreated b) 5 mins plasma-treated c) 10 mins plasma

treated and d) 15 mins plasma-treated AuNPs ......................................... 68

Figure 4.16: SERS of R6G on untreated (Black solid and inset) and 15 mins of

plasma-treated (Blue solid) ..................................................................... 70

Figure 5.1: Schematic illustration of plasma-assisted aerosolized NPs modified

sensor fabrication process and SEM images of morphological changes

during the sensor fabrication. .................................................................. 73

Figure 5.2: Schematic of stepwise fabrication of cortisol sensor ............................... 74

Figure 5.3: Electrochemical response of 5mM [Fe (CN)6]3−/4− using a) CV and

b) EIS of untreated and plasma-treated AgNPs modified electrodes. ....... 76

Figure 5.4: Electrochemical response of 5mM [Fe (CN)6]3−/4− using a) CV and

b) EIS of untreated and plasma-treated AuNPs modified electrodes. ....... 78

Figure 5.5: Evaluation of the power factor of the constant phase element of bare

Au, untreated AuNP modified, and plasma-assisted AuNPs modified

electrodes ............................................................................................... 80

Figure 5.6: EIS responses of I) untreated and III) plasma-assisted a) AgNPs,

b) DTSP SAM/AgNPs, c) Anti-Cab/DTSP SAM/AgNPs,

d) 0.012 µg/dl Cortisol/ Anti-Cab/ DTSP SAM/ AgNPs modified

SPCE; and Linear calibration plot for II) Anti-Cab/DTSP

SAM/untreated AgNPs and II) Anti-Cab/DTSP SAM/plasma-assisted

AgNPs modified SPCE. .......................................................................... 81

Figure 5.7: Reaction scheme for the conversion of PEI primary amines with DSP.

The resulting disulfide bonds are easily cleaved by reducing agents,

regenerated from [112] ........................................................................... 83

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Figure 5.8: EIS responses of I) untreated and III) plasma-assisted a) AuNPs,

b) DTSP SAM/AuNPs, c) Anti-Cab/DTSP SAM/AuNPs,

d) 0.012 µg/dl Cortisol/ Anti-Cab/ DTSP SAM/ AuNPs modified

SPCE; and Linear calibration plot for II) Anti-Cab/DTSP

SAM/untreated AuNPs and II) Anti-Cab/DTSP SAM/plasma-assisted

AuNPs modified SPCE. .......................................................................... 84

Figure 6.1: a) SEM image of AuNPs modified electrode, b) NPs outline detection

using image processing, and c) distribution of the size of NPs. ............... 91

Figure 6.2: TEM images of a) drop-casted, b) aerosolized, and c) plasma-assisted

aerosolized deposited AuNPs of the average size of 20nm. ..................... 91

Figure 6.3: AuNPs deposition model for untreated AuNPs [a, b, c] and plasma

treated AuNPs [d, e, f] for various inter-particel separation distance (S).. 93

Figure 6.4: CV responses of 20nm, 40nm, and 60nm of AuNPs modified electrodes

with a) drop-casting, b) aerosol deposition, and c) plasma-assisted

aerosolized deposition. TEM images of d) drop-casted, e) aerosol

deposition, and f) plasma-assisted aerosolized AuNPs. ........................... 95

Figure 6.5: EIS responses of a) drop-casted, b) aerosolized, and c) plasma-assisted

aerosolized AuNPs modified Au electrodes in 5mM of [Fe (CN)6]3−/4−,

and d) the variation in electron transfer resistance due to the deposition

methods and variation in size of NPs. ..................................................... 97

Figure 6.6: Boxplots of the spread of the distribution of a) ESA and b) ΔE of bare

Au electrodes, drop-casted, aerosolized, and plasma-assisted

aerosolized deposited AuNPs functionalized electrodes. ......................... 98

Figure 6.7: Variation of separation potentials for drop-casted, aerosolized, and

plasma-assisted AuNPs with the size of AuNPs .................................... 100

Figure 6.8: Correlation plot of the size of NPs and ESA for a) drop-casted, b)

aerosolized, and c) plasma-assisted aerosolized NPs modified

electrodes. d) Relative plot of ESA to bare Au electrodes, 20A,40A

and 60A-20nm,40nm and 60nm aerosolized deposited; 20d, 40d, and

60d- 20nm, 40nm, and 60nm drop-casted; 20P, 40P, and 60P- 20nm,

40nm, and 60nm plasma-assisted aerosolized AuNPs modified

electrodes ............................................................................................. 102

Figure 6.9: a) CV shows a stepwise surface modification in 5mM of

[Fe (CN)6]3−/4− and linear calibration plots with the sensitivity of

Anti-Cab/DTSP SAM functionalized on b) bare Au electrode,

c) 20nm, d) 40nm, and e) 60nm of drop-casted; f) 20nm, g) 40nm,

and h) 60nm of aerosolized; and i) 20nm, j) 40nm, and k) 60nm of

plasma-assisted aerosolized AuNPs modified electrodes. ...................... 105

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Figure 7.1: Summarized effects of plasma-assisted NP deposition methods ............ 108

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ABBREVIATIONS AND ACRONYMS

Nanoparticles NPs

Surface Plasmon Resonance SPR

surface-enhanced Raman scattering SERS

Current-Voltage CV

Electrochemical Impedance Spectrometry EIS

Deoxyribonucleic acid DNA

Silver Ag

Silver Nanoparticles AgNPs

Gold Au

Gold Nanoparticles AuNPs

Ultra Violet UV

Cold atmospheric Plasma CAP

Electrostatic Double Layer EDL

Self-Assembled Monolayer SAM

Dithiobis Succinimidyl Propionate DTSP

Argon Ar

Mass Flow controller MFC

Standard Cubic Centimeter per Minute SCCM

Scanning Electron Microscopy SEM

Transmission electron microscopy TEM

Optical Emission Spectroscopy OES

Hydroxyl group OH

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Nitrogen N2

Carbon dioxide CO2

Oxygen O2

Rapid Thermal Processing RTP

Deionized DI

Enhancement Factor EF

Atomic Force microscopy AFM

Reference Electrode RE

Counter Electrode CE

Working Electrode WE

Photoresist PR

Reactive ion etching RIE

Screen Printed Carbon Electrode SPCE

Branched Polyethylenimine BPEI

Silver Oxide AgO

Electrochemical Impedance Spectroscopy EIS

Screen-Printed Carbon Electrodes SPCE

Phosphate Buffer Saline PBS

Ethanolamine EA

Cyclic Voltammetry CV

Electroactive Surface area ESA

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1 INTRODUCTION

1.1 Motivation

The emerging wearable technology has integrated electronics into daily activities. The

awareness about health is growing in a society that leads to the demand in the healthcare

industry. However, it comes with the underlying need for innovations to keep it relevant. The

biosensors market has been expanding during the last decade. It was valued over $20 billion

in 2018[1]. Due to the technological advancements in biosensors, this value is expected to

reach around 80 percent compound annual growth rate in the next few years. The use of

biosensors delivers an innovative, flexible, user-friendly, and end-to-end service to the

modern health care system. Enhancement in technology has led to the awareness about a

healthy lifestyle along with the smart infrastructure, which is accelerating the demand for

wearable biosensors for continuous health monitoring.

There are a few sensors available in the market, such as blood pressure, heartbeat, and

pulse rate sensors. In wearable devices, health-related information is gathered via body-worn

wireless sensors and transmitted to the caregiver via an information gateway, such as a

smartphone, personal computers, and cloud computing. The schematic illustration of

wearable sensors with a health monitoring system has been shown in figure 1.1. Caregivers

can use this information to implement interventions as needed through continuous monitoring.

Hence, the development of biosensors is influencing the point-of-care diagnostics and

positively affects the health care market in forthcoming years.

Though the wearable sensors are advancing in the technology, there is a gap between

wearable devices and biosensors because of challenges in implanting biosensors in wearable

accessories. The performance of biosensors is the reflection of the optimization of their

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attributes, such as selectivity, sensitivity, stability, and reproducibility. The enhancement

in the sensitivity has been achieved by increasing the active surface area using

nanoparticle-functionalized electrodes due to the existence of edges where electric field

intensity is higher[2]. Efforts have been made to improve the performance of the biosensors

with the use of nanoparticles. However, the effective utilization of the properties of

nanoparticles has still lacked information about their behavior under certain conditions, which

hinders the methods of controlling their orientation in specific applications.

Figure 1.1: Illustration of wearable sensors with remote health monitoring system

regenerated with permission[3]

Nanoparticles exhibit higher surface to volume ratio and hence are chemically

reactive and are used as a catalyst in electrochemical sensing. Nanoparticles also play an

important role in optical sensing because of the excitation of the surface electrons.

Plasmonic nanostructures such as silver and gold nanoparticles enable amplification of the

scattering signal by electromagnetic and chemical enhancement[4]–[7]. The

electromagnetic enhancement occurs due to the collective oscillation of the free electrons

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on the surface of the NPs, while the chemical enhancement occurs due to the charge transfer

between molecules and the surface[8]. The surface architecture of surface-functionalized

electrodes with nanoparticles impacts on the response of the electrochemical biosensors.

The sensor response depends on the size, shape, and surface attachment of NPs [9]–[11],

as the optical and chemical properties of nanoparticles are dependent on their size and

shape. NP have broad spectra of applications in nanoscale electronics[12]. In most of the

sensing methods, the use of high-density NP causes polydispersity, and different

orientations on the electrode surface conceal the effects of these factors[13], [14].

Moreover, the orientation and tethered patterning of recognition molecules on the electrode

surface is always described using simple sketches, which fail to consider the non-idealities

in target-surface interaction and arrangement. The data obtained in these cases are averaged

over several phenomena that make it problematic to interpret.

Since the NPs are thermodynamically unstable in their free form due to high surface

energy, they tend to agglomerate. The uneven surface coverage, due to the present

deposition techniques, fails to provide the possible effective enhancement in the active

surface area. Furthermore, the commonly used NP deposition techniques for surface

modification are drop-casting, dip coating, spin coating[15], spray coating[16], and

molecular interface such as ligand exchange[17] face a challenge in controlling the NP size

core distribution and the polydispersity on the electrode.

Aerosolized nanoparticle deposition has been a well-known technique these days of

deposition of nanoparticles on the sensor substrates. However, it still fails to keep the

nanoparticles from agglomerating and form the clusters. There are a few techniques that

involve the plasma treatment in the synthesis of nanoparticles; however, there is a lack of

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understanding of the effects of plasma treatment on their morphology, optical, and

electrical properties. Since the electrochemical response depends on the nature of NP

distribution achieved during deposition, the variation in the electrode-electrode baseline

and the active surface area occur; hence, it affects the inter-electrode variability. In the

intent to improve the inter-electrode variability of electrochemical sensors, it is required to

obtain the uniform NP structure, controlled surface electron transfer, and understand the

NP-electrode surface interface.

The motivation of this research is to study the effects of the atmospheric cold plasma

treatment on plasmonic nanoparticles and quantify the NP coverage on the electrode

surface. To obtain the optimized method of nanoparticle deposition, first, there was a need

to understand the effects of plasma on morphology, optical properties, electrical properties,

and surface charge of nanoparticles.

In this research, the plasma-assisted morphological changes[18], optical and

electrical properties enhancement, and surface charge tuning of AgNPs and AuNPs have

been reported. The electroactive surface area was improved using the plasma-assisted

deposition method. The theoretical correlation based on the evaluation of the electroactive

surface area and morphological properties of AgNPs and AuNPs was established. The

nanoparticle deposition with optimization of aerosolized plasma system was carried out to

achieve the desired surface architecture. This deposition was done on the fabricated Au

electrodes on the silicon substrate, and these electrodes were used to detect cortisol.

The proposed technique of plasma-assisted surface charge tuning of NPs in this

research provided the distribution of NPs maintaining the repulsive barrier. The controlled

repulsive barrier led to the formation of a well-organized NP film on the electrode surface.

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It helped in providing the controlled, effective active surface area to enhance the

electrochemical response. The electrodes surface modified with the optimized parameters

have shown closely resembled NP distribution and hence nearly equal electroactive surface

areas. The proposed method has the potential to minimize the inter-electrode variability as

it involves a controlled surface modification process.

1.2 Objectives

i) Characterization of atmospheric plasma set-up and evaluation of its parameters

to understand their role in the deposition process. Optimization of the

deposition process.

ii) Understand the effects of atmospheric plasma treatment on optical,

morphological properties of AgNPs and AuNPs. Study of plasma treatment-

induced change in size, surface roughness, SPR, and SERS signals of Ag and

Au NPs.

iii) CV and EIS Characterization of NPs modified electrodes to investigate the

change in electrochemical performance of plasma-treated electrodes.

iv) Fabrication of microelectrodes using evaporated gold. Characterization of drop-

casted, aerosolized, and plasma-assisted aerosolized NP modified electrodes.

v) Comparison of electroactive surface area and inter-electrode variability of three

different sizes of nanoparticles for the chosen deposition techniques. Cortisol

detection and sensitivity analysis of as-fabricated electrodes.

1.3 Dissertation organization

Chapter 2 consists of a background and literature review. The sensing mechanism of

biosensors, the role of plasmonic NPs in biosensing, and current state-of-the-art of NPs

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6

deposition techniques with their advantages and shortcomings were presented. The theory

of the cold atmospheric plasma and its parameters were also discussed in this chapter. This

chapter was concluded with a description of various types of immunosensors.

Chapter 3 presents the plasma-assisted aerosolized deposition assembly and Ar

plasma characterization. This chapter also describes the methods of characterization, such

as SPR, SERS, surface charge measurement, SEM, and TEM imaging. Microfabrication of

electrodes was also explained with evaporation, photolithography, and lift-off procedures.

The mechanism of the electrochemical immunosensing of cortisol that is used in this work

was described.

Chapter 4 shows the study of the effects of plasma treatment on morphological and

optical properties of AgNPs and AuNPs. The mechanism of plasma-induced size reduction

was explained with SEM, TEM, and AFM characterization along with SERS signals. The

stability study of the plasma-assisted SERS substrate was also discussed.

Chapter 5 includes the study of the comparison of electrochemical properties of

untreated and plasma treatment on AuNPs and AgNPs modified SPCEs. Cortisol detection

using these electrodes were discussed in this chapter.

Chapter 6 presents the results of the electrochemical performances of 20nm, 40nm,

and 60nm of AuNPs modified Au electrodes deposited with drop-casting, aerosol spray,

and plasma-assisted aerosolized techniques. The comparison of CV and EIS responses of

these electrodes was carried out. The inter-electrode variability and correlation of ESA and

the size of NPs for all three methods of deposition were compared. Cortisol detection was

performed using all these electrodes with sensitivity evaluation.

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7

Chapter 7 summarizes the study of the effects of plasma on NPs and their

applications in optical and electrochemical sensing in this work. Future work and detailed

directions that can be considered to continue this work are also discussed in this chapter.

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8

2 BACKGROUND AND LITERATURE REVIEW

2.1 Biosensor

Biological recognition elements are converted into the signal output using biosensors

to detect the specific analyte. Biosensors consist of an analyte, bio-receptors, recognition

elements, and transducers. There are a few recognition methods that are usually used to

sense the analyte, such as immunosensors, cyto-sensors, geno-sensors, enzyme-based

sensors, and polymer-nanocomposite based sensors[19]. The bio-receptors such as

enzymes, antibodies, DNA, organelle, cells, and micro-organisms are selected based on the

recognition method. The schematic representation of the biosensing mechanism is shown

in figure 2.1.

Figure 2.1: Schematic representation of the sensing mechanism of biosensors

The recognition element captures the molecules of analyte selectively, which leads

to a change in the signal generated by the sensor. This change in the signal is then measured

and converted into the electrochemical, calorimeter, or optical signal using a suitable

transducer. The signal processing unit is added based on the mode of the measurement to

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9

convert the signal into the readable form. In the process of analyte detection, the possibility

of errors that get added is during sample preparation, due to environmental variations,

during biological binding due to poor affinity, transducer noise and total noise

accumulation in the measurements.

The advantages of decreasing scale in electrochemistry can be attributed to the

enhanced mass transport and allows increased sensitivity towards molecular level analysis.

The presence of NPs attributed to the enlarged electroactive surface area and fast electron

transfer kinetics as compared to bulk electrodes. It is the elementary need to determine the

electroactive surface area of the electro-catalysts during the electrochemical reactions.

However, in most of the studies, it is not considered. Efforts have been made to calculate

the electroactive area using the CV response of modified electrodes [20]. However, there

is a lack of understanding of the effects of the sensing area architecture on the electroactive

surface area. The orientations of elements used to modify the sensing area play an essential

role in the sensor response. NPs have a broad spectrum of applications in nanoscale

electronics[12]. These applications need to have the controlled deposition of NPs from

dispersion onto the electrode surface and understanding of the particle-particle, particle-

surface interactions[21].

2.2 Properties of NPs and their applications

Nanoparticles exhibit unique electrical, optical, chemical, and catalytic properties

and hence, play a vital role in biosensing. As the surface to volume ratio of NPs increases

as their size decreases, this larger surface area enables the solubilization of more

compounds and hence an increase in the dissolution rate. NPs have a high dissolution rate,

and surface to volume ratio that results in the enhancement of solubility and chemical

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10

reactions. Figure 2.2 shows the various applications of NPs and respective properties that

are used in biosensing.

Figure 2.2: Properties of NPs and their applications

NPs exhibit effective charge transfer in biological activities due to their

physiochemical properties. AgNPs are used in antimicrobials and antibacterial coating.

Also, their high extinction coefficient makes them suitable for fluorescence applications.

The electron band gap increases as the size of semiconductor NPs decreases, which means

more energy is required to excite the electron in NPs. In this case, the light of higher

frequency and lower wavelength would be absorbed; hence NPs are used in solar cells[22].

Magnetic NPs are used in data storage media, magnetic resonance imaging, and drug

delivery[23] as their properties can be modified using magnetic fields.

2.3 Plasmonic Nanoparticles

Plasmonic nanoparticles are the nanoparticles, unlike other materials whose electron

density can couple with wavelengths that are larger than the particle size. Plasmonic

materials are vital for applications such as molecular-level biosensing and nanoscale

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electronics[12]. Plasmonic nanostructures, such as AgNPs and AuNPs, enable the signal

amplification of the sensor through electromagnetic and chemical enhancement[4]–[7].

Being noble NPs, AgNPs and AuNPs provide high stability, simple chemical synthesis,

and tuneable surface functionalization, which make them valuable in the world of

biosensing.

AgNPs are responsible for the electromagnetic enhancement in the biosensing due to

the charge transfer between molecules and the electrode surface[24]. Nanoparticles exhibit

different properties depending on their morphology, e.g., AuNPs have a broad range of

optical spectra depends on their size. Au NPs suspension varies from the dark red color to

dirty yellow color depends on their sizes; hence, they are used in a colorimetric assay[25].

These optical properties are due to the oscillations of free surface electrons, which resonate

at a specific frequency called localized surface plasmon resonance.

Au NPs are most commonly used in electrochemical biosensing to immobilize the

different moieties such as antibodies, enzymes, nucleic acid, biocompatible polymers, or

other bio-receptors as they maintain their activities after immobilization[26], [27]. Such

functionalization of NPs for therapeutic applications has been increasing the application

area in targeting the specificity and increasing the biocompatibility of biosensing. The

morphology of the metal nanoparticles defines the response of a sample, which can be

altered by thermal annealing[28], [29]. The thermal annealing[30]–[32] of NP films leads

to a change in the mean NP diameter due to agglomeration, creating an observable red or

blue shift in the SPRs.

AgNPs have low sintering temperature and high stability, which make their

application area wider in bio-sensing devices, conductive ink, in textiles, and many

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more[33], [34]. Due to the rich electronic and catalytic properties, AuNPs have been used

to detect biomolecules through electrochemical signals[35]. AuNPs are used as labels in

immunosensing, and plasmonic sensing as SPR, and SERS. They display distinct physical

and chemical properties along with easy preparation and superior biocompatibility to

AgNPs, which make them an essential material in biological and chemical detection[36].

AgNPs are an ideal candidate for optical applications[10], [19] because of their high

extinction coefficient, their tunable optical properties through their size and shape, and

surface plasmon resonance (SPR) in the UV–visible region. These properties have been

exploited in applications such as solar energy harvesting, light-emitting diodes, printed

optoelectronic devices, and surface-enhanced Raman scattering (SERS)[8]. SERS is a

powerful analytical tool for sensing a low concentration of molecular biomarkers. SERS

allows amplification of Raman signals by inducing electromagnetic excitation of the

surface plasmons.

Ag NPs tend to form a natural oxide layer on their surface, which affects the

symmetry of the electro-dissolution in electrochemical reactions, SPR and SERS[37]. The

symmetrical partial surface oxidation is acceptable for maintaining the shape of Ag NPs

and their optical response[37].

2.4 Methods of Nanoparticle Deposition

The conventional methods of NP deposition are spin-coating a dispersion[38],

depositing a thin film by sputtering or evaporation, and then annealing to form the thin

film[39], [40]. However, they require more advanced instruments and still do not have

control over size and distribution over the surface. The schematic representation of NP

deposition techniques is as shown in figure 2.3.

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Figure 2.3: Schematic representation of various NP deposition techniques

The NP density varies in the films that are obtained using all these methods. The

drop-casting process provides the liquid media after the deposition, which leads to the

agglomeration of NPs. Cluster formation has been observed in Spin coated NP films[41]

and polydispersity, and insufficient surface coverage in dip coating, spray coating, and

aerosol spray. The ligand tethered NP attachment on the substrate involves the complicated

set-up to modify the ligands and NPs' surface charge and also, clusters, substrate defects,

and orientations of ligands are responsible for forming uneven films[21]. Moreover, the

uncontrolled NPs size can occur in the precursor growth of NPs[25]. The possible defects

in these methods are represented in figure 2.3.

In some of the techniques of NP deposition, such as vacuum evaporation[42] and

electrodeposition[43], the characterization of electrode interfaces is difficult[44]. Also, the

substrates being treated using these techniques must be tolerant of the elevated

temperatures and vacuum set-up.

Some of the commercially available inkjet printed [45], and electrodeposited[46]

electrodes also show the uneven and uncontrolled NP distribution. It is hard to control the

size of NPs in these cases. Plasma-assisted synthesis techniques of NPs have become a

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focus research area of material science and nanotechnology[47]. However, it lacks the

understanding of the plasma-induced modifications of NPs properties. Moreover, the

plasma synthesis processes used to control NP size are highly complex[48].

Figure 2.4 shows the NP distribution obtained using inkjet printing, drop-casting,

electrodeposition, hydrophobic coating, evaporation, and free-standing ultrathin film

formation using the filtration method.

Figure 2.4: a) Inkjet-printed, b) drop-casted, c) electrodeposited NPs on SPCE, d)

hydrophobic coating, e)evaporation-induced AuNP film formation, and f) ultra-thin

AuNPs film using filtration technique, adapted from [45], [46], [49]–[51]

The films formed using these techniques involve coalescence and agglomeration of NPs.

The recorded variability of NP inkjet-printed electrodes is 6%, of electrodes modified with

the electrodeposition method is around 5%, and that of drop cast NP deposited electrodes

is 10%. The molecular interface for the NP deposition also has limitations in controlling

the orientations of ligands on the surface. The surface roughness and defects can cause

random orientations and disturbed NP patterning.

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Microwave monitored AgNPs sintering at higher temperatures for SERS

enhancement has shown the amplification factor up to 108[52]. However, this method

forms clusters of NPs as a function of temperature. Plasma-assisted synthesis of NPs has

emerged as an essential research area of material science and nanotechnology[47].

Moreover, the plasma synthesis processes used to control NP size are highly complex[48].

In this research, a novel and simple approach of cold atmospheric plasma (CAP)

treatment was proposed to provide energy to the AgNPs substrate, similar to that provided

in thermal annealing but at room temperature. AgNPs on substrates by injecting aerosol in

flowing argon gas were deposited and then treated them with a low-temperature

atmospheric plasma jet. The aerosolized NP deposition assembly and plasma set-up are

derived from our previous work[53] and that of Dey et al.[54] with a few modifications.

The plasma technique used by Khan et al.[55] involves pulsed laser deposition and a

comparatively complex experimental assembly. By contrast, the set-up used in this work

contains a simple nebulizer to create NP aerosol for the deposition.

The NPs total potential is a function of their separation distance, and the interparticle

distance depends on the Van der Waals force, Electrostatic double layer (EDL), and

electrosteric interactions[56]. Based on the Van der Waals attraction or repulsion force, the

agglomeration and dispersion occur, respectively. The charged surface of NPs adsorbed

opposite charged ions in the stern potential region and formed the EDL.

2.5 Plasma

Plasma is energetically the fourth state of matter, which contains charged and neutral

particles. The particle (except electrons) energy in the plasma varies from 1 eV to 10 eV,

which is equivalent to the range, 104 K to 105 K, respectively. Plasma is referred to as gas

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discharges, and it can be produced by passing an electrical discharge through the gas.

According to the kinetic theory of gases[57], in ordinary neutral gas, only gravitational

forces act, which are negligible. Particles in the gas stream travel in straight lines with a

distribution of velocities. They collide with each other and with the wall of the container

during their journey. This collision defines the motion of the particles called a random

Brownian motion.

The cross-section for collision ‘a’ and mean free path λ,

a = πr2 (2.1)

Where, r - radius of particles as rigid spheres

d - density

𝜆 =1

𝑎𝑑 (2.2)

The average number of collisions per second (frequency of the collision) v can be

expressed as equation 2.3.

v = V/λ (2.3)

Where, V - the average velocity of the molecules in the gas.

From equation 2.3, the mean time between collisions can be expressed as,

t =1

v=

λ

𝑉, 𝑉 = (

kT

M)

1

2 (2.4)

Where, t - mean time between collisions

k - Boltzmann constant

T - Temperature

M - Mass of a molecule

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λ ∝1

p, λ = ct/p (2.5)

Where, ct - Constant depending on the gas

p - gas pressure

The larger is pressure, the smaller the mean free path of the collision that results in

the larger energy provided to the particles due to collision. In plasma, the motion collision

of the particles forms the positive and negative electric charges in small confined areas,

where these charges create long-range coulombic fields. It affects the motion of the

particles that are some distance from the charge concentrations.

A charged particle in a plasma moves along a path of the electric field. The overall

interaction of the particles in the plasma gives the characteristic collective behavior, and

each parameter defines the nature of the plasma stream. The collision-less plasma can be

created by keeping the pressure of the system low as it leads to a smaller number of

collisions and stronger long-ranges electromagnetic forces. Local concentrations of

charges in plasma are confined to volumes of small dimensions, λD, which is a

characteristic dimension of the plasma, called the Debye length.

For plasma to be stable, the dimensions of the system should be several orders of

magnitude larger than the Debye length. In order of tens of micrometers outside of these

small volumes, the charge density of ions is approximately equal to the density of electrons,

which makes the plasma electrically neutral. Due to the higher mobility of electrons, they

act faster to cancel the charge present in the plasma created by the applied electric field

than ions. This response is called Debye shielding, which gives plasma its quasi-neutrality.

Hence, the applied electrical potential develops mostly near the surfaces, over a distance

called the Debye length.

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𝜆𝐷 = (Ɛ0𝑘𝑇𝑒

𝑛𝑒 𝑒2 )

1

2 (2.6)

Where, Ɛ0 - the permittivity of the free space

e - the charge of the electron

Plasma is obtained when the required amount of energy, higher than the ionization

energy, is added to atoms of gas that cause ionization and production of ions and electrons.

Parallel to the ionization, the opposite process of recombination occurs to form neutral

atoms or molecules. Plasma is excited and sustained by providing gas, electromagnetic

energy in different forms, such as microwave, radiofrequency, and direct current.

2.5.1 Plasma parameters

As electrons make up the largest number of particles in the system, they transfer the

energy through collisions to the molecules of the gas. It causes the ionization and

dissociation of the gas molecules. Similarly, the ions absorb energy and cause chemical

reactions after collisions with the molecules. The motions of the plasma particles, which

induce the collisions, are of two types, elastic and inelastic. An elastic collision occurs

between electrons and heavy targets with negligible energy transfer and does not lead to

the excitation of the target. However, in an inelastic collision, the target goes to an excited

state, with the maximum amount of energy transfer.

Energy transfer Wtr = (2me

M) W (2.7)

Where, Wtr - Energy transfer from electrons to heavy targets

me - Mass of the electron

M - Mass of the heavy particle

W - Energy of the electron

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For an elastic collision of an electron with an argon atom, the fraction of transferred energy

is, Wtr

W~

1

40,000.

The electrons absorb energy through acceleration when the electric field is applied,

and transfer the same amount of energy by inelastic collisions with the neutral gas

molecules. The fraction of this transferred energy can be expressed as,

Wtr

W=

M

M+m (2.8)

Where, m- The mass of particle losing energy

In an inelastic collision, electrons transfer almost all their energy to a heavy particle

(m= me << M), creating an energetic plasma species. These are the particles responsible

for the creation and of sustained downstream plasma.

Energy transfer through inelastic collisions varies from 0.1 eV to more than 10 eV.

The energy required for excitation of molecules is less than 0.1eV, and ionization is more

than 10 eV. The density of the charged particles in the plasma is defined by the degree of

ionization of the gas. The degree of ionization, α defines as,

α= ni/n (2.9)

Where, ni - the number of ionized particles

n - the total number of particles present in the plasma

The critical ionization 𝑎𝑐~ 1.73𝑥1012 𝑎 𝑇𝑒2.

If α>>> ac, the charged particles behave as in a fully ionized gas.

Hence, to obtain stable atmospheric plasma, the energy should be less than the critical

ionization.

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2.5.2 Cold Atmospheric Plasma

Typically, the parameters and the range of their default values for the cold plasma

are as follows,

Electron Temperature, Te~ 1 eV, Electron density, ne ~ 1010 /cm-3, Debye Length, λD = 74

µm.

𝜆𝐷 = 6.93 ( 𝑇𝑒(°k)

𝑛𝑒 (𝑐𝑚−3)) (2.10)

Debye length depends on the electron temperature, the energy of the electrons which

get excited and responsible for collisional energy transfer, and electron density (ne).

The energy associated with the plasma is given by,

W = (3

2) (k𝑇𝑒) (2.11)

When the electric field is applied to the gas system, an electron multiplication process

takes place, which can be characterized by a macroscopic coefficient, aT that represents the

mean number of ion-electron pairs formed along a path of 1 cm. The coefficient, aT, is

called the first Townsend coefficient and is dependent on the electric field (E), the pressure

(P), and the nature of the gas as well, which helps in designing the plasma system.

When the applied voltage is low, the current produced by the collection of the

available free charges is negligibly small. When the applied voltage reaches a specific

threshold value, ions get accelerated and hit the cathode with their energy, which causes

the emission of the secondary electrons. The secondary electrons contribute to the

formation of more ions by collisions with the neutral particles of the gas. These ions also

are accelerated towards the cathode and produce more electrons and more ions.

Simultaneously, electrons created by ionizing collisions and by secondary processes are

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removed from the plasma by drift and diffusion to the walls of the gas system, by

recombination with positive ions, and in certain gases, by the formation of negative ions.

2.5.3 Plasma-Induced Surface Modification

When the surface comes in contact with the plasma, the electrons and ions recombine

and are lost from the plasma system after reaching the substrate surface, which has to be

modified. Electrons, due to the thermal velocities, reach the surface faster than the ions by

leaving the plasma with a positive charge. Thus, the surface achieves a negative potential

relative to the plasma. The Debye Shielding effect induces the potential developed between

the surface and the plasma that is enclosed to a layer of the number of Debye lengths. This

layer is called a plasma sheath that affects the penetration and repulsion of the electrons

through it. The sheath potential is given by[57].

For the planar surface:

Vs = (k𝑇𝑒

2e) ln (

me

2.3∗mi) (2.12)

Where, Vs - sheath potential

mi - a mass of the ion

The thickness of the plasma sheath is the thickness of the region where the electron

density is negligible and where the potential drop Vs occurs. It depends on the collisional

mean free path in the plasma and is affected by external biases applied to the surface.

2.6 Nanoparticle-based Electrochemical Immunosensing

Due to the rich electronic and catalytic properties, plasmonic NPs have been used to

detect biomolecules through electrochemical signals[35]. Although direct detection of

biomarkers using conventional electrochemical reactions is possible, various methods have

been successfully adapted where nanocomposites boost the sensitivity of biosensors.

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Electrochemical sensing involves a transducer which converts the biochemical signal into

an electrical signal.

There are various methods available to detect the analyte using electrochemical

immunosensing, including competitive, sandwich, and labeled immunoassay[19], [58].

Nanoparticles are used as labels, or as an underlying layer to bind biorecognition elements

for biosensing. In some cases, ligands such as Self Assembled Monolayer (SAM) are used

for binding, and in some methods, they are used to increase the electroactive surface area.

Figure 2.5 shows various techniques that involve NPs in immunosensing. Due to the strong

affinity of Au surface with amino groups and mercapto groups, AuNPs facilitate the

conjugation of biological ligands[59].

AgNPs provide improved electrochemical signals and can be oxidized easily; hence

they are suitable in the detection of tags in electrochemical sensing. However, they are

unstable and hard to functionalize. Also, most importantly, they have less biocompatibility

than AuNPs, which limits their application area in immunosening[19], [60].

Figure 2.5: Nanoparticle-based electrochemical immunosensing includes [a), and b)]

Competitive, c) sandwich, and d) NP as an underlying layer and Ligand-based

immunosensing. Adapted From [19]

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As nanoparticles serve as a fundamental layer in the fabrication of biosensors and

further layers of bio-receptors are attached to them, it is necessary to achieve a uniform

film on the sensing area.

In this work, the ligand-based electrochemical immunosensors have been fabricated

for the detection of cortisol with DTSP SAM to bind cortisol antibodies to the NP surface.

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3 EXPERIMENTAL PROCEDURE

In this work, the aerosolized deposition system combined with the cold atmospheric

plasma system using Argon gas has been designed and developed considering all the

parameters that are described in chapter 2. The designed plasma system has been used to

control the NP surface charge to obtain comparatively dispersed NP placement on the

electrode surface than drop-casting and aerosol spray methods. Zeta-potential

measurements were carried out to analyze the surface charge on NPs during deposition.

Based on the DLVO model[21], the force of Van der Waals attraction and the Electrostatic

double layer is dependent on average particle size, the distance between interacting

surfaces, absolute temperature, stern layer thickness, and zeta potential. The experimental

assembly for aerosolized deposition with surface charge tuning with plasma has been

developed in this work.

3.1 Aerosolized deposition and argon plasma assembly

Figure 3.1 shows the experimental set-ups used for NP deposition (Figure 3.1a and

3.1b) and plasma treatment (Figure 3.1c and 3.1d). For NP deposition, an ultrasonic

nebulizer was used to create an aerosol of the NP solution. The nebulizer was connected to

a quartz tube to provide a desired path to the aerosol. The ultrasonic nebulizer was modified

to have one inlet for Argon (Ar) gas and one outlet, which was connected to the quartz

tube, with an outer diameter of 6 mm and an inner diameter of 3mm.

A glass capillary with a 1-mm inner diameter was inserted into the quartz tube

(Technical Glass Products, Ohio). Another Ar inlet was connected to the midway point of

the quartz tube and was controlled by a mass flow controller (MFC). Teflon tape (Everflow

Supplies) was used as a moisture blocker between the quartz tube and glass capillary

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25

(placed in front of the MFC outlet) to absorb the excess moisture created from the aerosol

in the path before deposition.

A high-voltage supply (variable output of 1–20 kV; variable frequency of 20–60

kHz; current of 20–30 mA) to generate the plasma (Figure 3.1c and 3.1d), was connected

to two electrodes made from copper rings on the quartz tube. These tubular copper

electrodes were placed 1 cm apart. The MFC-controlled Ar gas flow was supplied to the

quartz tube to produce a stable Ar cold plasma.

The Ar flow rate was set using a gas flow controller at 2 L/minute. The external

power supply was used to deliver sinusoidal power (~10 kV, ~30 kHz) to the electrodes.

The high-voltage supply was turned on, and the impedance was adjusted in such a

way that the voltage and variable frequency reached a point where the downstream plasma

became stable. The voltage supply and gas flow were turned off after treatment.

biPolyethylenimine (BPEI)-functionalized Ag NPs and Citrate capped Au NPs of an

average size of 10 nm were purchased from Sigma-Aldrich and used to make a 0.02 mg/ml

solution in deionized water. The NPs solution was poured into the nebulizer to create the

aerosol.

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Figure 3.1: a) Schematic illustration and b) photograph of the experimental set-up for the

deposition of Ag NPs on the substrate. c) Schematic illustration and d) photograph of the

experimental set-up of the cold plasma unit. e) Ag NPs deposited on a flexible polyimide

film[61].

The stream of Ar gas of ~15 SCCM was supplied to the inlet of the nebulizer. NPs

aerosol was carried along the path through the quartz tube and capillary through the stream

of Ar. The Ar gas supplied to the midway of the quartz tube regulated the flow of the NPs

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27

for film deposition as required. The concentrated NPs were carried through the capillary.

They were deposited on the quartz slides for SPR detection, on silicon for the scanning

electron microscopy (SEM), and SERS characterization, and on the copper grids for the

transmission electron microscopy (TEM).

3.1.1 Argon plasma

Ar atomic structure: Ar has its atomic number- 18 and atomic mass- 39.948. It has

the electron distribution in its atomic structure as 1s2 2s2 2p6 3s2 3p6[62]. The energy

associated with each of the energy levels in the atom can be calculated as:

𝐸𝑛 = −13.6 𝑍/𝑛2 eV (3.1)

Where, n- number of the shell the electron present

Z- atomic number

The energy level diagram for the Argon atom is shown in figure 3.2.

Figure 3.2: Argon energy level diagram

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3.1.2 Plasma characterization

The free electrons are present in 3p- 3p, 4s levels of the Ar atom. The electron

transition mostly takes place between 4p and 4s atomic states. The applied external

potential acts on the neutral gas when applied, the electrons get energies by absorbing the

energy. The electrons then, due to collisions with other molecules, generate the ions and

then ions due to the collision with other molecules, generate the secondary electrons. The

current gets generated proportion to the applied electric field and electron-ion interactions.

The Argon plasma generated was diagnosed by using Optical Emission Spectroscopy

(OES). The electrons emit energy when they make transitions between different energy

levels in the atom. The transitions can be analyzed by obtaining the radiated emission

energy from the electrons at different wavelengths. The optical emission spectrum was

recorded for the Ar gas CAP, as shown in figure 3.3.

Figure 3.3: Optical emission spectra for Ar plasma. Inset: relative intensity of the Ar I

and Ar II transitions with respect to the relative higher-energy level[53].

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29

An optical emission spectrometer (Ocean Optics, HR2000+ES) was used to obtain

the intensity counts for the downstream plasma[63]. As the Ar plasma falls under the

visible spectra, the range of the wavelength at which we get the intensities of the radiated

energy is as shown in figure 3.3. From the OES of the Ar gas plasma, the intensities for

various transitions were detected. The ArI refers to the transition from 4p to 4s and ArII to

4d to 4p. The intensities for the Ar transitions were also measured[64] as,

Table 1. Ar Intensities and corresponding parameters

Wavelength I A (x107) E1 E2 G

695.3 Ar I 1.41E+04 0.63 11.54 13.32 3

705.5 Ar II 3.82E+03 0.38 11.54 13.3 5

725.9 Ar I 3.02E+03 0.18 11.62 13.32 3

750.8 Ar I 2.88E+03 4.45 11.82 13.27 1

762.2 Ar I 1.37E+04 2.45 11.54 13.15 3

772 Ar I 9.05E+03 0.51 11.54 13.15 3

801.2 Ar I 2.03E+03 0.92 11.54 13.09 5

811.2 Ar I 3.94E+03 3.31 11.54 13.07 7

826 Ar I 3.88E+03 1.53 11.83 13.32 3

842 Ar I 1.65E+03 2.15 11.62 13.09 5

852 Ar I 4.57E+02 1.39 11.82 13.28 3

912 Ar I 1324 1.89 11.54 12.90 3

Some of the intensities other than Ar were also identified such as,

At, 307.7 nm, 281.2 of OH; at 336.2 nm, 214.2 of N2; at 356.2 nm, 186.2 of CO2; at 776.2

nm, 804.6 of O2; at 921.2 nm, 248.2 of O2.

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The relative intensities were plotted against relative higher-level energies for the

respective transitions using the ratio method[65], as shown in the inset of Figure 3.3; the

relationship is given in Equation 1[66]:

ln (𝐼₁

𝐼₂) = −

𝐸₁−𝐸₂

𝑘𝑇+ ln (

𝐴₁𝑔₂ 𝜆₂

𝐴₂𝑔₂ 𝜆₁) (3.2)

Where, A - the transition probability

g - the statistical weight of the electron

λ - the wavelength associated with the transition

E - the higher-level energy for the transition

k - the Boltzmann constant

T - electron temperature

The relationship between the relative intensities and relative energies was plotted to

validate the above result. The slope of the fitted plot was calculated, as shown in the inset

of Figure 3.3, and the electron temperature was found to be T = 1.08 eV. Using the Saha–

Boltzmann method[67], [68], the electron density, ne, was calculated as:

𝑛𝑒 = 6.04𝑥1021𝑥 (𝐼₁

𝐼₂) 𝑥(𝑇)

3

2 𝑒 (𝑥𝑧−𝐸₁−𝐸₂

𝑘𝑇) (3.3)

Where, (2 𝜋𝑚𝑒𝐾

ℎ3)

3

2= 6.04 × 1021

xz - the ionization potential of Ar (assumed to be 15 eV)[69].

The electron density, 𝑛𝑒 = 1.9𝑥1011 cm-3, is the number of electrons that were

energized and excited into an ionized state.

The NPs were deposited on quartz and silicon substrates and then were treated with

plasma. Similarly, the deposited NPs were annealed thermally to compare the results

obtained with the proposed technique.

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3.1.3 Plasma treatment and thermal annealing of NPs

Samples for SPR were prepared by depositing Ag, Au NPs on quartz slides followed

by plasma treatment for 5, 10, or 15 minutes. For comparison between the plasma-treated

and thermally annealed substrates, sets of as-deposited slides were annealed using rapid

thermal processing (RTP) for the same duration at 250 and 400 °C in an Ar/O2

environment. The thermal energy for the gas at 400 °C was 0.058 eV, which was obtained

from the temperature of the gas used in RTP.

The characterization of optical properties of thermally and plasma-treated NPs was

carried out using SPR, and SERS.

3.2 Surface Plasmon Resonance

SPR occurs due to the excitation of conducting electron cloud present on the surface

of plasmonic NPs. In this technique, the free electrons get excited by incident polarized

light, and their collective oscillation is detected[70]. The interaction of electromagnetic

waves with the conducting electrons on the NP surface attributes to SPR, which leads to

strong scattering and absorption. SPR signals were measured for all samples using a UV–

visible spectrometer (Thermo Fisher, Evolution 300). The SPR absorbance peak depends

on the size, shape, and assembly of the metal nanoparticles and can be expressed as[71]–

[74],

𝐴 (𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒) = Ɛ 𝐶 (3.4)

Where, Ɛ - extinction coefficient

C - Concentration of a solution

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When a beam of light passes through the glass slab of the sample, some amount of it

gets scattered and transmitted when the remaining gets absorbed. Hence, the absorbance is

a measure of the density of NPs deposited on the substrate by experimental set-up.

The above equation can be rewritten in terms of particle density as,

𝐴 ∝ (𝑅2) 𝑁 (3.5)

Where, R - Radius of a spherical particles

N - Number of particles per unit area

Also, the position of UV Spectra Peak can be evaluated as,

𝑑 = 𝑙𝑛 (𝜆𝑠𝑝𝑟 – 𝜆0)/ 𝐿2 (3.6)

Where, d - particle size (diameter)

λspr - position of the absorbance peak

As the position of the absorbance peak depends on the particle diameter, change in

particle size accounts for a shift in the UV visual spectra towards blue range or red range,

depends upon the positive or negative change in the size. Due to the surface to volume

ratio increment in NPs, the number of scattering electrons at the surface increases. This

phenomenon leads to the reducing lifetime of the oscillation and hence increase the spectral

width. The LSPR (Localized Surface Plasmon Resonance) occurs at a specific wavelength

of the incident light. When the source of this wavelength is used to excite the molecules in

SERS, the signal enhances, while the respective SPR signal would be blank. Hence, these

two methods are not interchangeable. The collected signal in SPR passes through the set

of monochromators to separate the intensities. These intensities are plotted as a function of

wavelength.

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3.3 Surface charge measurements

The electric charge on the NP surface after plasma treatment was analyzed by

performing zeta potential measurements using a particle zetasizer (Malvern Instruments).

Measurements were made from a solution of NPs and averaged before and after treatment

in the liquid. Also, the NPs were deposited on the silicon substrates and treated as described

in section 3.1.3 and then scraped out from the substrate, and the suspension was prepared

in DI water. The zeta potential of the suspension was recorded immediately after the

treatment. The readings were taken for 15 runs and were averaged for five different

samples.

3.4 Surface-enhanced Raman spectroscopy

Raman signals are inherently weak; the method to amplify weak Raman signals is to

employ surface-enhanced Raman scattering (SERS). SERS uses nanoscale roughened

surfaces typically made of Au or Ag NPs. Laser excitation of these roughened metal

nanostructures resonantly drives the surface charges creating a highly localized

(plasmonic) light field. When a molecule is absorbed onto the enhanced field at the surface,

a significant enhancement in the Raman signal can be observed. R6G was used in this

work, as it has been used extensively as a model dye for SERS characterization. It is an

extremely strong fluorophore when excited by visible radiation.

SERS involves sample illumination using a laser beam. The electromagnetic

radiation from the illuminated sample is collected and dispersed onto a detector, as shown

in figure 3.4. This scattered photon shifts to a different energy level and, therefore, at a

different frequency. This energy difference is equal to that between the initial and final

states of the molecule. If the final state is higher in energy than the initial state, the scattered

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photon shifts to a lower energy level so that the total energy remains the same. This shift

in frequency is called the Stokes shift; similarly, if the final state is lower in energy, the

scattered photon shifts to a higher frequency, which is called an anti-Stokes shift, or upshift,

as shown in figure 3.4.

Figure 3.4: Schematic representation of SERS measurement technique, adapted

from[75].

Raman shifts are typically reported in wavenumber, which have units of inverse

length, as this value is directly related to energy. The shift in the Raman spectrum can be

calculated as follows,

𝛥𝑣 = (1

𝜆0) − (

1

𝜆1) (3.7)

Where Δv is the Raman shift expressed in wavenumber, λ0 is the excitation wavelength,

and λ1 is the Raman spectrum wavelength. It is expressed with cm−1.

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The SERS signals of the samples were recorded using a LabRAM HR spectrometer.

The intensities were recorded at 30 selected points and then averaged. The spectral position

was calibrated at the 520.5 cm−1 peak of Si/SiO2. Signals were recorded at 532-nm

excitation wavelength with a 5 µm diameter of the region under the laser beam. Gaussian–

Lorentzian peak detection was used after subtracting the noise from the signal in 15

acquisitions. NPs were deposited on silicon substrates, and the SERS signal for R6G was

measured with and without plasma treatment. R6G (Sigma Aldrich) was diluted to 5 µM

and placed directly on the untreated and treated NP films. A 10-µl probe solution was used

to create a drop on the substrates. This drop was spread into the circle with a 4-mm

diameter.

The peak intensities of the R6G directly on the bare silicon substrate were detected

after spreading it in a 4-mm-diameter circle and letting it dry to calculate the enhancement

factor. The EF for the recorded signals was calculated based on the methods used in Meyer

et al.[76] and Li et al.[77]. The enhancement factor can be calculated as,

𝐸𝐹 = (𝐼𝑆𝐸𝑅𝑆 /𝑁𝑆𝐸𝑅𝑆)/(𝐼𝑁𝑅𝑆/𝑁𝑁𝑅𝑆) (3.8)

𝑁𝑆𝐸𝑅𝑆 = 𝑁𝐴 𝑛𝑆𝐼𝑟𝑟/𝑆𝑑𝑖𝑓 (3.9)

𝑁𝑁𝑅𝑆 = 𝑑 𝑆𝐼𝑟𝑟 𝑁𝐴ℎ/𝑀 (3.10)

Where, ISERS and INRS- The peak intensities of the SERS (Surface Enhanced Raman

Scattering) and NRS (Normal Raman Scattering), respectively.

NSERS and NNRS correspond to the number of probe molecules excited in the

SERS and NRS tests.

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NA refers to the Avogadro's constant, n correspond to the molar quantity of

the probe molecule (5μM)

SIrr - The irradiation area under the laser beam (5μm)

Sdif - The diffusion area of the substance to be tested on the substrate. For

10μL of volume and 4mm circle of the drop of R6G, the diffusion area is

50.24mm3)

d - The packing density of R6G molecules in the surface of the substrate

h - The laser confocal depth (26μm)

M - The molecule weight of R6G (479).

3.5 Imaging

The aerosolized Au and Ag NPs were deposited with thermal and plasma treatment

onto silicon substrates for SEM imaging (JOEL, SEM 7000l) at an operating voltage of 20

kV and with a 9-μA probe current. The SEM images were processed using ImageJ software

to obtain the Feret diameter distribution of the untreated and treated NPs. The TEM images

of the Ag NPs were captured using a Philips CM200 TEM at a 200-kV excitation voltage.

Atomic force microscopy (AFM) images were recorded for samples prepared on

silicon substrates using AFM WORKSHOP TT, and the results were processed using

Gwyddion software tools. AFM is usually used to measure the topography and surface

roughness. It records the deflection and oscillation amplitude of the beam as the tip of

cantilever moves along the surface to scan. The cantilever was used in this project was in

vibrating mode. The surface roughness and texture of the untreated and plasma-treated

samples were plotted.

The electrode response for closely placed NPs deposition by avoiding interparticle

attraction using the designed deposition system has been statistically analyzed. The NP

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distribution using drop-casting, aerosolized, and plasma-assisted aerosolized NP

deposition were compared. Plasma tunes the surface charge of NPs during deposition on

microelectrodes that were fabricated, as described in section 3.6. The effects of these

methods on the electroactive surface area and inter-electrode variability have been

compared.

3.6 Microelectrodes Fabrication

The microelectrodes were fabricated using photolithography patterning on silicon

substrates, followed by evaporated Au deposition. The electrode was designed to use for

three-electrode electrochemical systems by considering the standard proportions using

LayoutEditor, as shown in figure 3.5.

Two layers of the fabrication were performed for gold evaporated reference (RE),

counter (CE), and working electrodes (WE), an insulation layer on top to make a microwell

to limit the electrolyte during electrochemical measurements. The photomask of the design

shown in figure 3.5 was made using Heidelberg uPG-101 mask maker.

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Figure 3.5: Layout of the electrode design

The standard procedures of photolithography, physical evaporation, and lift-off were

performed to fabricate the electrodes on silicon (Si) wafers. The process flow of

photolithography, metal deposition, lift-off is shown in figure 3.7. The silicon wafers must

undergo a standard cleaning to begin the patterning process. The silicon wafers were

cleaned with Acetone for 2 minutes, Isopropyl alcohol for 1 minute to rinse off the acetone

residues, and deionized (DI) water for 5 minutes, followed by drying with dry air.

The spin coater is used to apply PR layers onto the wafer due to the resulting high

uniformity of the coatings. When a wafer is to be spin-coated, it is first placed on the

rotating panel where the vacuum chuck secures it into place. The photoresist is then

dispensed onto the wafer as it rotates. Figure 3.6 shows the schematic of the spin coating

technique.

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Figure 3.6: The schematic of a photoresist spin coater set-up, as regenerated from[78].

Photoresist AZ5214E, which is an image reversal photoresist, was used to coat the

silicon wafer using a spin coater. The wafer thickness and type of orientation are considered

when deciding the parameters that should be used to spin coat photoresist onto a wafer.

In this project, AZ5214E was used as a positive photoresist for patterning on the Si

wafers. The optimized parameters for spin coating were used. The rotating speed was set

to 500rpm for 10 sec with the ramp of 100rpm/sec. It was then switched to 4000 rpm for

40 sec with the ramp of 1000rpm/sec to obtain a thickness of 1.40µm[79].

Moving on from the coating step, the next part of the backside patterning is the

exposure and development of the wafer. The coated wafer was baked at 110̊C for 1 min,

followed by exposure to 17mJ/cm2 UV intensity of OAI mask aligner for 6 sec. The PR

was used as a positive PR; the regions on the wafers that had not been exposed to UV light

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became insoluble in the developer. The exposed region was removed for further deposition

of the metal films.

Figure 3.7: Fabrication steps for microelectrodes

The exposed wafer was developed in AZ 400k developer with a 1:4 ratio of developer

and deionized water. The developed wafers were treated using reactive ion etching (RIE)

to descum the unwanted material deposited on the wafers during the process.

RIE was first conditioned using O2 gas at 100 watts, 400mTorr for 100 sec. The

physical evaporation of chromium (Cr) of the thickness of 20nm and gold (Au) of the

thickness of 100nm was done using an E-beam evaporator.

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Figure 3.8: Picture of evaporator a) and b) planetary fixture

E-beam evaporator is a physical vapor evaporation technique, used for the thin film

deposition. E-beam is focused on the crucible of the material, which has to be deposited on

the wafer by keeping the substrate planetary fixture rotating to obtain the uniform

deposition on the wafer. The deposition was done on the entire surface of the wafers, as

shown in the process flow in figure 3.7. The Cr/Au deposited wafers were kept in acetone

overnight for lift-off to remove the PR from the masked regions.

These deposited films on silicon were used as three electrodes and contact pads for

connection purposes. Figure 3.8 shows the picture of the E-beam evaporator with the dome

on which the substrates were attached. The average thickness of the metal deposition was

measured using the Dektak profilometer as 115.6nm. The microwells on the circular area,

which covers the CE, RE, and WE, as shown in figure 3.5, were created using PR on the

fabricated electrodes.

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Also, the electrodes prepared with these methods were used to detect cortisol using

ligand-antibody-based electrochemical immunosensing.

3.7 Electrochemical Immunosensing of cortisol

The electrochemical immunosensor composed of AgNPs and AuNPs on the activated

SPCE and gold microelectrodes surface as a current collector, along with biotin-conjugated

cortisol antibody covalently cross-linked on the surface of the nanoparticles using DTSP.

The sulfur binding of DTSP is attached to the surface of AgNPs and AuNPs. Further, the

amine group of the antibodies binds to the DTSP ligand, as shown in figure 3.9. This self-

assembled monolayer (SAM) of antibody on the nano-substrate provides irreversible

binding sites for the cortisol present in any given sample. This irreversible binding of the

protein structures limits ion transport to the sensor surface (Figure 3.9).

Figure 3.9: Schematic of Antibody/DTSP SAM/NPs binding and stepwise fabrication of

cortisol immunosensor

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In this sensing approach, the electroactive species (ions) on the sensor surface is

proportional to the cortisol concentration. By quantifying the electroactive species

concentration interacting on the sensor surface, the amount of cortisol concentration can

be determined.

The electroactive probe used was Fe2+/3+ ion, and its change in concentrations on the

electrode surface was monitored through its oxidation reaction given in equation 3.11.

Fe2+ + e- Fe3+ (3.11)

For electrochemical measurements, a voltammogram was used for monitoring

oxidation peak current (Ipa) of Fe2+ oxidation E0 in the potential window 0 to 0.5 V.

In this approach, the electron transfer is generated in the electrolyte, [Fe (CN)6]3−/4−

is blocked during the surface modification. Further, the binding of cortisol molecules with

Antibodies prevents the electron transfer from the electrolyte to the electrode surface. This

blocking leads to a reduction in the redox current. This current is proportional to the

molecules getting attached to the antibodies. Hence, the resultant current is correlated to

the concentration of the cortisol present.

3.8 Cyclic Voltammetry

Cyclic voltammetry is an electrochemical tool in biosensing, which is used to analyze

oxidized and reduced species[80]. The binding of antigen and antibodies is estimated as a

function of the redox current in the electrolyte. In CV, a varying potential range is applied

to the three-electrode system, and the corresponding current is recorded.

The oxidation peak and the reduction peak response can be explained using the

Nernst equation. This equation shows the proportionality of the potential of an

electrochemical cell (E) to the standard potential of a species (E0). The relative activities

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of the oxidized (Ox) and reduced (Red) analyte in the system at equilibrium is given as

equation 3.12.

𝐸 = 𝐸0 + 𝑅𝑇

𝑛𝐹ln

(𝑂𝑥)

(𝑅𝑒𝑑) (3.12)

Where, R - universal gas constant

F - Faraday’s constant

n - the number of electrons

T - temperature

A one-electron reduction system in [Fe (CN)6]3−/4−, the Nernst equation can be

rewritten as equation 3.12 to replace the activities with their concentrations. The standard

potential can be substituted with formal potential E0’ and n is equal to 1.

𝐸 = 𝐸0′ + 2.3026 𝑅𝑇

𝐹 𝑙𝑜𝑔10

[𝐹𝑒3+]

[𝐹𝑒2+] (3.13)

This equation can be modified to predict the change in the system proportional to the

concentration of the chemical species and the electrode potential. In the three-electrode

system, the voltage is applied between RE and WE to measure the current from CE to RE.

The system maintains its equilibrium by providing the current from counter to reference to

regulate the voltage drop across working and reference electrodes. This phenomenon can

be utilized for biosensing where the surface of the electrode is functionalized with detecting

molecules, such as an enzyme, antibodies, DNA, aptamers, and many more.

3.9 Electrochemical Impedance Spectroscopy

This method of characterization of chemical processes in terms of electrical

measurements. This tool exploits Faraday’s law,

𝑄 = 𝑛 𝑧 𝐹 (3.14)

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Where, Q - charge passing through a cell

n - the amount of substance

F - the Faraday’s constant

z - the number of electrons needed per conversion

The connection can be established with [Fe (CN)6]3−/4− in this work to measure the

electrochemical impedance of the three-electrode system using fabricated electrodes.

It measures the impedance of the electrochemical cell to an applied potential, which

depends on the frequency. The complex response of the system usually interpreted from

the Nyquist plot. The equivalent circuit (Randle’s circuit) for the biosensor is shown in

figure 3.10 with the equivalent Nyquist plot.

Figure 3.10: Equivalent circuit and Nyquist plot of the biosensor

The semicircle in the Nyquist plot represents the parallel combination of electron

transfer resistance (Rct) and double-layer capacitance (Cdl) in Randle’s equivalent

circuit[81]. The linear portion is due to Warburg impedance (Zw), indicates the ion

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exchange/diffusion in the electrolyte of the cell. It is used to characterize the surface

architecture in the functionalization process of the electrodes.

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4 EFFECTS OF COLD ATMOSPHERIC PLASMA TREATMENT ON

MORPHOLOGICAL, AND OPTICAL PROPERTIES OF PLASMONIC

NANOPARTICLES

The use of plasma processes in nanomaterial synthesis is limited by a lack of

understanding of the effects of plasma treatment on their morphology and other properties.

Here, the impact of atmospheric plasma treatment on the morphology and optical properties

of Ag and Au NPs have been studied. In this work, the analysis of AgNP and AuNP

morphology and their correlating optical properties have been explored.

The aerosol injection technique that has been discussed in chapter 3 was used to

deposit well-dispersed AgNP films, and these films were treated by CAP. The size

reduction observed due to the surface plasma treatment of AgNPs was caused by the

symmetric partial oxidation. These deposited films were used as SERS substrate to detect

R6G peaks.

The intensity of the SERS signal of R6G from plasma-treated AgNP substrate was

~120 times as high as that of the untreated substrate, and that of AuNPs was ~95 times as

high. This enhancement is attributed to the surface roughness, presence of nanogaps, and

size reduction after the treatment of NPs on the substrates. The morphological changes are

explained by a redox reaction induced by the energy and concentration of reactive species

in the CAP environment. This surface reaction accounts for the difference in the effective

electric charge on NPs surface that was quantified using zeta potential measurements.

4.1 Surface Plasmon Resonance (SPR)

The SPR absorption peaks of the untreated and thermally or plasma-treated Ag NPs

films deposited on quartz substrates are shown in Figure 4.1a, 4.1b, and 4.1c. The SPR

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peak of the untreated substrates was observed at 426 nm, which was blueshifted to 415,

393, and 361 nm, after plasma treatment for 5, 10, and 15 minutes, respectively.

Figure 4.1: SPR of untreated, 5 mins, 10 mins, and 15 mins a) plasma-treated; b)

thermally treated at 250̊ C and c) thermally treated at 400 ̊C and d) Blueshift of a), b) and

c) shows the blueshift in SPR spectra after the thermal and plasma treatment[82].

Thermal annealing resulted in a blueshift of 7, 17, and 25 nm at 250°C, and 9, 28,

and 45 nm at 400°C at 5, 10, and 15 minutes, respectively. After 15 minutes, no significant

peak shift was observed for thermal annealing. Moreover, the strength of the SPR

absorbance was reduced for the treated samples. An almost linear plot of the blueshift after

plasma or thermal treatment is shown in Figure 4.1d, where the plasma treatment resulted

in a similar resonance response to thermal annealing. From Figure 4.1d, we note that there

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was a more significant shift in the SPR peak for plasma treatment than for thermal

annealing for the same duration of the process.

The blue shift and the reduced intensity of the SPR peak in figure 4.2 suggest that

plasma treatment reduced the size of the NPs and increased the distance between adjacent

particles[83], [84]. Because of the size reduction, the surface-to-volume ratio increased.

This increase led to the rise in the number of scattering electrons at the NP surface, which

is responsible for the reduced lifetime of the oscillations and increased spectral width[85].

Similar experiments were performed on Au NPs deposited quartz substrates. The

results are as shown in figure 4.2. The absorbance was decreased as the Au NPs were

treated with the plasma. The blueshift of 5nm, 40nm, and 55 nm was observed after 5 mins,

10 mins, and 15 mins of the plasma treatment, respectively.

Figure 4.2: a) SPR results of plasma-treated Au NPs and b) Blueshift in SPR after the

plasma treatment

Our results differ from those of studies that use sintering processes of drop-casted

NPs[48], [86], because of the deposition technique. After aerosol deposition, the lack of

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liquid media prohibits NP migration and agglomeration. Besides, the process is

independent of the substrate and takes place at a low temperature. Hence, it allows NPs to

be processed individually, which contributes to the size reduction and blue shift in the SPR

signal. To confirm this statement[21], we repeated the experiments using drop-casting as

the deposition method; this led to coalescence and a redshift in the SPR signal. The results

for the plasma-treated drop-casted NPs are as shown in figure 4.3.

Figure 4.3: SPR and redshift of plasma-treated drop-casted a), c) AgNPs and b), d) Au

NPs

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The redshift in plasma-treated drop-casted AgNPs and AuNPs supports the

coalescence phenomena. The SPR of AgNPs was shifted from 8nm for 5 mins, 68nm for

10 mins, and 79 nm for 15 mins of the treatment. The redshift in plasma-treated drop-casted

AuNPs was 8nm for 5 mins, 28nm for 10mins, and 60nm for 15mins of the treatment.

Hence, as the plasma-assisted deposition method maintains the interparticle distance, it

differs from other techniques.

4.2 SEM Imaging

The AgNPs were deposited on silicon substrates using the same deposition

conditions applied for the SPR study. The substrates were then thermally annealed at

400 ℃ or plasma-treated for 15 minutes. Figure 4.4a, 4.4b, and 4.4c show the SEM images

of the untreated, thermally annealed, and plasma-treated samples, respectively. The size of

the NPs was reduced, and the NPs were well dispersed after both treatments. The SEM

images were processed in ImageJ to identify the NP boundaries (Figures 4.4d, 4.4e, and

4.4f) and to determine their Feret’s diameter (Figures 4.4g, 4.4h, and 4.4i).

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Figure 4.4: SEM images of AgNP films deposited on silicon substrates a) before

treatment, b) after 15 mins thermal annealing, and c) after 15 mins plasma treatment. The

SEM images processed in ImageJ to identify nanoparticle boundaries [d), e) and f)]; and

Feret’s diameter distributions [g), h) and i)] corresponding to untreated, thermally treated

and plasma-treated AgNPs. The left shift in the AgNPs size distribution was attributed to

the size reduction after the treatments. The plasma-treated AgNPs exhibited a more

uniform film than other samples[82].

Untreated NPs have an average Feret’s diameter of ~15 nm with the largest particle

size of 44 nm because of NP agglomeration during deposition. After thermal annealing,

the mean Feret’s diameter was reduced to 6 nm, with the diameter of most particles falling

in the range of 5–7 nm. Similarly, for the plasma-treated sample, the particle distribution

was further narrowed with a mean diameter of 5 nm. The SEM images were captured for

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AuNPs deposited untreated and 15 mins of plasma-treated samples that can be seen in

figure 4.5. The comparatively uniform NPs films were observed, similar to AgNPs films.

Figure 4.5: SEM images of AuNP films deposited on silicon substrates (a) before

treatment, (b) after 15 mins of plasma treatment. The SEM images processed in ImageJ to

identify nanoparticle boundaries [(c) and (d)]; and Feret’s diameter distributions of (e)

untreated and (f) plasma-treated AuNPs film.

The Feret’s diameter distribution after plasma treatment was narrowed down to the

average diameter of ~7nm. The distribution of plasma-treated AuNPs was broader than that

of AgNPs film, as shown in figure 4.5i and 4.5f. The further mechanism of plasma-induced

size reduction of Au and Ag NPs has been studied in the next sections.

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4.3 Surface charge measurements

Figure 4.6: Zeta potential distribution of (a) untreated and (b) plasma-treated (15

minutes) Ag NPs. The surface charge changes from +25.1 to −25.9 mV after

treatment[82].

The zeta potential changed from +25.1 ± 4 mV of the untreated sample to −25.9 ± 6

mV after 15 minutes of plasma treatment, as shown in Figure 4.6. The magnitude of the

zeta potential defines NP stability due to interparticle electrostatic repulsion. The high

magnitude of the untreated Ag NPs is due to the BPEI coating, which imparts a positive

charge. Similarly, the high magnitude after the treatment (with reversed polarity) ensures

the NPs remain stably dispersed. The observed change in the zeta potential and the possible

mechanism behind it has been discussed in section 4.5.

Zeta potential measurements were repeated for AuNPs before and after plasma

treatment, as shown in figure 4.7. The AuNPs have citrate capping on them as a

stabilization agent, and that gives NPs a negative zeta potential[87].

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Figure 4.7: Zeta potential measurements for a) untreated and b) plasma-treated AuNPs.

The surface charge changed from -20.9 mV to -43.9mV.

Zeta potential of untreated AuNPs was recorded as -20.9mV and that of plasma-

treated AuNPs as -43.9mV.

4.4 TEM and AFM imaging

Figure 4.8a and 4.8b show the TEM images of untreated and plasma-treated Ag NP

samples prepared on copper grids. The untreated NPs were agglomerated, but the plasma-

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treated NPs were well organized and dispersed. The NP size was reduced after the plasma

treatment and a surface oxide formed (Figure 4.8b). The diffraction pattern that is shown

in Figure 4.8(insets) reveals that the untreated NP film was polycrystalline with various

diffraction rings.

The diffraction pattern of the NP film tended towards single crystallinity because of

the un-piling of NPs as a result of plasma treatment. The surface roughness was quantified

for untreated and plasma-treated (15 minutes) Ag NPs, as shown in the inset of Figure 4.8c.

The plots of the roughness of untreated and treated samples show the increase in the

amplitude and the number of peaks after treatment for the same projected area. The

topology of the selected area (indicated by the white arrow) for the untreated sample is

shown in the inset of Figure 4.8d.

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Figure 4.8: TEM images and diffraction patterns (insets) of (a), (c) untreated and (b), (d)

plasma-treated Ag NPs. AFM images of (e) untreated and (f) plasma-treated Ag NP film;

the roughness and texture are shown in the insets[82].

The untreated sample shows a smooth topology, and the treated sample shows a

rough surface. The mean roughness changed from 1.12 to 1.4 nm, and the route mean

square roughness changed from 2.2 to 3.2 nm.

4.5 Mechanism of plasma-induced reduction in AgNP size

Our proposed mechanism of Ag NP surface modification due to the plasma treatment

is presented in Figure 4.10. The BPEI coating on the NPs provides electrosteric repulsion

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between particles and prevents agglomeration[88]. Because the BPEI electrosteric

stabilization involves electrostatic and steric bonding between the particle and polymer,

NPs acquires a positive surface charge[89] (as confirmed by zeta potential measurement in

Section 4.3). Figure 4.9 shows the structure of BPEI and positively charged AgNPs

formation using BPEI coating.

Figure 4.9: Representation of positively charged AgNPs using BPEI

Negatively charged ions are adsorbed on the surface of positively charged NPs in the

Stern potential region to form an electric double layer (EDL)[90]. It is believed that as the

thermal annealing process starts, the EDL that is built around the surface of the NPs breaks

and leaves a net positive surface charge. Because the BPEI coating has a flashpoint at 110

℃, the NP–polymer electrosteric bonds are broken by further annealing at 250 and

400 ℃[90].

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Figure 4.10: a) Proposed mechanism by which plasma treatment modifies Ag NP films.

The bottom panel b) is the SERS spectra of the untreated and plasma-treated NPs at 532-

nm excitation, which validates the hypothesis that removal of the BPEI coating and

subsequent surface oxide formation results in the observed size reduction[82].

In the case of plasma treatment, the energetic electrons (average energy ~1 eV)

provide enough energy to break the weak bonds between the BPEI polymer and the surface

of the NPs[91]. The Raman spectrum in Figure 4.10b shows intense peaks at 123.8 and

142.2 cm−1 attributed to the vibrational mode of the Ag lattice[92]. These peaks are

enhanced after plasma treatment, which validates our hypothesis that the coating is

removed from the NP surface during treatment.

The Raman spectrum shows an intense band of amine bending at 1600 cm−1, which

can be observed after 5 minutes of plasma treatment owing to the breaking of the amine

group of the BPEI coating from the NP surface[93]. The shift of this band to 1555 cm−1

attributed to the protonation of an amino group of PEI. Also, the bands at 1442 cm−1 and

1062 cm−1 are of the amine group of PEI. The Raman peaks at 791 cm−1 and 885 cm−1 are

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due to vibrations of the ethylene groups present[93]. The band at 942 cm−1 was enhanced

after plasma treatment until 15 minutes, which indicates the breaking of BPEI bonding and

removal of the coating from the NP surface. After 15 minutes, the intensity of this band

decreased owing to the formation of the surface oxide layer.

The SERS of BPEI is very weak at 532 nm excitation; hence, this excitation

wavelength was used to detect R6G peaks (as shown in Section 3.5) to study the structural

changes of the surface of the Ag NPs. Further, a new EDL is formed owing to the ions and

electrons present in the downstream plasma. For the CAP set-up used in this work, the

Debye length is ~ 75 µm[94]. Therefore, the potential developed between the surface and

the plasma is confined in the plasma sheath. It is much larger than the EDL formed on the

NP surface, as shown in figure 4.11. In the case of plasma treatment, the energetic (In cold

plasma, Te>Ti, which is the energy of electrons is higher than the ions present in the plasma)

electrons (average energy ~1 eV) provide enough energy to break the weak bonds between

the BPEI polymer and the surface of the NPs.

The electrons present in the plasma environment recombined with the surface charge

in the EDL, and then EDL broke. The surface redox reaction within the plasma shield is

shown in figure 4.11.

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Figure 4.11: Schematic representation of AgNPs surface reaction within the plasma

sheath.

In the case of plasma treatment, the energetic (In cold plasma, Te>Ti, which is the

energy of electrons is higher than the ions present in the plasma) electrons (average energy

~1 eV) provide enough energy to break the weak bonds between the BPEI polymer and

the surface of the NPs.

The energy provided by plasma treatment was sufficient to keep the surfaces of the

NPs separated by distances equal to the repulsive barrier. The plasma-treated NPs formed

an organized NP film on the substrate as they settled during deposition, under attractive

Van der Waals forces. Although some positive surface charges on Ag NPs are nullified by

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electrons, Ag+ also combines with the reactive oxygen species present in the plasma to

form AgO and Ag2O on the surface. Some Ag+ ions are also reduced by the plasma

electrons.

The Raman spectra in Figure 4.10 show a sharp peak at 240 cm−1, which is attributed

to the stretching of Ag–O bonds as the reactive oxygen species present in the plasma

environment (as shown in chapter 3) react with the surface of the Ag NPs[95]. The peaks

at 217 cm−1, 300 cm−1, and 490 cm−1 are due to O2 anions in the AgO and AgO2 lattice.

This phenomenon is evidenced by the formation of an oxide layer on the NP surface[92],

[96].

The low intensities of all these peaks show the partial oxide formation until 15

minutes of the treatment. After 15 minutes, higher intensities were observed, which are

attributed to asymmetric oxide layer formation on almost the entire NP surface. The ratio

of intensities of the Ag lattice vibrations at 123.8 cm−1 and 142.2 cm−1 to AgO at 240 cm−1

increased after 20 minutes of plasma treatment, indicating a higher coverage of the oxide

layer. Hence, changes in morphology and optical properties were observed until 15 minutes

of treatment.

The lower intensity of the SPR absorbance peak after plasma treatment, as shown in

Figure 4.1, can also be explained as a consequence of the decrease in the net positive charge

on the Ag NP surface after treatment[97]. The reduction in the NP size is also due to the

surface oxide layer. The negative charge density developed on the NPs is desorbed when

plasma species provide the appropriate activation energy leading to the sublimation of the

surface oxide[98].

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The minimum energy required to decompose the Ag2O layer is 1.25 eV, and the

reduction potential of Ag+ is 0.8 V[99]. The average electron energy in the downstream

plasma is 1 eV (see chapter 3), which is sufficient to remove the BPEI coating, form a

surface oxide, change the surface charge, and ultimately reduce the particle size. However,

the plasma energy is slightly lower than that required for decomposition of the Ag2O oxide

layer formed after 20 minutes of treatment, as shown in the Raman spectra in Figure 4.10.

4.6 SERS characterization of the plasma-treated and thermally treated AgNP film

Figures 4.12a and 4.12b show the SERS signals for R6G on the untreated sample and

after plasma treatment for 3, 5, 10, 15, 20, and 25 minutes. Prominent R6G peaks were

detected at 612 cm-1, 772 cm-1, 930 cm-1, 1084 cm-1, 1127 cm-1, 1187 cm-1, 1310 cm-1, 1364

cm-1, 1419 cm-1, 1507 cm-1, 1575 cm-1, and 1648 cm−1 for the untreated and plasma-treated

samples. The spectra also show additional peaks at 658 cm-1, 1084 cm-1, 1127 cm-1, 1275

cm-1, and 1595 cm−1 for the plasma-treated samples.

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Figure 4.12: SERS response of R6G on as-deposited and plasma-treated Ag NP films

on a silicon substrate. The R6G response is enhanced until 15 minutes of plasma

treatment. It is saturated because of the complete surface coverage with the oxide layer

after 15 mins of the treatment. The inset in (b) shows the enhancement factor of R6G as a

function of treatment time[82].

The spectra for samples treated from 3 to 15 minutes showed the sharpest lower-

intensity peaks. These peaks were suppressed in the untreated sample. Moreover, the

SERS signal at 1364 cm−1 after 15 minutes of treatment was ~120 times as high as that of

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the untreated film. The SERS enhancement shows the promising improvement of SERS

performance of plasma-treated Ag NP films.

The uneven redox reaction occurring on the NP surface, leading to the increased

surface roughness and hotspots created at nanogaps[100], also contributed to the SERS

enhancement. The untreated NPs agglomerated, resulting in lower SERS enhancement than

for the plasma-treated samples[100]. The inset of Figure 4.12b shows the change in

intensities of the SERS peaks for the untreated and treated samples.

The EF was calculated for the R6G peak at 612 cm−1 as 7.8 × 1014, and for 1364 cm−1

was calculated as 3.4 × 1014 for 15 minutes of plasma treatment. The EF was reduced after

20 minutes of treatment because of asymmetric or entire surface oxidation, as described in

Section 4.5. The enhancement in the Raman peaks of the R6G signal until 15 minutes of

plasma treatment with a subsequent decay validate the mechanism discussed in Section

4.5.

The thermally treated AgNPs at 400ͦC are compared with the plasma-treated. The

slight SERS enhancement (of ~ 5 folds) was observed after the treatment, but the intensity

of SERS peaks of R6G was not enhanced after further treatment, as shown in figure 4.13.

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The enhanced intensities R6G peaks after the treatment were observed to be almost

stable until 15 mins of thermal treatment, and after 20 mins, they have decreased. The

distinct peaks of R6G were observed even after 25 mins of the treatment. However, in

plasma treatment, the intensities were enhanced up to ~120 folds, and after 20 mins of

treatment, no distinct peaks were observed.

4.7 Stability of Plasma treated AgNPs film

The stability studies were performed for 15 mins of plasma-treated Ag NPs film and

the system found stable for around 96 hours (4 days), as shown in figure 4.14. A total of 5

silicon samples with plasma-treated NPs deposition were fabricated, and SERS signals for

R6G were recorded at the time, and the response was averaged out. The signal decayed

gradually after 96 hours after the treatment.

Figure 4.13: SERS response of R6G molecules on thermally treated AgNPs at 400֯C

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Figure 4.14: SERS response for R6G on 15 mins plasma-treated AgNPs recorded at a

different time interval at room temperature. The samples were stored at room temperature

(25̊C)

The results found that the plasma-treated AgNPs films were stable for 4 days and

hence have the potential to serve as efficient SERS substrates.

4.8 Mechanism of Plasma Induced reduction in AuNPs size

SERS responses of citrate capped AuNPs before and after plasma treatment were

recorded, as shown in figure 4.15. SERS signal for untreated AuNPs is shown in figure

4.15a.

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Figure 4.15: SERS of a) untreated b) 5 mins plasma-treated c) 10 mins plasma treated

and d) 15 mins plasma-treated AuNPs

Broad Raman peaks of citrate capping were identified at 770cm-1 and 850cm-1 in

untreated AuNPs[101]. The intensity of these Raman peaks was decreased after the plasma

treatment. The prominent Raman peaks at 718.3 cm-1, 899 cm-1, 1164 cm-1, 1220 cm-1,

1565 cm-1, and 1674 cm-1 are associated with AuNPs and increased intensity of which

attributes to the removal of citrate from the surface[102], [103].

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Based on the literature, the energy required to break the bonding between citrate

capping and AuNPs surface is 9kCal/mole, which is 0.387eV[104]. Hence, plasma

treatment could easily break this bond. Further plasma treatment induced redox reaction

on the surface of AuNPs, which can be seen in figure 4.15c and 4.15d, where Au-Ox peaks

at 491 cm-1 and 635 cm-1 to 677 cm-1 are present[105]. Raman peak at 590 cm-1 in figure

4.15d is attributed to the surface oxide species[106].

The reduction in the size of AuNPs due to the plasma treatment occurred as a result

of the removal of citrate capping and partial surface oxide formation. The energy required

for the Au oxidation reaction to happen is 1.42V[106], which is more than that needed for

AgO formation. However, the energy that is provided in plasma treatment is around 1eV,

which explains the broader size distribution and a comparatively smaller change in size

than AgNPs and partial surface oxide formation.

4.9 SERS characterization of the plasma-treated and thermally treated AuNP film

SERS responses of R6G were recorded on untreated and plasma-treated AuNPs films

to study the plasma-induced enhancement of optical properties of AuNPs, as shown in

figure 4.16. Prominent R6G peaks were detected at 612 cm−1, 658 cm−1, 772 cm−1, 930

cm−1, 1084 cm−1, 1127 cm−1, 1187 cm−1, 1275 cm−1, 1310 cm−1, 1364 cm−1, 1419 cm−1,

1507 cm−1, 1575 cm−1, 1595 cm−1, and 1648 cm−1 for the untreated and plasma-treated

samples.

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Figure 4.16: SERS of R6G on untreated (Black solid and inset) and 15 mins of plasma-

treated (Blue solid)

The intensity of peak at 1364 cm−1 was 1367 a.u., and it has been enhanced to

1.278x105 a.u., which is ~95 folds enhancement. The optical properties of AuNPs have

been improved after the plasma treatment due to the nanogaps present in AuNPs films,

removal of citrate capping, and size reduction.

4.10 Summary of chapter 4

In this work, the effects of low-temperature atmospheric plasma treatment of AgNP

and AuNP films were studied. The SEM and TEM imaging showed the reduction of the

nanoparticle size and well-dispersed film formation without any agglomeration after the

plasma treatment. The similar effects as of thermal annealing were achieved by the plasma

treatment at room temperature without processing the substrate at elevated temperature.

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The novel redox reaction, which results in the morphological changes in the AgNPs,

and AuNPs were found to be induced by the plasma treatment. The reactive oxygen species

in the plasma oxidized the outer surface layer of AgNPs and AuNPs. Plasma treated NPs

were well dispersed and had an uneven surface roughness, which provided unique features

for their use as ideal SERS substrates. The results obtained for the plasma treatment at

room temperature are comparable to the thermal treatment at higher temperatures. This

unique property can be used for processing metal nanoparticles at room temperatures for

designing sensitive SERS substrates on flexible materials such as polymers, papers, and

textiles. The surface charge tuning of AgNPs and AuNPs using the adapted plasma

treatment can be used in the electrochemical sensing as it increases the activity and affinity

towards the specific charged molecules.

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5 PLASMA INDUCED ENHANCEMENT IN ELECTROCHEMICAL PROPERTIES

OF PLASMONIC NANOPARTICLES

This chapter presents the study of plasma-induced enhancement of electrical

properties of Ag and Au NPs at room temperature. In this work, the enhancement in the

electrochemical performance of AgNPs and AuNPs due to plasma-assisted electrode

surface modification is demonstrated. The distribution of plasma-assisted aerosolized

deposited NPs in the previous section showed the promising architecture for surface

modification of the electrochemical sensor. The uniform film formation after the

aerosolized plasma-assisted deposition has improved the electrochemical sensing, which is

validated by detecting the cortisol molecules. Cortisol is a vital stress hormone, and its

level in many physiological functions such as fat mobilization for metabolism, immune

suppression, cardiovascular disease, autoimmune disorders, infectious diseases, and mental

illness makes it an essential biomarker in health-monitoring[107]. The enhancement in

electrochemical properties induced by plasma treatment was studied by analyzing CV, and

electrochemical impedance spectroscopy (EIS) of untreated and plasma-treated NPs

surface-modified electrodes.

5.1 Fabrication of AgNPs and AuNPs modified electrodes

The Zensor SPCEs from eDAQ were used to deposit AgNPs and AuNPs films using

drop-casting (untreated) and the experimental set-up described in chapter 3. SPCEs were

polished with 0.3 microns of alumina powder, applying circular motions on the polish

paper to smoothen the surface before NPs depositions. This deposition was confined to the

working electrode surface of SPCE. The Ag and AuNPs were deposited on SPCE with

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drop-casting, and plasma-assisted aerosolized NPs method for 5 mins by placing the

electrode below the plasma assembly, as shown in figure 5.1.

The SEM images of untreated SPCE with drop-casted NPs and aerosolized plasma-

assisted deposited NPs are shown in figure 5.1. A comparatively uniform NP distribution

is seen as drop-casted NPs.

Figure 5.1: Schematic illustration of plasma-assisted aerosolized NPs modified sensor

fabrication process and SEM images of morphological changes during the sensor

fabrication.

CV of these NPs modified electrodes were recorded for 10 μl Phosphate Buffer

Saline (PBS) (pH 7.4) containing 5 mM [Fe (CN)6]3-/4− to compare the electrochemical

properties.

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5.2 Surface functionalization of AgNPs and AuNPs based sensors for cortisol

measurement

DTSP, Sodium Borohydride, a monoclonal antibody (Anti-Cab), and cortisol were

purchased from Sigma-Aldrich. For further fabrication of DTSP SAM, antibody, and

cortisol detection, the established protocol from Khalid et al. [108] was used. PBS of pH

7.4 was prepared for this work, as it is suitable to presume higher biological activities

during electrochemical immunosensing[59]. Figure 5.2 shows the stepwise fabrication of

the cortisol sensor.

Figure 5.2: Schematic of stepwise fabrication of cortisol sensor

The binding of DTSP towards plasma-treated AuNPs and AgNPs was possible due

to the direct Sulphur bond with Ag and Au surface. The surface of Au NPs has citrate

capping, DTSP replaces citrate capping, and then antibodies bind with DTSP. DTSP was

reduced using Sodium Borohydride, the solution of which was prepared in DI water. The

NPs modified electrodes were then modified with DTSP-SAM fabrication by immersing

electrodes in the 2mg/mL solution of DTSP in acetone for 2 hours. Electrodes were cleaned

with acetone and DI water to remove the unbounded DTSP from the surface of the

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electrodes. Anti-Cab were then immobilized to form a bonding of its amino group with a

succinimidyl group of DTSP. The Ant-Cab/SAM/NPs modified electrodes were incubated

at room temperature for 2hours, followed by rinsing with PBS. Ethanolamine (EA) was

used as a blocking agent to block the nonbinding sites by incubating electrodes for 10 mins.

EA/Anti-Cab/SAM/NPs electrodes were then washed with PBS.

5.3 Electrochemical Measurements

The electrochemical measurements were conducted using a CHI 1230 analyzer using

surface-modified Zensor SPCEs to compare the electrochemical response of untreated and

plasma-treated Ag and AuNPs. 5mM Ferro/Ferricyanide K3[Fe (CN)6])/ K4[Fe (CN)6] (1:1,

5 mM) was prepared in a Phosphate buffer solution (pH-7.4) (PBS). The cyclic

voltammograms (CV) were obtained from a 5µl of K3[Fe (CN)6])/ K4[Fe (CN)6] at

50mV/sec scan rate. The standard cortisol solutions of 0.012 µg/dl, 0.037 µg/dl,

0.111µg/dl, 0.333 µg/dl and 1 µg/dl were prepared with Hydrocortisone-H0888 (cortisol)

that was purchased from Sigma Aldrich. The cyclic voltammograms were recorded after

adding 5ul of the different concentrations between -1 V to +1 V potential swing to detect

the corresponding oxidation and reduction current values using CHI 1230. The

Electrochemical Impedance Spectroscopy (EIS) was carried out for untreated and plasma-

treated AgNPs, AuNPs, and SAM/Antibody modified electrodes using Metrohm Autolab

analyzer in 5mM [Fe (CN)6]3−/4− containing 0.1M KCl solutions.

5.4 Electrochemical Performance of AgNPs modified electrode

The electrochemical performance of untreated and plasma-treated AgNPs modified

electrodes in 5mM K3[Fe (CN)6])/ K4[Fe (CN)6] (1:1) is shown in CV curves in figure 5.3.

As the redox reaction of [Fe (CN)6]3−/4− are quasi reversible, it was used to characterize the

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electrochemical properties of modified SPCEs[109]. The oxidation and reduction current

peak values of untreated AgNPs were lower as compared to that of plasma-treated AgNPs

functionalized SPCEs.

Figure 5.3: Electrochemical response of 5mM [Fe (CN)6]3−/4− using a) CV and b) EIS of

untreated and plasma-treated AgNPs modified electrodes.

The peak oxidation current, Ipa for plasma-treated AgNPs modified electrode, is

increased by 16.78%. This increase in peak current corresponds to the increase in the

electroactive surface area after the plasma treatment.

The formal potential of the Fe2+/3+ redox reaction was observed at E0’ 0.369 V. The

oxidation peak potential was observed for untreated AgNPs modified electrode at Epa

0.202 V and for plasma-treated AgNPs modified electrode at 0.052 V. The redox peaks for

untreated AgNPs modified electrodes were obtained at 0.202V and 0.016V. They were

shifted for plasma-treated AgNPs modified SPCEs to 0.047V and -0.094V, respectively.

This reduction in the difference of oxidization and reduction peaks showed that the rate of

electron transfer is increased.

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The increase in the oxidation peak signifies the increase in the active sites on the

plane of the working electrode. The uniform NP film on the electrode surface enables an

increased conductivity of the electrode. The untreated AgNPs modified SPCE showed a

prominent oxidation peak for Ag (Ag0 to Ag+) at 0.047V[110]. However, the shift and

suppressed Ag peak at -0.093V for plasma-treated AgNPs showed a smaller amount of

energy required to oxidize AgNPs as they were partially oxidized. The result indicates that

the current peak was enhanced because of plasma treatment. This electrochemical activity

enhancement of AgNPs is a result of the improved effective AgNPs sites formed on the

surface area of electrodes[111], [112].

Nyquist plots were used to analyze the EIS data, as shown in figure 5.3b with the

Randles equivalent circuit inset. The analyzed results provide the information on the

impedance changes of the electrode surface as the modification was carried out. The

semicircle observed in the plot is due to the parallel combination of electron transfer

resistance (Rct) and double-layer capacitance (Cdl), suggesting that the bare (black) and

AgNPs modified SPCEs (red) have a low conductive nature, which opposes the interfacial

charge transfer[81]. The linear portion, due to Warburg impedance (Zw), indicates the ion

exchange/diffusion in the electrolyte. The decrease in the diameter of the semicircle in

AgNPs and plasma-treated AgNPs indicates the higher conductivity and accelerated

electron transfer between the electrode surface and [Fe (CN)6]3−/4− than the bare electrode.

The increase in the electrochemical response was observed after the deposition of AgNPs

and plasma-treated AgNPs on SPCE. Rct of bare SPCE is ~ 0.25MΩ, of AgNPs, modified

SPCE is 4kΩ, and that of plasma-treated AgNPs modified SPCE is 1.2kΩ. The plasma-

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treated AgNPs modified electrode has the lowest and hence exhibit the highest conductivity

as compared to other electrodes.

5.5 Electrochemical Performance of AuNPs modified electrode

Figure 5.4 shows the electrochemical response of bare (black), AuNPs modified

SPCE (red), and plasma-treated AuNPs modified SPCE (blue).

Figure 5.4: Electrochemical response of 5mM [Fe (CN)6]3−/4− using a) CV and b) EIS of

untreated and plasma-treated AuNPs modified electrodes.

The peak oxidation current of 5mM of [Fe (CN)6]3−/4− for bare SPCE was observed

as 45µA, for untreated AuNPs modified SPCE as 49.5 µA, and that of for the plasma-

treated AuNPs modified SPCE as 64µA. This 29.3% of the increase in current attributed

to the enhancement in electrochemical properties of electrodes prepared by the plasma-

assisted NP deposition method. Moreover, it shows that improvement in the electroactive

surface area occurred due to plasma.

The separation potential of oxidation and reduction peaks was observed to be

decreased after the modification of SPCE. The separation potential for untreated AuNPs

modified SPCE is 0.577V, and it has been reduced to 0.173V for plasma-treated AuNPs

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modified SPCE. Similar to AgNPs, the CV showed an increase in the rate of reaction for

plasma-treated AuNPs.

Based on the EIS response of the same electrodes, as shown in figure 5.4, the radius

of the semicircle was reduced after the modification of SPCE. The value of Rct in the

equivalent Randle’s circuit was reduced from 0.23MΩ for bare SPCE to 8kΩ for untreated

AuNPs modified SPCE, and it was reduced to 280 Ω for plasma-assisted AuNPs modified

SPCE. The plasma-treated AuNPs modified SPCE has the lowest Rct, which is signified

for the highest conductivity.

The double-layer capacitor (Cdl) for the bare Au electrode was calculated as 16.7nF,

for AuNPs modified electrode, its 9.2μF, and for plasma-treated AuNPs modified

electrode, its 25 μF. The increase in the value of Cdl indicates the increase in charge transfer.

Due to the induced NPs on the surface of the electrodes, the active sites were increased. It

has led to the improved charge transfer and decreased in the impedance. Hence, at higher

frequencies, the value of the capacitor increased, and the capacitance was decreased.

The Constant phase element (CPE) is an imperfect capacitance, and it can be calculated as,

1 / Z = Y = Q° ( j ω)n (5.1)

where Q° has the numerical value of the admittance (1/ |Z|) at ω =1 rad/s. The units of Q°

are S·sn. The value of ‘n’ for the double layer capacitor is '1'. The logarithmic values of

imaginary parts were plotted against corresponding frequencies to find out the value of 'n'

for the above EIS responses[113], the slope of which gave the value of 'n,' as in figure 5.5.

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Figure 5.5: Evaluation of the power factor of the constant phase element of bare Au,

untreated AuNP modified, and plasma-assisted AuNPs modified electrodes

The value of the slope decreased after modification of electrodes with AuNPs, and

it decreased further for plasma-assisted AuNPs modified electrodes. The decrease in the

n attributed to the decrease in the phase and hence, the impedance. The impedance was

decreased for modified electrodes as the charge transfer increased. The value of

impedance is lowest for plasma-assisted AuNPs modified electrode.

5.6 Electrochemical detection of cortisol using modified SPCEs

Cortisol sensors using DTSP SAM and cortisol antibodies were fabricated as

described in section 5.2. EIS responses of each step of fabrication on AgNPs modified

SPCEs were recorded, as shown in figure 5.6. Figure 5.6 I a represents EIS of untreated

AgNPs and figure 5.6 I b, c and d are DTSP SAM/ untreated AgNPs, Anti-Cab/DTSP

SAM/untreated AgNPs and 0.012 µg/dl of cortisol/ Anti-Cab/DTSP SAM/untreated

AgNPs.

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Figure 5.6: EIS responses of I) untreated and III) plasma-assisted a) AgNPs, b) DTSP

SAM/AgNPs, c) Anti-Cab/DTSP SAM/AgNPs, d) 0.012 µg/dl Cortisol/ Anti-Cab/ DTSP

SAM/ AgNPs modified SPCE; and Linear calibration plot for II) Anti-Cab/DTSP

SAM/untreated AgNPs and II) Anti-Cab/DTSP SAM/plasma-assisted AgNPs modified

SPCE.

Rct of EIS for each step of fabrication has increased. This increase associated with

the resistance to the electron transfer in the electrolyte induced by the surface-modified

layers.

The peak current decreased as the concentration of the cortisol increases and showed

the linear relationship between the cortisol concentration and electroactive species surface

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coverage. The sensitivity of the cortisol sensor on untreated AgNPs is calculated as

6.08x10-6 A/(µg/dl).

Similarly, EIS response for the cortisol sensor using plasma-assisted AgNPs is shown

in figure 5.6 III. The increase in the radius of the semicircle was observed after each step

of the surface modification. EIS response of plasma-assisted sensor has shown more

prominent results as compared to the untreated sensor as broad semicircles were seen in

the sensor with untreated AgNPs. As the fabrication goes on, the surface blocks the electron

probe; hence an increase in the Rct is a validation of a fabrication process in plasma-assisted

AgNPs modified SPCE.

The weak binding of DTSP SAM on untreated AgNPs was indicated by the weak

EIS response for untreated AgNPs. Untreated AgNPs have BPEI coating on their surface,

and it was assumed that binding was taking place between the amine group of BPEI and

DTSP, as shown in figure 5.7[114]. Further, the sulfur bound with the AgNPs surface in

the presence of NaBH4. However, as BPEI coating is thick and binding of AgNPs surface

with DTSP is through its layer, weak affinity of surface modification results in the weak

response.

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Figure 5.7: Reaction scheme for the conversion of PEI primary amines with DSP. The

resulting disulfide bonds are easily cleaved by reducing agents, regenerated from [114]

The linearity of the calibration curve of the cortisol sensor on plasma-assisted AgNPs

modified SPCE was increased with the sensitivity of 6.32x10-6 A/(µg/dl). The standard

deviation has also been decreased in plasma-assisted AgNPs modified SPCE.

Furthermore, the cortisol sensor fabrication was done on AuNPs modified SPCE, and

plasma-assisted AuNPs modified SPCEs. The EIS response after each step of surface

modification is shown in figure 5.8. The sequential increase in the Rct with every stage of

surface architecture alteration shows the absorption of DTSP SAM and antibodies on the

NPs sites provided on modified SPCEs.

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Figure 5.8: EIS responses of I) untreated and III) plasma-assisted a) AuNPs, b) DTSP

SAM/AuNPs, c) Anti-Cab/DTSP SAM/AuNPs, d) 0.012 µg/dl Cortisol/ Anti-Cab/ DTSP

SAM/ AuNPs modified SPCE; and Linear calibration plot for II) Anti-Cab/DTSP

SAM/untreated AuNPs and II) Anti-Cab/DTSP SAM/plasma-assisted AuNPs modified

SPCE.

The Rct was strengthened from 8kΩ to 13kΩ for DTSP SAM on untreated AuNPs. It

was further increased to 15kΩ for the Anti-Cab/DTSP SAM/untreated AuNPs

functionalized electrode. Rct is 17kΩ for 0.012µg/dl of cortisol/Anti-Cab/DTSP

SAM/untreated AuNPs modified SPCE. The linear regression curve for various

concentrations of cortisol is shown in figure 5.8 II.

The sensitivity of Anti-Cab/DTSP SAM/untreated AuNPs modified SPCE was found

as 9.38x10-6 A/(µg/dl). In case of the cortisol sensors that were fabricated on plasma-

assisted AuNPs modified SPCE, Rct was 300Ω for plasma-assisted AuNPs, 10kΩ for

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DTSP SAM/plasma-assisted AuNPs, 21kΩ for Anti-Cab/DTSP SAM/plasma-assisted

AuNPs, and 30kΩ for 0.012 µg/dl of cortisol/Anti-Cab/DTSP SAM/plasma-assisted

AuNPs modified SPCE.

From figure 5.8 IV of the response of the concentration of cortisol, the sensitivity of

Anti-Cab/DTSP SAM/plasma-assisted AuNPs modified SPCE was evaluated as 1.138x10-

5A/(µg/dl) with improved linearity. The responses of plasma-assisted AuNPs in the

fabrication of the cortisol sensor have shown promising results, which can be used to

enhance the detection. An active surface alteration for cortisol sensors on AuNPs was

observed due to the replacement of citrate capping with the thiol group of DTSP, which

has a strong affinity towards the surface of AuNPs[115], [116]. Also, this binding took

place directly on the surface on AuNPs, unlike AgNPs, as explained before. Moreover, the

ultrathin layer of citrate and ionic interaction of AuNPs with antibodies resulted in efficient

detection.

After the NPs are treated with the plasma, the oxygen species present react with them

to form a surface oxide, and the size of NPs gets decreased. Ultimately, it increased the

surface energy of NPs to enhance the electrochemical properties. The particles then

arranged themselves to provide better surface coverage. Therefore, the study confirmed the

effective cortisol detection using the plasma-treated NPs modified SPCE.

The plasma-assisted AuNPs showing more enhanced sensitivity towards the cortisol

molecules than the others, proved the added surface excitation to NPs by plasma treatment.

The effective detection of cortisol was observed for AuNPs than AgNPs due to the weak

binding of SAM and antibodies on the AgNPs surface. Hence, further studies were carried

out with the same protocol of cortisol sensor fabrication that was used using AuNPs.

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5.7 Summary of chapter 5

AuNPs and AgNPs deposition using plasma treatment is successfully demonstrated

at room temperature. The CV and EIS results showed the enhancement in the electrical

conductivity in plasma-treated NPs modified electrodes. The plasma treatment induced the

increase in the electrical sensitivity of the modified electrodes, which was suitable for

effective electrochemical detection. Besides, this method offers the approach in sensor

fabrication towards improvising the biomolecule detection. The desired material deposition

and plasma treatment can be achieved on the flexible substrates for various applications as

electrochemical sensors, supercapacitors, batteries, and POC systems.

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6 UNDERSTANDING THE RELATION BETWEEN NANOPARTICLE

DISTRIBUTION AND ELECTRODE RESPONSE

Surface architecture-based electrochemical biosensors work on a complex structure

that is created to capture probes. A small defect in the architecture can cause an error in

probe-target interaction. In such electrochemical sensors, the primary assumption that is

made is that the sensor has an ideal architecture. Nevertheless, these sensors drive in the

detection of analytes as the focus is on the probe-target interaction. The underlying surface

properties due to the resultant architecture are yet to be considered while analyzing the

detection phenomenon. Hence, the assessment of the dependence of the sensing process on

the non-idealities present is required to achieve reliable measurements.

Considering NPs as a fundamental layer in surface modification, this work focuses

on the NP distribution on the sensing surface. In this work, the correlation of NP

distribution with electroactive surface area, separation potential of oxidation and reduction

peak, and size of NPs are achieved. Three methods of deposition of three different sizes

of AuNPs as 20nm, 40nm, and 60nm were chosen as drop-casting, aerosol deposition, and

plasma-assisted aerosolized deposition.

In this work, evaporated Au electrodes are fabricated on a silicon substrate, as

described in chapter 3. The enhancement in the sensitivity of biosensors to improve their

performance has been achieved by increasing the active surface area using nanoparticle

(NP)-functionalized electrodes as they exhibit higher electric field intensities [2]. Based on

the previous results, plasma-assisted NP functionalized electrodes displayed higher

electrochemical performance. This improved electrochemical response is due to plasma-

assisted surface activation of NPs. However, as the improved NP coverage was observed

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in plasma-assisted NP deposition, this might also be the reason for the enhancement of the

electrochemical properties, electroactive surface area, and sensitivity.

The sensor response depends on the size, shape, and surface attachment of

nanoparticles [9]–[11]. In most of the sensing methods, the use of high-density NP causes

polydispersity, and defects due to orientation on the electrode surface that conceal the

efficacy of these factors in biosensing[13], [14]. In the intent to achieve a more reliable

surface structure, Au electrodes were fabricated using photolithography, RIE, and

evaporation techniques. The surface roughness and effective surface area in SPCEs depend

on the polishing process and hence vary from the electrode to electrode. Though defects

are still present in the microfabrication process, the surface was assumed to be less distinct

than other electrodes because of the bulk processing.

The inter-electrode variation of fabricated electrodes without any surface

modification has also been taken into consideration in this work. These NP functionalized

electrodes were then modified using DTSP SAM and cortisol antibodies to make them

selective towards cortisol molecules. The stepwise NP surface functionalization using

SAM and antibodies has been described in chapter 5. The enhancement in the ESA and

sensitivity was observed for plasma-assisted AuNPs functionalized electrodes.

6.1 Methods

Gold electrodes on Si/SiO2 substrate are fabricated using the evaporation technique,

as explained in chapter 3. Au NPs of an average size of 20 nm, 40nm, and 60nm were

purchased from Sigma Aldrich. AuNPs were then deposited on fabricated electrodes using

drop-casting, aerosolized, and plasma-assisted aerosolized deposition[53]. These methods

of deposition have been explained in chapter 3. Cyclic voltammetry responses of Au

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electrodes with and without surface modification were recorded using a CHI1023

Instrument. These results were obtained using a cyclic voltammogram obtained in 5mM

[Fe (CN)6]3−/4−. 5mM Ferro/Ferricyanide K3[Fe (CN)6])/ K4[Fe (CN)6] (1:1, 5 mM) was

prepared in a Phosphate buffer solution (pH-7.4) (PBS). CV curves were obtained from a

3µl of K3[Fe (CN)6])/ K4[Fe (CN)6] at 50mV/sec scan rate.

The standard cortisol solutions of 0.012 µg/dl, 0.037 µg/dl, 0.111µg/dl, 0.333 µg/dl

and 1 µg/dl were prepared with Hydrocortisone-H0888 (cortisol) that was purchased from

Sigma Aldrich. CV curves were recorded after adding 3µl of the different concentrations

between -1 V to +1 V potential swing to detect the corresponding oxidation and reduction

of current values using CHI 1230. EIS responses were carried out for AuNPs modified

electrodes using Metrohm Autolab analyzer in 5mM [Fe (CN)6]3−/4− containing 0.1M KCl

solutions.

6.2 Electroactive surface area from the electrochemical response

ESA of the three-electrode electrochemical cell can be calculated using its CV

response. Using Randles and Sevcik equation electrochemical characterization of [Fe

(CN)6]3−/4− oxidation reaction can be done on the working electrode as the oxidation of [Fe

(CN)6]3−/4− has good reversibility[117].

[Fe (CN)6]4− Oxidation [Fe (CN)6]

3− (6.1)

The oxidation reaction is controlled by the diffusion of Ferrocyanide ions. Hence,

the peak current obtained from this reaction is proportional to the electroactive surface area

of the working electrode. This proportionality can be evaluated from Randles and Sevcik

equation as[118],

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𝐼𝑝 = 268600 𝑛3/2𝐷1/2𝐶𝑉1/2𝐴 (6.2)

Where, Ip - the peak oxidation current in A

n - the number of electrons transferred in the redox reaction

A - the electroactive surface area in cm2

D - the diffusion coefficient

C - the concentration in mol/cm3

V - the scan rate in V/sec.

Here, n=1, D=7.20x10-6 for Ferrocyanide, C=5x10-6 mol/cm3 as 5mM of [Fe

(CN)6]3−/4−, and V=0.1V/sec. In this work, the peak current was obtained from the CV

response. Hence, the ESA can be evaluated as,

𝐴 = 877.51 𝐼𝑝 cm2 (6.3)

ESA for the electrodes with and without surface modification was calculated to

establish the correlation between the deposition method, size of NPs, and ESA.

6.3 Image quantification of AuNPs modified electroactive surface area

The particle density and their size distribution were quantified using SEM and TEM

image analysis. Real surface area can be evaluated using image processing as follows.

The average number density of AuNPs ρ was estimated by dividing the particle

number that was found using software ImageJ by the image area, as shown in figure 6.1.

The average diameter of AuNPs was used to calculate the area of AuNPs as AAuNP = 4π r2.

Ageo is the geometrical area of the working electrode. The surface area is estimated as[119],

A= ρ AAuNP Ageo cm2 (6.4)

The geometric area of the Au electrode without surface modification is calculated as

Ageo is 0.0335cm2 of a working area.

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Figure 6.1: a) SEM image of AuNPs modified electrode, b) NPs outline detection using

image processing, and c) distribution of the size of NPs.

From the image quantification, ρ= 1.12x1011 AuNPs/cm2, and AAuNP = 8.04x10-12

cm2/AuNPs, and therefore, an effective surface area that was calculated theoretically is,

A= 0.028 cm2.

6.4 TEM images

TEM images of 20nm AuNPs deposited by drop-casting, aerosol, and plasma-

assisted aerosol are shown in figure 6.2 a, b, and c, respectively.

Figure 6.2: TEM images of a) drop-casted, b) aerosolized, and c) plasma-assisted

aerosolized deposited AuNPs of the average size of 20nm.

From TEM images, it can be seen that the distribution of NPs varies drastically for

the deposition methods. In drop-casted AuNPs, agglomeration was observed, where for

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aerosolized deposition, partial surface coverage was seen. This random arrangement of

NPs made the quantification of particle density using image processing difficult. However,

in plasma-assisted AuNPs deposition, the comparatively uniform monolayer of NPs with

size reduction was observed. The phenomenon of plasma-induced size reduction of NPs

has been explained in chapter 4.

The electrodes were functionalized with 20nm, 40nm, and 60nm sized AuNPs to

analyze the effects of the surface coverage obtained using drop-casting, aerosol deposition,

and plasma-assisted aerosolized deposition methods.

6.5 Nanoparticle deposition model

The Nanoparticle deposition depends on the EDL force of repulsion between the two

adjacent particles. The arrangement obtained after the deposition depends on this repulsive

barrier. The interparticle EDL interaction is given by the equation 6.5,

𝑊𝐸𝐷𝐿 = 2𝜋 𝑟 Ԑ0 Ԑ𝑟 ᴪ𝑝2

𝑒𝑥𝑝(−𝑘 𝑆) (6.5)

Where, WEDL - Electrostatic double layer

S - Inter-particle distance (Center to center)

ψp - diffuse potential (zeta potential)

Ԑr - the relative permittivity of the medium

r - the radius of the particles

k - the inverse Debye screening length

The probability of finding a particle next to another particle at distance S is,

𝑃𝑝 = 𝑒𝑥𝑝(𝑊𝐸𝐷𝐿/𝑘𝐵𝑇) (6.6)

Where, kB - Boltzmann's constant

T - the absolute temperature

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Based on the zeta potential and NPs size measurements, the probability of having the

adjacent particle at the separation distance was plotted for untreated (aerosolized

deposition) and plasma-assisted deposition, as shown in Figure 6.3, considering the

extended RSA model of NPs deposition. The separation distance was varied to analyze the

probability of having the agglomeration.

Figure 6.3: AuNPs deposition model for untreated AuNPs [a, b, c] and plasma treated

AuNPs [d, e, f] for various inter-particel separation distance (S).

The plots showed that the probability of having the next particle at 7nm for NPs

10nm of the radius is 96%, which implied the probability of having the agglomeration. The

probability decreased as the size of NPs increased. However, the overall range of

probability is between 86-96%. The further increase in the probability with the increase in

the separation distance implies the definite presence of the particle at the specific separation

distance. In the plasma-assisted deposition, the probability of having the adjacent particle

at distance 7nm, for NPs of 10nm of the radius is 78%, 20nm of the radius is 65%, and 30

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nm of the radius is 48%. The probability increased as the separation distance increased but

not as compared to that of untreated deposition. Hence, the probability of having

agglomerated NP arrangement is lower in plasma-assisted deposition than that of the

aerosolized untreated NPs deposition. Based on the Images, zeta potential results, and this

model, it is proved that the arrangement we get for the plasma-treated NP deposition is

uniform with keeping the separation distance.

6.6 Electrochemical characterization of AuNPs modified Au Electrode

6.6.1 CV characterization

AuNPs of the average size of 20nm, 40nm, and 60nm were deposited on the Au

electrode and CV responses of these electrodes in 5mM of [Fe (CN)6]3−/4− were compared

for drop-casted, aerosol deposition, and plasma-assisted aerosolized deposition of AuNPs.

Figure 6.4a showed the averaged CV response for 20nm, 40nm, and 60nm of AuNPs

modified electrodes using drop-casting.

The peak current was increased after 20nm of NPs deposition, as can be seen in figure

6.4b. This increase in peak current was evaluated as 9% of that of the bare electrode.

Further, it was reduced to 0.5%, and then it was increased by 18.5% of the bare electrode

for 40nm and 60nm of NPs, respectively. In the aerosolized deposition, the enhancement

in the peak current with the change in the NPs size was 15.3%, 12.5%, and 7.6% of bare

electrodes. However, for plasma-assisted aerosolized deposition, it was observed as 94%,

41.5%, and 30% of that of the bare electrode. The enhancement factor that was obtained in

plasma-assisted NPs modified electrodes showed the pattern of increase in ESA for 20nm,

40nm, and 60nm AuNPs modified electrodes. There is no such pattern observed in the

drop-casted, and the smaller enhancement was observed in aerosolized deposition than the

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electrodes prepared by plasma-assisted deposition. The increase in peak current

corresponds to the increase in ESA, as expressed in equation 6.2.

Figure 6.4: CV responses of 20nm, 40nm, and 60nm of AuNPs modified electrodes with

a) drop-casting, b) aerosol deposition, and c) plasma-assisted aerosolized deposition.

TEM images of d) drop-casted, e) aerosol deposition, and f) plasma-assisted aerosolized

AuNPs.

Hence, the consistent increase in ESA was observed in the electrodes with plasma-

assisted deposition of AuNPs, whereas the random increase and decrease in ESA were

observed in the drop-casted deposition. In the aerosolized deposition, the ESA was

decreased as the size of NPs increased.

The separation of oxidation and reduction peak, ΔE in the CV response, was

observed as 0.45V for 20nm, 0.37V for 40nm, and 0.508V for 60nm drop-casted AuNPs

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modified electrodes. This ΔE was reduced to 0.242V for 20nm, 0.38V for 40nm, and

0.428V for 60nm for aerosolized AuNPs modified electrodes. ΔE was improved to 0.221V

for 20nm, 0.30V for 40nm, and 0.287 for 60nm for plasma-assisted electrodes.

This decrease in separation between Faradaic peak potential after the NP deposition

is attributed to the facilitation of the electron transfer reaction, kinetic, and thermodynamic

at the electrode surface[79]. To establish the correlation between the size, ESA, and ΔE of

NPs modified electrodes, 15 electrodes for each method and size were fabricated.

6.6.2 EIS characterization

EIS responses of AuNPs modified electrodes are shown in figure 6.5. The semicircle

of EIS decreased for the NP modified electrodes, which suggested the increase in the

electrochemical activities due to the presence of NPs on the electrode surface. This increase

in the conductivity also indicates that the ESA of the electrodes was increased after NP

deposition.

The value of electron transfer resistance for the bare Au electrode is 40 kΩ, as seen

in figure 6.5. In drop-casted electrodes, the value of Rct varies as the size of NPs varies. Rct

for 20nm of AuNPs is 22kΩ, for 40nm 28.5kΩ, and 60nm is 19kΩ. Since the distribution

of NPs that was obtained using drop-casting has no control over the resultant film

formation on the electrode, this variation occurred. Similarly, the average deviation of the

value of Rct for aerosolized AuNPs deposited electrodes was 6kΩ, and that of for the

plasma-assisted AuNPs was reduced to 0.6kΩ. The variation of Rct confirmed the effects

of the deposition method and NP size variation on the conductivity of the electrodes.

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Figure 6.5: EIS responses of a) drop-casted, b) aerosolized, and c) plasma-assisted

aerosolized AuNPs modified Au electrodes in 5mM of [Fe (CN)6]3−/4−, and d) the

variation in electron transfer resistance due to the deposition methods and variation in

size of NPs.

Considering the ideal distribution of NPs on the confined surface area, ESA should

vary as the size of NPs, as based on the theoretical calculations, when the size of NPs

increases, the density per unit area decreases to achieve the monolayer of NPs. Hence,

plasma-assisted distribution showed promising results in enhancing the effective ESA of

the electrodes.

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6.7 Inter-Electrode Variability of methods of deposition

ESA and ΔE for 15 electrodes of each size for three deposition methods were

quantified and compared, as shown in figure 6.6.

Figure 6.6: Boxplots of the spread of the distribution of a) ESA and b) ΔE of bare Au

electrodes, drop-casted, aerosolized, and plasma-assisted aerosolized deposited AuNPs

functionalized electrodes.

The quantification of the spread of ESA in bare Au electrodes showed the median

ESA at 0.012cm2, and the spread is between 0.0085 cm2 and 0.018 cm2. It showed that the

ESA for bare electrodes is less than the theoretical value. Though the controlled

microfabrication process was used, the inter-electrode variability was observed. This

variability might be due to the surface defects present on the electrodes.

The boxplot of drop-casted AuNPs modified Au electrodes has a median value of

ESA at 0.0165 cm2, which validated the increase in ESA after AuNPs deposition. However,

the spread of this plot was from 0.006 cm2 to 0.028 cm2. Therefore, though there was an

increase in the ESA after drop-casting, the inter-electrode variability was also increased.

Simultaneously, the median value of ESA for aerosolized deposition was noticed at 0.0165

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cm2, which was the same as drop-casted electrodes. However, the spread of aerosolized

deposited AuNPs electrodes was between 0.012 cm2 and 0.022 cm2, which was narrower

than the drop-casted one; therefore, it improved the inter-electrode variability. Plasma-

assisted AuNPs modified electrodes displayed a median value of ESA at 0.02 cm2, and the

spread was observed between 0.019 cm2 and 0.021 cm2. Hence, improved inter-electrode

variability and ESA were observed for plasma-assisted AuNPs deposited electrodes.

The spread of ΔE for bare Au electrodes lay between 0.42mV to 0.93mV with a

median value of 0.8mV. It varies from 0.23mV to 0.7 mV with a median value at 0.5mV

for drop-casted, from 0.22mV to 0.48mV with a median value at 0.35mV for aerosolized,

and from 0.25mV to 0.3mV with a median at 0.27mV for plasma-assisted AuNPs modified

Au electrodes. The variability was improved after plasma-assisted deposition, and the

decrease in ΔE indicated the potential of this method.

The architecture of the bare electrode for 20, 40, and 60nm was different as separate

electrodes were used. Hence, the architecture variation for bare added up with the variation

due to plasma-assisted 60nm NP modified electrode. The separation potential for a set of

15 electrodes for different sizes of AuNPs is plotted in figure 6.7. It shows the increase in

the separation potential with an increase in the size of NPs.

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Figure 6.7: Variation of separation potentials for drop-casted, aerosolized, and plasma-

assisted AuNPs with the size of AuNPs

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The boxplot showed the variability and spread of the separation potential for plasma-

assisted 20, 40, and 60nm AuNPs modified electrodes. The spread was decreased the

plasma-assisted electrodes as compared to that of drop-casted and the aerosolized

deposition. The pattern can be seen in the change in ΔE in AuNPs modified electrodes with

the plasma-assisted deposition.

6.8 The relation between the Size of NPs and ESA

In the efforts to establish the correlation between the size of NPs and ESA of

respective modified electrodes, the size of NPs was plotted against ESA for all three

methods, as shown in figure 6.8.

The relation between the size of NPs (d) and ESA for drop-casted electrodes is shown

in figure 6.8a as

d= -0.64 ESA, which shows a low negative correlation. The lower negative

correlation that was obtained for the aerosolized deposition method is d= -0.39 ESA. Also,

the range of variation in the ESA is between 0.0095 cm2 to 0.03 cm2 for drop-casted

deposition.

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Figure 6.8: Correlation plot of the size of NPs and ESA for a) drop-casted, b)

aerosolized, and c) plasma-assisted aerosolized NPs modified electrodes. d) Relative plot

of ESA to bare Au electrodes, 20A,40Aand 60A-20nm,40nm and 60nm aerosolized

deposited; 20d, 40d, and 60d- 20nm, 40nm, and 60nm drop-casted; 20P, 40P, and 60P-

20nm, 40nm, and 60nm plasma-assisted aerosolized AuNPs modified electrodes

For aerosolized deposition, this range of variation was narrowed down from 0.01 cm2

to 0.022 cm2. It was further narrowed down from 0.018 cm2 to 0.0215 cm2 for the plasma-

assisted deposition.

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The strong positive correlation, as expressed in equation 6.5, was achieved for the

plasma-assisted deposition method, which shows that with the increase in size, the ESA

increases.

𝑑 = 0.83 𝐸𝑆𝐴 (6.7)

However, the increase in ESA with the size of NPs was ~4% of the mean value,

which is due to more surface coverage with larger NPs. Plasma -assisted AuNPs modified

electrodes showed a strong correlation with smaller variations in overall ESA with the

change in the size of AuNPs. This variation occurred, possibly with the interparticle

distance variation due to the plasma-induced size reduction, as discussed in chapter 4.

Hence, to study the variation of ESA for the same size of NPs, the size of the NP was kept

constant, and the deposition method varied.

The dot plot of ESA against various sizes of NPs for all three methods is shown in

figure 6.8d. It can be seen that ESA for plasma-assisted NPs of 20nm, 40nm, and 60nm

overlapped and showed almost negligible variation, while other methods showed

substantial variation in ESA. Hence, the inter-electrode variability was improved using

plasma-assisted AuNPs deposition on electrodes by achieving nearly equal ESA.

6.9 Cortisol detection using AuNPs modified Au electrodes

The surface of bare Au electrodes, drop-casted, aerosolized, and plasma-assisted

aerosolized AuNPs modified electrodes using DTSP SAM, and cortisol antibodies were

functionalized, as explained in chapter 5. The decrease in the oxidation peak current in the

CV in figure 6.9a is a validation of the stepwise modification of surface architecture using

DTSP SAM and antibodies.

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The linear calibration for the bare Au electrode is shown in figure 6.9b, which shows

the sensitivity of 5.55x10-7A/(µg/dl). The calibration plots for different concentrations of

cortisol for the electrodes modified with all three deposition methods with 20nm, 40nm,

and 60nm of AuNPs are shown in figure 6.9c, 6.9d, 6.9e, 6.9f, 6.9g, 6.9h, 6.9i, 6.9j, and

6.9k.

The sensitivity of the cortisol sensor was increased to 1.41x10-6A/(µg/dl), 1.56 x10-

6A/(µg/dl), and 2.104 x10-6A/(µg/dl) for 20nm, 40nm and 60nm of sensor fabricated with

drop-casted AuNPs modified electrodes respectively. It was further improved to 2.41 x10-

6A/(µg/dl), 2.15 x10-6A/(µg/dl), and 2.37 x10-6A/(µg/dl) for cortisol sensor with 20nm,

40nm, and 60nm of aerosolized AuNPs functionalized electrodes respectively.

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Figure 6.9: a) CV shows a stepwise surface modification in 5mM of [Fe (CN)6]3−/4− and

linear calibration plots with the sensitivity of Anti-Cab/DTSP SAM functionalized on b)

bare Au electrode, c) 20nm, d) 40nm, and e) 60nm of drop-casted; f) 20nm, g) 40nm, and

h) 60nm of aerosolized; and i) 20nm, j) 40nm, and k) 60nm of plasma-assisted

aerosolized AuNPs modified electrodes.

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Furthermore, the sensitivity of plasma-assisted aerosolized AuNPs modified

electrodes was enhanced to 3.49 x10-6A/(µg/dl), 2.56 x10-6A/(µg/dl), and 2.45 x10-6

A/(µg/dl) corresponding to 20nm, 40nm, and 60nm.

The enhancement in the linearity and sensitivity of the response of the cortisol sensor

in plasma-assisted electrodes was a result of controlled ESA and hence controlled binding

sites as compared to other methods.

6.10 Summary of chapter 6

The peak current in electrochemical responses has been enhanced after the deposition

of Au NPs. The decrease in separation between Faradaic peak potential after the NP

deposition validates the higher electron transfer at the electrode surface for plasma-assisted

AuNPs deposited electrodes. The enhancement in the overall response of the electrode and

the active surface area was achieved with comparatively even distribution of nanoparticles

during plasma-assisted aerosolized deposition.

The NPs were sonicated properly during deposition using nebulizer in the assembly,

and the plasma provided the surface excitation and surface charge tuning during plasma-

assisted deposition. The promising results shown in this work have improved the variability

of the sensors by quantifying the NP coverage on the electrode surface. The supervised

interparticle and NP-electrode interaction helps improve biosensing as demonstrated in this

work by achieving the desired surface architecture during optimization of the sensing

process. This work would make the data interpretation more reliable.

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7 SUMMARY AND FUTURE WORK

7.1 Summary

In summary, a novel technique of plasma-assisted NPs deposition, which could

maintain the inter-particle repulsive barrier to obtain the uniform monolayer of NPs was

reported. This barrier was achieved due to the sonication NPs during deposition using

nebulizer in assembly and providing the plasma for surface excitation and surface charge

tuning of NPs during plasma-assisted deposition.

The NP films obtained from the plasma treatment at room temperature are

comparable to the thermal treatment at higher temperatures. This unique property can be

used for processing metal nanoparticles at room temperatures for designing sensitive SERS

substrates on flexible materials such as polymers, papers, and textiles. The intensity of the

SERS signal of R6G from plasma-treated AgNPs substrate was ~120 times as high as that

of the untreated substrate, and that of AuNPs was ~95 times. However, the enhancement

in thermal annealed NPs SERS substrate was ~7 folds.

The significant enhancement in plasma-assisted SERS substrate is attributed to the

surface roughness, presence of the nanogaps, and size reduction after the treatment of

nanoparticles on the substrates. These surface modifications due to the atmospheric plasma

of plasmonic NPs were studied, and the decrease in the size of NPs was quantified through

SEM, TEM, and AFM imaging. The summarized study of the effects of the plasma

treatment on the NPs is shown in figure 7.1.

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Figure 7.1: Summarized effects of plasma-assisted NP deposition methods

Analytical modeling of the effects of plasma on optical and electrochemical

performances of electrodes that were made by the proposed method was done to establish

a relation between deposition parameters and the response of the electrodes. The surface

charge tuning of NPs induced by the plasma and its mechanism was studied using SERS

signals captured after each step of the plasma. The microelectrodes were fabricated using

photolithography, Cr/Au evaporation, and lift-off techniques.

The enhancement in electrochemical properties induced by plasma treatment was

studied by analyzing the CV and EIS of untreated and plasma-treated NPs surface-modified

electrodes. A comparative study of electrochemical performances of microelectrodes

functionalized with 20nm, 40nm, and 60nm AuNPs was performed. Improved

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electrochemical properties with enhancements in the electroactive surface area and charge

transfer were reported in plasma-assisted NPs modified electrodes.

The inter-electrode variability of electrodes with each combination of deposition

methods and the size of NPs was evaluated. The improved inter-electrode variability was

achieved. These electrodes were used to detect cortisol, and the enhancement in the

sensitivity was achieved in plasma-assisted NPs modified electrodes. The surface charge

tuning of AgNPs and AuNPs using the adapted plasma treatment can be used in the

electrochemical sensing as it increases the activity and affinity towards the specific charged

molecules.

7.2 Future Work

The plasma-assisted deposition techniques have become the area of interest in

nanotechnology. However, this field contained a lack of knowledge regarding the effects

of plasma on the morphology, optical, and electrical properties of NPs. In this work, the

plasma-induced modification of the properties of NPs was studied. The relation between

the NPs distribution and the response of their functionalized electrode was also established.

However, with regard to continuing the current work, the following points should be taken

into consideration:

1. The preliminary work in understanding the mechanism of plasma-induced

excitation in AuNPs and AuNPs has been done. A similar mechanism for other NPs

than plasmonic like oxide and polymer NPs would be interesting to explore.

2. The morphological and optical effects of plasma treatment on non-spherical

particles such as stars, rods, are worth being studied. The energy optimization can

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be done for different substrates like polymers, papers, textiles, as this work has been

performed on silicon, quartz, and thin-film metal.

3. The comparison of plasma treatment with thermal annealing promises to achieve

similar effects at room temperature. The energy comparison in the two processes

can be made to match their impacts on the substrate modifications.

4. The findings that SERS measurement becomes ineffective after long plasma

treatment due to Ag-oxide formation on the AgNP after 20min, which damps the

signal, is a significant result, which shows the limitation of the current plasma

process. This behavior can be considered further for other NPs to obtain their point

of saturation.

5. The preliminary relation between the size of NPs and ESA for deposition methods

has been established in this work. The dataset of 15 of each variation was used

towards the lowest requirement to build a correlation. However, to achieve a strong

correlation with the optimized system, a larger dataset is recommended.

6. The study of NPs arrays by varying the duration of the treatment has yet to be done

considering the inter-particle distance. The mapping of NPs can be used to build a

prediction model, from which the real physical area of the NP deposited electrode

can be evaluated and related to the ESA.

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APPENDICES

I The binding force between the nanoparticles and the surface:

The Van der Waals force of attraction between the particles and the substrate,

𝑊𝑉𝑑𝑊 = −𝐴𝑟/6𝐷

Where A- The Hamaker constant (the number densities of the two interacting particles

and the magnitude of this constant implies the strength of the WvdW force between a

particle and a substrate.)

.

From the literature, the Hamaker constant is, A= 1.5 × 10−20 J.

D is the distance between the particle and substrate and

r = 10 nm the particle radius.

- WvdW = −Ar/6D

- W2=((-1.5e-20 r)/6 r);

- Pp=exp(-Wvdw/(1.6e-19)) = 1;

As D=r (the distance between the center of the particle and the substrate), the force of

attraction between particles and substrate is strong. 'Pp' is the probability of having a

secure binding between the particle and the substrate. Also, from the extended RSA

model shown in the figure below, it can be seen that as D decreases, the WvdW tends to

increase drastically.

Page 142

123

Figure: RSA model of NPs deposition, regenerated from [21].

II EIS of bare and NPs modified Electrodes (data of Figures 5.3b and 5.4b)

EIS Bare AgNP AgNPP AuNP AuNPP Z' Z" Z' Z" Z" Z' Z" Z' Z"

7258.9

45

213.65

83

9549.8

27

5.18190

5

10.580

03

0.2494

99

8630.6

74

8630.7

96

183.96

26

7258.9

44

213.64

75

9549.8

43

5.34690

3

10.724

64

0.3157

56

8630.6

73

8630.7

95

183.91

93

7258.9

42

213.63

38

9549.8

62

5.55322

9

10.905

47

0.3997

79

8630.6

72

8630.7

94

183.86

44

7258.9

4

213.61

65

9549.8

87

5.81176

5

11.132

06

0.5065

46

8630.6

7

8630.7

92

183.79

48

7258.9

37

213.59

47

9549.9

17

6.13234

9

11.413

03

0.6407

88

8630.6

68

8630.7

9

183.70

75

7258.9

33

213.56

7

9549.9

56

6.53475

2

11.765

71

0.8116

3

8630.6

65

8630.7

87

183.59

66

7258.9

28

213.53

19

9550.0

04

7.03856

8

12.207

27

1.0284

83

8630.6

62

8630.7

84

183.45

62

7258.9

22

213.48

8

9550.0

63

7.66200

1

12.753

66

1.3004

76

8630.6

58

8630.7

8

183.28

08

Page 143

124

7258.9

14

213.43

16

9550.1

38

8.45379

2

13.447

61

1.6505

82

8630.6

52

8630.7

75

183.05

6

7258.9

05

213.36

13

9550.2

31

9.43089

3

14.303

98

2.0883

6

8630.6

46

8630.7

68

182.77

65

7258.8

93

213.27

29

9550.3

47

10.6483

3

15.370

99

2.6408

52

8630.6

37

8630.7

6

182.42

64

7258.8

78

213.16

14

9550.4

92

12.1732

2

16.707

48

3.3415

65

8630.6

27

8630.7

49

181.98

64

7258.8

58

213.02

04

9550.6

75 14.0913

18.388

61

4.2336

98

8630.6

14

8630.7

36

181.43

3

7258.8

34

212.84

43

9550.9

02

16.4795

4

20.481

89

5.3574

75

8630.5

97

8630.7

2

180.74

64

7258.8

04

212.62

32

9551.1

88

19.4762

7

23.108

65

6.7831

14

8630.5

77

8630.6

99

179.89

25

7258.7

67

212.34

79

9551.5

44

23.2219

9

26.392

22

8.5833

24

8630.5

52

8630.6

74

178.84

13

7258.7

2

212.00

27

9551.9

96

27.9581

8

30.544

71

10.880

84

8630.5

21

8630.6

44

177.54

35

7258.6

63

211.58

37

9552.5

51

33.7907

3

35.659

71

13.733

34

8630.4

85

8630.6

07

176.00

06

7258.5

9

211.05

21

9553.2

73

41.3612

4

42.301

64

17.461

54

8630.4

4

8630.5

62

174.09

77

7258.5

04

210.41

61

9554.1

67

50.7295

8

50.526

49

22.100

28

8630.3

88

8630.5

1

171.90

96

7258.4

209.65

34

9555.2

94

62.5119

9

60.882

05

27.956

12

8630.3

3

8630.4

51

169.43

05

7258.2

77

208.75

36

9556.7

19

77.3746

6

73.967

45

35.357

17

8630.2

67

8630.3

87

166.74

77

7258.1

35

207.70

78

9558.5

48 96.3629

90.730

78

44.813

95

8630.2

06

8630.3

23

164.04

85

7257.9

78

206.55

63

9560.8

71

120.321

6

111.97

19

56.728

33

8630.1

56

8630.2

69

161.80

67

7257.8

15

205.35

84

9563.8

67

150.889

5

139.24

75

71.888

41

8630.1

4

8630.2

46

160.81

32

7257.6

68

204.28

04

9567.6

9 189.281

173.83

19

90.869

89

8630.1

91

8630.2

86

162.49

09

7257.5

73

203.58

33

9572.7

25

238.674

6

218.94

05

115.23

2

8630.3

74

8630.4

5

169.38

6

7257.6

03

203.80

36

9579.2

92

300.999

1

276.92

98

145.94

53

8630.7

91

8630.8

36

185.65

81

7257.8

81

205.84

2

9587.9

74 379.88

352.07

09

184.85

19

8631.6

18

8631.6

12

218.41

46

7258.6

14

211.22

34

9599.5

25

479.000

4

449.23

03

233.96

31

8633.1

33

8633.0

42

279.06

11

7260.1

27

222.33

75

9614.8

72

601.296

9

573.07

43

295.23

76

8635.7

58

8635.5

37

385.36

42

Page 144

125

7263.0

4

243.72

9

9636.0

56

754.700

4

733.42

76

373.72

14

8640.3

03

8639.8

83

570.12

97

7268.2

64

282.08

46

9665.1

22

941.895

3

932.50

55

472.39

06

8647.8

91

8647.1

89

872.44

99

7277.3

45

348.70

83

9705.5

91

1171.38

5

1173.8

08

596.66

49

8660.4

03

8659.3

26

1343.3

07

7292.8

99

462.59

17

9762.9

88

1457.24

7

1464.2

27

753.92

63

8680.9

4

8679.3

97

2020.9

64

7318.8

01

651.14

43

9844.2

54

1809.42

8

1813.5

66

949.84

92

8713.8

77

8711.7

97

2859.3

67

7360.9

38

953.46

7

9959.1

96

2233.99

1

2238.7

1

1190.4

72

8765.5

54

8762.8

47

3720.5

35

7431.4

01

1441.8

61

10128.

94 2774.64

2774.9

9

1493.9

65

8848.6

62

8844.9

17

4567.4

93

7541.1

62

2148.3

09

10365.

75

3554.33

1

3407.5

52

1850.0

16

8972.4

78

8966.1

57

5528.0

06

7717.9

68

3130.7

21

10710.

96

5337.65

8

4159.6

35

2278.7

14

9161.4

25

9147.1

95

7107.7

81

8004.7

04

4344.5

44

11219.

89

11230.5

2

5069.1

69

2786.7

38

9447.2

54

9409.4

2

9892.4

07

8442.1

71

5554.3

58

11928.

24

31202.4

6

6392.3

48

3339.0

47

9846.3

81

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44

14306.

6

9110.3

49

6692.5

13

12916.

11

96455.9

9

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04

3921.1

91

10392.

05

10164.

44

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86

10085.

88

7914.6

79

14230.

4

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2

19278.

66

4483.4

79

11088.

52

10616.

17

28980.

14

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06

9298.8

21

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09

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2

47203.

21

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44

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36

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22

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37

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63

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8

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79

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6

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95

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36

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84

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31

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36

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77

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83

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92

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06

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83

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6

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3

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83

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08

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34

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01

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75

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04

Page 145

126

31263.

49

8908.0

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67

363904

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15

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1

44040.

94

III Separation Potential (data of figure 6.7):

ΔE (Size of NPs) Bare Au Drop-casted Aerosolized Plasma-assisted

20 0.45 0.238 0.28 2.29E-01

20 0.534 0.268 0.245 2.62E-01

20 0.545 0.251 0.24 2.23E-01

20 0.521 0.498 0.22 2.23E-01

ΔE (Size of NPs) Bare Au Drop-casted Aerosolized Plasma-assisted

40 0.545 0.4 0.475 2.73E-01

40 0.433 0.337 0.375 2.67E-01

40 0.459 0.569 0.352 3.05E-01

Page 146

127

ΔE (Size of NPs) Bare Au Drop-casted Aerosolized Plasma-assisted

60 0.526 0.518 0.39 3.25E-01

60 0.567 0.504 4.41E-01 3.21E-01

60 0.515 0.58 3.20E-01 3.00E-01

IV Peak oxidation current for modified electrodes (data of figure 6.4d)

Drop-casted (Ip)

Aerosolized (Ip)

Plasma-assisted (Ip)

1.97E-05 1.93E-05 1.88E-05

2.16E-05 2.23E-05 2.79E-05

1.98E-05 2.13E-05 2.59E-05

2.33E-05 2.06E-05 2.39E-05

Page 147

128

VITA

APURVA SONAWANE

Born, Maharashtra, India

2006-2010 B.E., Electronics

University of Pune

Maharashtra, India

2015-2016 M.S., Electrical Engineering

Florida International University

Miami, Florida

Jan 2016- Nov 2016 Computer Support Specialist

Florida International University

Miami, Florida

2017 -2020 Doctoral Candidate

Florida International University

Miami, Florida

2017-2018 Teaching Assistant

Florida International University

Miami, Florida

2019-2020 System Engineer Intern

Engineering Resources Group., Inc.

Pembroke Pines, Florida

PUBLICATIONS AND PRESENTATIONS

1. A. Sonawane, P. Manickam, S. Bhansali, 2019. Stability of enzymatic biosensors

for wearable applications. IEEE Reviews in Biomedical Engineering 10, 174-186

2. A. Sonawane, M. Mujawar, S. Bhansali, 2019. Atmospheric Plasma Treatment

Enhances the Biosensing Properties of Graphene Oxide-Silver Nanoparticle

Composite. Journal of The Electrochemical Society 166 (9) B3084-B3090

3. P Manickam, V Kanagavel, A Sonawane, SP Thipperudraswamy, A Sonawane

Electrochemical Systems for Healthcare Applications. Bioelectrochemical

Interface Engineering, 385-409.

Page 148

129

4. A. Sonawane, M Mujawar, S. Bhansali, 2019. Cold Atmospheric Plasma

Annealing of Plasmonic Silver Nanoparticles. ECS Transactions 88 (1), 197-201

5. SK Pasha, M Mujawar, A Sonawane, S Bhansali Thin Film FETs on Flexible

Substrates: a Case for Biosensing Application. Meeting Abstracts, 145-145.

6. A Sonawane, M Mujawar, S Bhansali, 2020. Plasma Assisted Control of

Nanoparticle Distribution for Enhancing the Electrochemical Activity of

Electrodes. 237th ECS Meeting with the 18th International Meeting.

7. A Sonawane, M Mujawar, S Bhansali, 2020. Effects of Cold Atmospheric Plasma

Treatment on the Morphological and Optical Properties of Plasmonic Silver

Nanoparticles. Nanotechnology 31 365706 (11pp).

8. A Sonawane, S Nasim, P Shah, M Mujawar, S Ramaswamy, G Urizar, P

Manickam, S Bhansali 2020. Detection of salivary cortisol using Zinc Oxide and

Copper porphyrin composite using electrodeposition and plasma-assisted

deposition. ECS Journal of Solid State Science and Technology. (Accepted

manuscript).

9. A Sonawane, M Mujawar, P Manickam, S Bhansali, Plasma-Induced Enhancement

in Electronic Properties of Gold Nanoparticles: Application in electrochemical

biosensing of cortisol Applied Surface Science (Submitted manuscript)