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ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

Daniel Kantanka Sarfo MPhil (Nuclear and Radiochemistry)

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

School of Chemistry, Physics and Mechanical Engineering

Faculty of Science and Engineering

Queensland University of Technology

2019

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS i

Keywords

Aminodibenzo-18-crown-6, Aminobenzo-18-crown-6, Gold nanostructures, Zinc

Oxide nanowires, Lead (II) ions, Mercury (II) ions, SERS substrate fabrication,

Surface - enhanced Raman Spectroscopy (SERS), Electrochemical detection, Ultra-

trace detection, dual sensing

ii ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

Abstract

The detection of toxicants in different matrices have attracted much attention

due to the detrimental effects they pose on humans and wildlife. As a result, analytical

techniques have been developed and used by monitoring and regulatory bodies for

their detection and identification. However, most of these techniques which require

the use of bulky, expensive and sophisticated instruments, and hence highly skilled

personnel for their operation. In addition, their bulky nature makes field measurements

unattractive. In some cases, the techniques are either not sensitive or less selective and

rely on tedious pre-sample preparation protocols. The scarcity of detection techniques

that address these drawbacks provided the motivation for this current research.

Therefore, this study is aimed at fabricating user-friendly, sensitive, selective

and field deployable conductive nanostructured substrates for the dual detection of

toxicants at trace and high concentrations by surface enhanced Raman spectroscopy

(SERS) and electrochemistry.

To achieve this aim, Au nanostructures (AuNS) of high density were

electrodeposited onto a solid Au platform yielding abundant hotspots without the use

of capping agents, surfactants or templates. The solid Au platform was used due to its

ability to generate a surface plasmon polariton (SPP). This SPP couples with the

surface plasmon resonance (SPR) of AuNS and leads to multiplicative Raman signal

enhancement. To demonstrate a real world application of the fabricated substrate,

aminodibenzo-18-crown-6 (ADB18C6) was coupled to mercaptopropionic acid

(MPA) using a 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) coupling

reaction. The resultant crown ether derivative was then used to functionalise the AuNS

substrate for the selective detection of Hg(II) ions. The functionalised substrate was

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS iii

able to selectively detect and quantify Hg(II) ions in water, against Pb(II) and Cd (II),

with a limit of quantification (LOQ) and limit of detection (LOD) of 2.1 pM and 0.51

pM, respectively, by SERS. The LOQ and LOD were 1000 folds below the

Environmental Protection Agency (EPA) and World Health Organisation (WHO)

defined levels for Hg(II) ions in water. Electrochemical detection of 1 µM Hg(II) was

also demonstrated on the fabricated substrate.

To explore the possibility of using the AuNS substrate to detect different toxic

metal ions and its reuse abilities, it was repeatedly used to selectively detect Pb(II) ions

in water. This was achieved by using aminobenzo-18-crown-6 (AB18C6) to chelate

Pb(II) ions from Pb(II) contaminated water. The Pb(II)- AB18C6 complex was then

immobilised onto the AuNS substrate and Pb(II) ion detection at a LOQ of 2.2 pM and

a LOD of 0.69 pM were achieved, which are five orders of magnitude lower than the

maximum allowed by the US Environmental Protection Agency. The used substrate

was electrochemically cleaned by cyclic voltammetry and reused for the detection of

Pb(II) ions.

Building on the previous knowledge established in the study on the fabrication

of a reusable AuNS substrate with a surface that can easily be modified to detect

different toxicants, disposable AuNS substrates were designed using indium tin oxide

(ITO) and carbon fiber (CF) as solid supporting platforms. Here, before AuNS were

electrodeposited unto these substrates, a thin, planar layer of gold was coated on the

ITO using E beam evaporation, while ZnO nanowires were grown on a CF using a

hydrothermal method. Whilst the thin Au layer improved the electromagnetic SERS

effect on the nanostructured ITO, the ZnO nanowires increased the charge transfer

effect as well as the AuNS population, and hence the hotspot density on the CF

substrate. The AuNS bare ITO, the AuNS ITO with a thin planar gold layer and the

iv ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

ZnO nanowire bearing CF were used to detect melamine at an LOD of 0.12 pM, 0.19

fM and 57.0 pM respectively. Electrochemical detection of 0.1 µM melamine was also

demonstrated on the nanostructured ITO substrates with an LOD of 0.05 µM.

In this study, reusable, disposable as well as conductive and plasmonic

nanostructured substrates which can be used for both electrochemical and SERS

detection of toxicants has been presented. A cheaper and simple electrochemical

method with high throughput for the fabrication of electrochemical and SERS based

substrates has also been demonstrated. The SERS and electrochemical based substrate

and detection technique developed in this work provide a relatively cheaper, faster yet

highly sensitive, selective and field deployable means of detecting and identifying

toxicants within a wide range of concentration. The research outcome is potentially

useful to, among others, toxicant monitoring bodies as well as forensics and

environmental law enforcement agencies.

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS v

List of Publications and Presentations

Journal articles

D. K. Sarfo, A. Sivanesan, E. L. Izake and G. A. Ayoko, Rapid detection

of mercury contamination in water by surface enhanced Raman

spectroscopy, RSC Advances, 2017, 7, 21567-21575 (Q1 Journal).

D. K. Sarfo, E. L. Izake, A. P. O’Mullane and G. A. Ayoko, Molecular

recognition and detection of Pb (II) ions in water by aminobenzo-18-

crown-6 immobilised onto a nanostructured SERS substrate, Sensors and

Actuators B: Chemical, 2018, 255, 1945-1952 (Q1 Journal).

D. K. Sarfo, E. L. Izake, A. P. O’Mullane, T. Wang, H. Wang, T.

Tesfamichael and G. A. Ayoko, Fabrication of dual function disposable

substrates for spectroelectrochemical nanosensing, Sensors and

Actuators B: Chemical, 2019, 287, 9-17(Q1 Journal).

Presentations

D. K. Sarfo, A. Sivanesan, E. L. Izake and G. A. Ayoko, Development

of a field deployable SERS nanosensor for the detection of Hg (II),

Nanotechnology and Melecular Science HDR Symposium, 16th February

2016, Brisbane, Queensland, Australia.

D. K. Sarfo, A. Sivanesan, E. L. Izake and G. A. Ayoko, A selective and

in situ approach for the detection of Mercuric ions in the Environment,

RACI Analytical and Environmental National Symposium, 18-20th July

2016, Adelaide, Australia.

vi ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

D. K. Sarfo, A. Sivanesan, E. L. Izake and G. A. Ayoko, Selective

nanosensors for ultra trace detection of Hg(II) and Pb(II) ions, 6th

EuCheMS chemistry congress, 11-15th September 2016, Spain.

D. K. Sarfo, A. Sivanesan, E. L. Izake and G. A. Ayoko, A selective and

in situ approach for the detection of Mercuric ions in the Environment,

QACS 2016-Qld Annual Chemistry Symposium, 25th November 2016,

Brisbane, Queensland, Australia (Prize winner-best oral presentation)

D. K. Sarfo, E. L. Izake, A. P. O’Mullane and G. A. Ayoko, Crown ether

nanosensors for the rapid in-field detection of heavy metals, RACI

National Centenary Conference, 23rd - 28th July 2017, Melbourne,

Australia.

D. K. Sarfo, E. L. Izake, A. P. O’Mullane and G. A. Ayoko, A reusable

nanostructured substrate for the selective detection of Pb(II) ions, 2nd

Queensland Annual Chemistry Symposium - QACS 2017, 27th

November 2017, Brisbane, Queensland, Australia (Prize winner-best

oral presentation).

D. K. Sarfo, E. L. Izake, A. P. O’Mullane and G. A. Ayoko, A reusable

and selective crown ether sensor for the detection of Pb(II) ions in water.

7th EuCheMS Chemistry Congress, 26th - 30th August 2018, Liverpool,

UK

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS vii

Table of Contents

Keywords .................................................................................................................................. i

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

Table of Contents ................................................................................................................... vii

List of Figures ......................................................................................................................... ix

List of Tables ..........................................................................................................................xv

List of Abbreviations ............................................................................................................ xvi

Statement of Original Authorship ....................................................................................... xviii

Acknowledgements ............................................................................................................... xix

Chapter 1: Introduction ............................................................................................ 1

Background ...............................................................................................................................1

Research objectives ...................................................................................................................2

Research methodology ..............................................................................................................3

Accounts of scientific progress linking scientific papers ..........................................................4

Implications of research outcome .............................................................................................5

Chapter 2: Literature Review ................................................................................... 7

Analytical techniques for the detection of toxicants .................................................................7

Electrochemistry as a tool for detecting toxicants ....................................................................9

Fabrication of nanostructured substrates on conductive solid platforms for surface enhanced Raman spectroscopic (SERS) sensing ....................................................................................11

Detection of toxicants by SERS ..............................................................................................39

Implications from review ........................................................................................................44

Chapter 3: Fabrication of crown ether functionalized Au nanostructured substrate for trace detection and quantification of Hg(II) ion ............................. 47

Preface ....................................................................................................................................49

Abstract ...................................................................................................................................50

Introduction .............................................................................................................................50

Experimental ...........................................................................................................................53

Results and discussions ...........................................................................................................59

Conclusions .............................................................................................................................70

Acknowledgements .................................................................................................... 70

Chapter 4: A reusable Au nanostructured substrate for selective detection and quantification of Pb(II) ions by SERS .................................................................... 71 Preface ........................................................................................................................ 73 Abstract ...................................................................................................................... 74 Introduction ................................................................................................................ 74

viii ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

Materials and methods ............................................................................................... 77 Results and discussions .............................................................................................. 82 Conclusions ................................................................................................................ 92 Acknowledgements .................................................................................................... 92

Chapter 5: Fabrication of disposable and dual function substrates for SERS and electrochemical detection of toxicants .................................................................... 93 Preface ........................................................................................................................ 95

Abstract ...................................................................................................................... 96 Introduction ................................................................................................................ 97 Experimental .............................................................................................................. 99 Results and discussion .............................................................................................. 104 Conclussions ............................................................................................................. 115 Acknowledgements .................................................................................................. 116

Chapter 6: Summary, Conclusions and Recommendations for future work ... 117

Bibliography ........................................................................................................... 123

Appendices .............................................................................................................. 142

Appendix A (Chapter 3) ........................................................................................... 142 Appendix B (Chapter 4) ....................................................................................................... 146

Appendix C (Chapter 5) ....................................................................................................... 148

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS ix

List of Figures

Fig. 1.0 Flow diagram of research progress and associated

publications…………………………………………………………………………...3

Fig. 2.1 Schematic illustration of (A) surface plasmon polariton and (B) localised

surface plasmon……………………………………………………………………..12

Fig. 2.2 Schematic representation of SERS phenomenon for an analyte on Au

nanoparticles………………………………………………………………………...13

Fig. 2.3 Number of publications per year from 2009 – 2018 period searched through

Web of Science using the keywords “SERS Substrate and fabrication” and “SERS and

nanostructures”………………………………………………………………….…..17

Fig. 2.4 Field Emission scanning electron microscope (FSEM) images of GNPs

immobilised on ITO by electrophoresis at ≤ 2mins (a and b), 8 mins (c) and (d) High

resolution image at 8 mins. Red circles indicate sub-10-nm gaps between neighbouring

nanoparticles………………………………………………………………………...19

Fig. 2.5 A schematic illustration of the preferential nucleation and oriented growth for

cross-linking Ag nanoplate arrays: (a) random-oriented Ag seeds laying on the ITO

substrate, and Ag nuclei are preferentially formed on some <110>-oriented seeds (see

arrow’s marks); (b) oriented growth of the nuclei along the fastest <110> within (111)

plane under a low deposition current density; (c) cross-linking Ag nanoplate array

structure is formed and standing on the substrate

vertically…………………………………………………………………………….20

Fig. 2.6 (a–c) Scanning electron microscope (SEM) images of Ag microhemispheres

at different magnification. The inset in (a) is the SEM side view of the

microhemispheres. (d and e) Cross-sectional SEM views of a microhemisphere. (f)

x ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

The size distribution of the Ag micro-hemispheres in (a). (g and h) Differently

magnified SEM images of an imperfect Ag microhemisphere. (i) TEM image of fan-

shaped pieces broken off from an imperfect Ag micro-hemisphere as shown in (g). The

inset in (i) is the SAED pattern from the black circle. (j) SERS spectra of 10−11 M R6G

from five randomly chosen individual micro-

hemispheres……………………………………….....................................................21

Fig. 2.7 A schematic illustration of the seed mediated two step electrochemical

approach for deposition of AuNPs on ITO and its utilization for the detection of 4-

Mercaptobenzoic acid (p-MBA)…………………………………………………….22

Fig. 2.8. SEM images of (a) Au nanoparticles grown on a PANI membrane (doped by

citric acid) by immersing the PANI membrane in 10 mM AuCl3 aqueous solution for

10 s, and the Ag nanostructures produced by immersing the Au nanolayer-supported

PANI membrane in 50 mM AgNO3 aqueous solution for (b) 10 s, (c) 30 s, (d) 1 min,

(e) 10 min, and (f) 60 min. The scale bar is 500

nm…………………………………………………………………………………...25

Fig. 2.9 SEM images of Ag nanostructures produced on hydrazine treated PANI films

at a reaction time of 30 s (a), 1 min (b), 2 min (c) and 5 min (d), with lactic acid present

and 30 s (e), 1 min (f), 2 min (g) and 5 min (h), with succinic acid present in the AgNO3

solution. Scale bar: 3 mm…………………………………………………………….27

Fig. 2.10 SEM images of Ag nanostructures produced on the camphorsulfonic-acid-

doped PANI membranes, with (a, b) succinic acid, (c, d) lactic acid, and (e, f)

camphorsulfonic acid present in the AgNO3 solution………………………………27

Fig. 2.11 FE-SEM images of gold nanoparticles patterned onto SWCNT films by

electrochemical deposition. The deposited charges for (A), (B), and (C) were 1 mC,

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS xi

10 mC, and 30 mC, respectively. The scale bars in the left, middle and right columns

are 100 mm, 10 mm and 1 mm, respectively………………………………………..30

Fig. 2.12 ZnO-mesoporous-NSs grafted on CFs (a) Top-view SEM image. The lower-

left inset is an optical photo of a piece of ZnO-mesoporous-NSs@CFC. (b) Magnified

SEM observation on two adjacent CFs grafted with ZnO-mesoporous-NSs. (c) Close-

up SEM view of a few ZnO-mesoporous-NSs. (d) TEM image of a single ZnO-

mesoporous-NS and its corresponding selected area electron diffraction

pattern…………………………………………………….........................................32

Fig. 2.13 (a) Scanning electron micrograph of an array of nanoholes patterned into a

Si wafer after Cr deposition, removal of PLGA posts, and reactive ion etching. (b)

Aspect of whole wafer patterned with nanoholes…………………………………...34

Fig. 2.14 Schematic procedure to fabricate the 3D SERS substrate and the related SEM

images, (a) Top view of texturized Si and side view (the inset) (b) ZnO NRs on

texturized Si, (c) plasmonic structures of ZnO NRs with Ag decoration…………...36

Fig. 2.15 Fabrication strategy for periodic spherical nanoparticle arrays. a) Monolayer

PSs were formed on silicon wafer by self-assembling process. b) The monolayer PSs

were heated on electric-plate at 120 °C for 25 s. c) 0.5 M Fe (NO 3) 3 with addition of

20 × 10 −3 M Triton X-100 was dropped onto the surface of monolayer PSs. d) After

drying at 110 °C for 30 min, the samples were annealing at 400 °C for 2 h to remove

the template of PSs, and regular network-structured arrays with prism-like protrusions

among three neighboring holes were formed. e) After magnetron sputtering deposition

at 50 W for certain time, hexagonal periodic spherical nanoparticle arrays were

formed………………………………………….……………………………………37

xii ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

Fig. 2.16 FE-SEM images of periodic spherical nanoparticle arrays using regular

network-structured arrays as sputtering deposition template (b) is expanded image of

(a) Scale bars: 500 nm………………………………………………………………38

Fig. 3.1, UV-visible spectra of (A) ADB18C6 and (B) ADB18C6 in different

concentrations of Hg (II): (a) 20 (b) 40, (c) 60 and (d) 80 µM……………………...60

Fig 3.2 Fluorescence spectra of ADB18C6 in different concentrations of Hg (II): (a)

0, (b) 20, (c) 40, (d) 60 and (e) 80 µM………………………………………………61

Fig. 3.3 (A) UV Absorption spectra and (B) Fluorescence emission spectra of

ADB18C6 crown ether after reaction with Hg (II), Cd (II) and Pb (II) ions

respectively………………………………………………………………………….61

Fig. 3.4 Cyclic voltammogram of bare nanostructured gold substrate (Bare Au), TCE-

functionalized gold substrate (Au/TCE) and TCE-functionalized gold substrate after

complex formation with Hg (II) [(Au/ TCE /Hg)]…………………………………....63

Fig. 3.5 SERS spectra of ADB18C6 and TCE on nanostructured gold substrate…..65

Fig. 3.6 SERS spectra of TCE and TCE -Hg (II) complex………………………….67

Fig. 3.7 A linear correlation plot of the Raman intensity (at 1501) versus the logarithm

of Hg(II) concentration and corresponding SERS spectra(Insect) from (a) 1x10-11 M

to (f) 1x10-6 M………………………………………………………….....................67

Fig. 4.1. (A) SEM images of gold nanostructures electrochemically deposited onto a

flat solid Au material at different magnifications (B) Coupling of the SPP (from Au

solid base material) and SPR (from gold nanostructures) for high SERS

signals……………………………………………………………………………….83

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS xiii

Fig. 4.2. Fluorescence spectra of AB18C6-Pb(II) complex at different concentrations

of Pb (II) ions………………………………………………………………………..84

Fig. 4.3. UV-visible absorption spectra of mono and divalent ions after interaction with

AB18C6………………………………………………………………………..……85

Fig. 4.4 A: SERS spectra of (i) nanostructured gold substrate, (ii) AB18C6 crown ether

and B: (iii) AB18C6-Pb(II) complex………………………………………….……..86

Fig. 4.5. Cyclic voltammograms between 0.10 and -1.40 V at a scan rate of 0.1 V/s for

10 µM AB18C6-Pb(II) functionalised substrate………………………………..........88

Fig. 4.6 (a) Raman spectrum of the AB18C6-Pb(II) complex on the nanostructured

gold substrate before electrochemical desorption, (b) Raman spectrum of the substrate

after desorption, (c) Re-emergence of the AB18C6-Pb(II) complex Raman fingerprint

after depositing a new aliquot of the complex…………………………..……………89

Fig. 4.7 A plot of the Raman intensity (at 820 cm-1) versus the logarithm of Pb(II)

concentration (1x10-12 M to 1x10-6 M) and the corresponding SERS spectra from 750

to 900 cm-1(Inset)……………………………………………………………………90

Fig. 5.1 SEM images of electrodeposited Au nanostructures on bare ITO at (a) 400s

(b) 600s and (c) 900s and on gold-coated ITO at (d) 400s (e) 600s (f) 900s

respectively………………………………………………………………………...105

Fig. 5.2 SEM images of (a) bare CF (b) ZnO NWs-coated CF (c) electrodeposited Au

nanostructures on bare CF (d) electrodeposited Au nanostructures on ZnO NWs-

coated CF (e) Energy Dispersive X-ray spectrum (EDX) of gold nanostructured ZnO

NWs-coated CF…………………………………………………………………….106

Fig. 5.3 SERS spectra of 0.1µM quinolinethiol on (a) Au/ITO and (b) Au/Au-

ITO…………………………………………………………………………………107

xiv ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

Fig. 5.4 (a) SERS spectra of 0.1 µM QT on Au/ZnO-CF at different deposition times

(brown spectrum = clean Au/ZnO-CF) (b) SERS spectra of 0.1 µM QT on (i) Au/ZnO-

CF substrate (red line) and (ii) Au/CF substrate (green line) at 600 s deposition

time……………………………………………………………………..………….107

Fig. 5.5 Schematic diagram showing enhanced Raman scattering resulting from

plasmon coupling between neighbouring Au nanostructures and Au - ZnO

junctions……………………………………………………………………………109

Fig. 5.6 SERS spectra of melamine in water on (a) Au/ITO (b) Au/Au-ITO (c)

Au/ZnO-CF and SERS spectra of melamine extracted from milk on (d) Au/Au-ITO.

The black spectra represent the Raman spectra of blank water (a –c) and milk (d)

samples. The red spectrum (d) is for melamine standard at the same extraction

conditions…………………………………………………………………………..112

Fig. 5.7 Voltammograms of (a) bare ITO (i), Au/ITO (ii), Au/ITO with 0.1 µM

Melamine (iii) and (b) gold coated ITO (i), Au/Au-ITO (ii), Au/Au-ITO with 0.1 µM

melamine (iii). CV scans were carried out in 0.1MKOH………………………….114

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS xv

List of Tables

Table 2.1: A list of some SERS applications using ITO based substrates………….23

Table 2.2: Some selected SERS applications using Carbon based substrates……...32

Table 2.3: Some selected SERS applications using Si wafer based substrates……..38

Table 2.4: Detection of some organic toxicants by SERS………………………….41

Table 3.1: SERS wavenumbers/cm of ADB18C6 and TCE and their band

assignments………………………………………………………………………….65

Table 3.2: Comparison of proposed Hg(II) sensing method with some reported

methods…...................................................................................................................69

Table 4.1: SERS wavenumbers/cm of Pb(II) - AB18C6 complex and AB18C6 and

their band assignments………………………………………………………………87

Table 4.2: Comparison of the proposed Pb(II) sensing method with some reported

methods……………………………………………………………………………...91

Table 4.3: Recovery studies with spiked drinking water (n = 3)…………………......91

Table 5.1: A comparison of the three fabricated substrates………………………..115

xvi ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

List of Abbreviations

AuNS Gold nanostructures

Au/ITO Nanostructured bare ITO

Au/Au-ITO Nanostructured gold-coated ITO

Au/ZnO-CF Nanostructured ZnO NWs-coated CF

ADB18C6 Aminodibenzo-18-crown-6

tADB18C6 thiolated aminodibenzo-18-crown-6

AB18C6 Aminobenzo-18-crown-6

AAS Atomic Absorption Spectrometry

CT Charge Transfer effect

CF Carbon fibre

CV Cyclic voltammetry

DEA Diethanolamine

EM Electromagnetic enhancement effect

EDC 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide

FSEM Field Emission Scanning Electron Microscope

GC-ECD Gas chromatography coupled to electron capture detection

GC-APLI-MS Gas Chromatography coupled with Atmospheric Pressure Laser

Ionization and Mass Spectrometry

HPLC-DAD High-Performance Liquid Chromatography with Diode-Array

Detector

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS xvii

HMT Hexamethylene tetraamine

ICP-MS Inductively Coupled Plasma - Mass Spectrometry

ITO Indium tin oxide

LC-MS/MS liquid chromatography-tandem mass spectrometry

LOQ Limit of quantification

LOD Limit of detection

MPA Mercaptopropionic acid

NWs Nanowires

NHS N-hydroxysuccinimide

NAA Neutron Activation Analysis

QT Quinolinethiol

SERS Surface enhanced Raman spectroscopy

SPP Surface Plasmon polariton

SPR Surface Plasmon resonance

SWCNTs Single-Walled Carbon Nanotubes

SEM Emission Scanning Electron Microscope

ZnO NWs ZnO Nanowires

xviii ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the best

of my knowledge and belief, the thesis contains no material previously published or

written by another person except where due reference is made.

Signature:

Date: April 2019

QUT Verified Signature

ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS xix

Acknowledgements

I am exceedingly grateful to the Almighty God for blessing me with strength,

perseverance and wisdom from the beginning of this journey to the final compilation

and submission of this thesis. I would also like to express my heartfelt gratitude to the

following for their indispensable contributions towards the successful completion of

this work:

My supervisors; Prof. Godwin A. Ayoko, Dr Emad Kiriakous and

A/Prof. Anthony P. O’Mullane for their patience, encouragement and

professional expertise that saw me through this PhD journey. I thank

them for the valuable time they spent on reading, editing and shaping all

the papers published in this work as well as the compilation of this thesis.

Dr. Arumugam Sivanesan and Dr. Llew Rintoul for their training and

assistance on the use of Raman spectrometers and electrochemical

devices. Dr Sivanesan, your positive impact on my research skills will

forever be remembered. Though it was for a brief moment, it carried me

all the way to the end.

The Ghana Atomic Energy Commission (GAEC) for the study leave

granted and the Queensland University of Technology (QUT) for their

support through the QUT Postgraduate Research Award (QUTPRA) and

the QUT International HDR Tuition Fee Sponsorship.

The staff of Central Analytical Research Facility (CARF) for their

training and support. I thank the Science and Engineering Faculty of

QUT for making access to CARF possible through their generous

funding.

xx ULTRA TRACE DETECTION OF TOXICANTS USING NANOSENSORS

Last but not least, I will like to acknowledge fellow members of the

Raman group for their assistance.

Finally, I dedicate this thesis to my family, especially my wife and

parents. Their endless prayers and motivation kept me going despite the

many difficulties. I am indebted to them for their matchless love and

treasured support.

Chapter 1: Introduction 1

Chapter 1: Introduction

In this chapter, the background, context of this research and its purpose are

provided. The significance of the research, its objectives and methodology have been

outlined. Finally, an account of scientific progress leading to and linking the scientific

publications generated from this work as well as an outline of the remaining chapters

of this thesis are given.

1.1 Background

There has been an evolution of various analytical techniques for the detection,

monitoring and control of toxicants such as azo dyes, dioxins, polycyclic aromatic

hydrocarbons [1], persistent organic pollutants, microtoxins and heavy metals [2].

Among these methods are liquid chromatography/electrospray ionization mass

spectrometry, linear ion trap quadrupole orbitrap mass spectrometry [3], Neutron

Activation Analysis [4], chromatography-tandem mass spectrometry [5],

Spectrofluorimetry [6], gas chromatography coupled to electron capture detection [7],

Atomic Absorption Spectrometry [8] and X-ray Fluorescence [9]. The increased

interest in these toxicants is due to their abundance, environmental persistence, their

ubiquitous nature and the toxicity they pose to the environment, animals and humans.

For instance, in humans, toxicities/adverse health effects ranging from neurotoxicity

to moderate and hepatic renal toxicity [10, 11]; memory loss to mental retardation [12];

kidney and brain damage as well as heart failure [13, 14] are possible when exposed

to these toxicants through ingestion of contaminated food and drinking water,

inhalation of ambient air (with high concentrations of toxicants) and their absorption

through the skin [15, 16]. Although most of these analytical methods have enormous

advantages, the associated cost for chemicals, equipment and human resources as well

as the tedious sample preparation protocols and associated lengthy analysis time make

their applications, in many cases, uneconomical. In addition, these analytical

techniques are not field-deployable, making on site analysis difficult. Hence, there is

a continual search for techniques that are cost effective, sensitive, field deployable,

capable of selective and rapid analysis with efficient and easy-to-operate procedures.

2 Chapter 1: Introduction

Surface enhanced Raman scattering (SERS) and electrochemical based

nanosensor techniques provide a fast, efficient and cost-effective method for detection

and analysis of toxicants [17, 18]. The development and availability of portable and

handheld electrochemical and Raman spectrometers extends the advantages of these

techniques as they offer alternative routes for on-site measurements [19, 20].

Furthermore, the fabrication of portable solid based nanostructured substrates with

high throughput is possible by electrochemical means. Here, nanostructures are

immobilised onto conductive platforms via applied voltages to generate substrates that

can easily be handled as well as transported from one place to another. The conductive

and plasmonic nature of these substrates and the ease with which they can be handled

and transported can be explored to make them attractive for on-site detection of

toxicants using electrochemical and SERS based techniques. This research work

therefore aims at designing selective, quick, sensitive, user friendly, cheap, rugged,

safe and field deployable conductive and nanostructured substrates, for ultra-trace

detection of toxicants using SERS and electrochemical based techniques. Suitable

molecular recognition molecules that have the ability to selectively bind target

toxicants onto metallic nanoparticles were also identified and used.

1.2 Research objectives

The overall objective of the study was to fabricate nanostructured substrates

for both SERS and electrochemical based detection of toxicants by developing Au

nanostructures onto conductive solid support platforms. The developed substrates will

then be characterised and tested with common toxicants for their detection capabilities

on a laboratory scale.

The specific aims of this thesis are to:

Prepare nanosensor platforms suitable for both SERS and electrochemical

detection.

Characterise the fabricated platforms using techniques such as scanning

electron microscopy and energy-dispersive X-ray spectroscopy.

Identify suitable recognition molecules for the selective detection of toxicants.

Analyse toxicants at ultra-trace levels using handheld devices.


Develop field deployable, reusable and disposable platforms for toxicant

detection.

1.3 Research Methodology

The research plan used to meet the above objectives of this study, the thesis

structure and the outputs of this research work are described in the following flow

chart (Fig. 1.0).

Fig. 1.0 Flow diagram of research progress and associated publication

Chapter 1: Introduction

Chapter 2: Literature Review

Fabrication of Au nanostructured substrates for trace detection of toxicants

Chapter 3: Fabrication of crown

ether functionalized Au nanostructured

substrate for trace detection and

quantification of Hg(II) ion

(Paper 1)

Chapter 4: A reusable Au

nanostructured substrate for selective detection and quantification of Pb(II) ions by SERS

(Paper 2)

Chapter 5: Fabrication of dual function disposable substrates for SERS and electrochemical

nanosensing

(Paper 3)

Chapter 6: Conclusions and recommendations for future work

Detection of toxicants by SERS and electrochemistry using Au nanostructured

substrate


1.4 Account of scientific progress linking the scientific papers

The literature review within this research (chapter 2 of the thesis) indicates the

ongoing need for new sensitive and selective methods for rapid and in-field detection

of toxicants. Hence, a nanostructured substrate was fabricated by electrochemically

developing Au nanostructures onto a solid gold disc. The fabricated substrate was

robust and can be transported easily from one place to another making it a good

candidate for field analysis. In addition, the substrate can be used for the detection of

toxicants at both high and ultra-trace concentrations by electrochemical and SERS

techniques. This widens the detection range for sample analysis. The practical

application of the fabricated Au nanostructured gold disc was demonstrated by

utilising it for the detection of Hg(II) ions in water. Mercury is known to be toxic to

both humans and wildlife and can be found in soil, water, sediment and air. This work

has been detailed in paper 1 (submitted and published) and in chapter 3.

The possible reuse and amenability of the fabricated substrate for the detection

of other toxicants was also explored. To arrive at this, the developed substrate was

modified for repetitive detection of Pb(II) ions in water to demonstrate its versatility

and reusability. The detection of Pb(II) ions is important due to their toxicity after

finding their way into humans, animals and other wildlife through the food chain. The

experimental details and associated results relating to the user friendly, versatile and

reusable substrate (hence, making it cost-effective) for the detection of Pb(II) ions by

SERS, have been presented in the published journal article in chapter 4 of this thesis.

To improve on the substrate already designed and described previously, we

researched the possible use of cheaper solid supporting platforms onto which the Au

nanostructures could be deposited. This was done with the aim of fabricating new cost-

effective, disposable and dual SERS/electrochemical nanosensors for detection of

toxicants. In chapter 5, a detailed description of the method for the fabrication of Au

nanostructured indium tin oxide (ITO) and carbon fibre dual sensing platforms has

been documented. Greatly improved detection sensitivities of these substrates were

achieved using a thin layer of plane gold and ZnO nanowires. The utilisation of these

new substrates for cyclic voltammetry (CV) and SERS detection, using melamine as a

demonstrator toxicant, is presented.


Limit of quantification (LOQ) and limit of detection (LOD) were calculated

and used to demonstrate/estimate the sensitivities of all the substrates fabricated and

hence, their capacities to detect concentrations of analytes at trace levels. The number

of significant figures reported do not indicate the accuracy of the LOD and LOQ

measurements.

This thesis has been written in six chapters. These chapters describe the

significance and objectives of the study as well as the state of knowledge and the gap

in literature relating to the detection of toxicants at ultra-trace levels using nanosensor

techniques that are rapid, user friendly, selective, sensitive and field deployable.

Furthermore, the fabrication and characterisation of reusable and disposable dual

sensing Au nanostructured substrates and their application for the detection of various

toxicants have also been discussed. Recommendations on possible future work that

can emerge from this study have also been made.

1.5 Implications of research outcomes

The outcome from the research work in this thesis have contributed to

knowledge by providing an easy-to-use and cost-effective yet field deployable,

selective and highly sensitive way of detecting and quantifying toxicants compared to

some of the already existing techniques.

A new SERS and electrochemical method for the selective detection of Hg(II)

ions in water using a thiolated crown ether derivative to form a self-assembled

monolayer on a nanostructured gold disc was identified. In addition, a cost-effective,

simple and rapid three-step SERS method for the selective detection and quantification

of Pb(II) ions in water involving Pb(II)-crown ether complexation, immobilisation

onto a nanostructured gold disc and detection was also identified. These techniques

were able to detect and quantify Hg(II) and Pb(II) at ultra-trace levels (i.e. at pico-

molar concentrations). As a result, early detection of toxicants at stages where their

concentrations are not likely to cause harm is possible. This, combined with the field

deployable nature of these methods, make them potentially useful for research and

regulatory bodies in their quest to study, monitor and control toxicants. Besides, the

ability to reuse the nanostructured gold disc substrate for multiple detection makes the

methods presented in this work cost-effective.


In addition, methods for fabricating disposable nanostructured substrates with

enhanced detection sensitivities using the concept of surface plasmon resonance-

surface plasmon polariton (SPR-SPP) coupling and noble metal-semiconductor

nanocomposite have been shown. The possibility of using these nanostructured

substrates for both SERS and electrochemical applications were also demonstrated.

Hence, with these nanostructured substrates, routine quantification of toxicants at high

concentration can be done using the relatively easy-to-use and inexpensive

electrochemical technique whilst analysis requiring molecular identification of

unknown compounds at ultra-trace concentrations can be done by the SERS technique.

Chapter 2: Literature Review 7

Chapter 2: Literature Review

This chapter provides a review of literature on analytical techniques for the

detection of toxicants, electrochemistry as a tool for detecting toxicants, surface

enhanced Raman spectroscopy (SERS) at electrodes and detection of toxicants by

SERS. The final section of this chapter discusses the implications from this review.

2.1 Analytical techniques for the detection of toxicants

In order to mitigate the negative effects posed by toxicants on humans and

wildlife, researchers have used various analytical techniques such as atomic absorption

spectrometry (AAS), neutron activation analysis (NAA), inductively coupled plasma

- mass spectrometry (ICP-MS), liquid chromatography-tandem mass spectrometry

(LC-MS/MS) and high-performance liquid chromatography with diode-array detector

(HPLC-DAD) to detect and quantify toxicants.

For instance, Kar et al [21] assessed the situation of heavy metal pollution in 96

surface water samples from the Western part of Bengal using AAS. They found

concentration range of 0.025-5.49, 0.025-2.72, 0.012-0.370, 0.012-0.375, 0.001-0.044

and 0.001- 0.250 mg/L respectively for Fe, Mn, Zn, Ni, Cr and Pb and 0.001-0.003

and 0.003-0.032 mg/L for Cd and Cu respectively. Using Instrumental Neutron

Activation Analysis (INAA) and ICP-MS, Veado et al also investigated metal

pollution in the tissues of tilapia from the Pampulha Lake of Brazil [22]. In addition,

LC-MS/MS has been used in Tokyo by Nakada et al in a work aimed at removing

some pharmaceuticals and personal care products and endocrine-disrupting chemicals

from a municipal sewage treatment plant [23]. Most of the targeted chemicals were

detected at ng/L levels. A quadrupole-based ICP-MS method has also been used by

Hsu and co-workers to assess the variations of Cd/Pb and Zn/Pb ratios in Taipei

8 Chapter 2: Literature Review

aerosols. They reported detection limits of 0.2 ng/m3 for Pb, 0.5 ng/m3 for Zn and 0.01

ng/m3 for Cd [24]. Though AAS, INAA, ICP-MS and LC-MS/MS are good analytical

techniques and, in some cases, have excellent sensitivities, they usually consist of

bulky instrumentation making field measurements unattractive.

The use of hyphenated techniques such as HPLC-MS/MS, HPLC-DAD, Liquid

chromatography/electrospray ionization tandem mass spectroscopy (LC-ESI-MS/MS)

and Gas Chromatography coupled with Atmospheric Pressure Laser Ionization and

Mass Spectrometry (GC-APLI-MS) has also been explored by researchers in the study

of toxicants in different matrices with the aim of arriving at higher sensitivities. For

instance, López-Fernández et al [25] developed and used an HPLC-MS/MS technique

with hexane followed by a clean-up step using Supelclean™ Envi-Carb II/primary–

secondary amine(PSA) cartridges to analyse 7 types of neonicotinoids insecticide

residues in dietary bee pollen. A detection limit (LOD) and quantification limit (LOQ)

ranging from 0.2-2.2 μg/kg and 0.4-4.3 μg/kg respectively were achieved. In other to

reduce matrix effects, Lian and co-workers synthesised a melamine-imprinted polymer

and used it to pre-concentrate melamine from aquaculture feed before analysis by

HPLC-DAD [26]. This approach yielded recoveries ranging from 84.6% to 96.6% with

LOD and LOQ of 0.1 μg/g and 0.35 μg/g, respectively. Recently, Reichert et al [27]

demonstrated how to simultaneously detect over 100 pesticides and 30 mycotoxins in

coffee using LC-ESI-MS/MS without a clean-up process prior to analysis. In 2013,

40 polycyclic aromatic hydrocarbons in environmental samples were studied by Stader

et al [28] using GC-APLI-MS. In that work, the LODs obtained for each PAH in a

standard mixture ranged from 5–100 fg/mL. According to the authors, this

demonstrated high sensitivity of GC-APLI-MS towards PAHs. Although these

hyphenated techniques have shown good efficiency and sensitivity with reduced


sample analysis time, they require sophisticated instrumentation and their costs of

operation are relatively expensive. In addition, they are usually not field-deployable

and required highly trained personnel for their operation.

2.2 Electrochemistry as a tool for detecting toxicants

Electroanalytical techniques are known for their low cost, high sensitivity and

simplicity. They are easy to operate with relatively rapid data acquisition time. There

are various techniques such as cyclic voltammetry, differential pulse voltammetry,

chronoamperometry and stripping voltammetry. This allows a researcher to select the

most appropriate technique for analysis. These advantages, coupled with the

availability of portable and handheld electrochemical devices, make on site analysis

possible [17, 29, 30]. For these reasons, Asadollahi-Baboli’s group [31] successfully

applied square wave voltammetry and chemometrics to simultaneous measure

tetracycline and cefixime in biological fluids. They reported concentrations in the

ranges of 10-5 and 10-3 mol/L for tetracycline and cefixime respectively. Differential

pulse polarography and differential pulse anodic stripping voltammetry has also been

used by Ersnt et al to study Cd, Zn and Pb trace metal species and their stability

constant [32]. In that study, it was concluded that cathodic and anodic voltammetric

techniques can be used to characterise trace metal interactions in an aquatic

environment. Wiyaratn et al also developed a general purpose electrochemical sensor

for detecting organohalogens [33]. They reported that, 1-chloropropane, chloroform,

carbon tetrachloride, iodoethane, and bromoethane can be detected with a detection

limit of 0.1 nM by differential pulse voltammetry. Though electrochemical techniques

have successfully been used by these authors, the selectivity, and in some cases,

sensitivities from these studies were observed to be relatively poor.


Due to this, modifying the surfaces of electrodes with nanoparticles and

recognition molecules to enhance the sensitivity and selectivity of electrochemical

methods has been explored by researchers [30]. Metallic nanoparticles exhibit

exceptional properties that are not usually seen in bulk material. Among these are

improved selectivity, high signal-to-noise ratio, catalytic activity, high effective

surface area and mass transport [34, 35]. Since modification of nanoparticle

morphology can lead to improved selective detection of analytes, whilst change of

nanoparticle size can also lead to a shift in peak potential, control of electrochemical

responses is made possible with the use of nanoparticles by varying the properties of

particles when accumulated on electrodes[34]. Du et al used an acetylcholinester-

modified multiwalled carbon nanotubes-chitosan composite for electrochemical

detection of triazophos [36]. According to these authors, the use of multiwalled carbon

nanotubes enhances sensitivity in the detection of triazophos due to their ability to

facilitate electron transfers at low potentials. A detection limit of 0.01 µM was

achieved using cyclic voltammetry. Methyl parathion and chlorpyrifos pesticide has

also been detected using single-walled carbon nanotubes (SWCNTs) coated by single-

stranded DNA (ssDNA) with a thiol head group [37]. The ssDNA wrapped SWCNTs

were then immobilised onto an Au electrode to form a Au/ssDNA-SWCNT layer. The

electrode was used to detect methyl parathion and chlorpyrifos at a detection limit of

1 pM by square wave voltammetry. This was done after electrochemical

polymerisation of aniline onto the Au/ssDNA-SWCNT substrate. Wang et al has also

detected parathion by square wave voltammetry at a detection limit of 3 ng/mL using

a ZrO2/Au nano-composite filmed electrode. The chemisorption of parathion onto the

ZrO2/Au nano-composite film was based on the affinity of nano-ZrO toward the

phosphate groups on parathion [38]. Electrochemical detection techniques have also


been used in the early diagnosis of various diseases. For instance, Sun et al [39] has

developed a cost effective and rapid method for the detection of cancer via

electrochemistry after functionalising a gold nanostructure-modified glassy carbon

electrode with a thiolated TLS11 aptamer. The sensor had a detection limit of

15 cells mL−1 with a wide detection range of 1×102-1×107 cells mL−1.

The availability of disposable platforms for electrochemical studies also

provides a quick and cost-effective analytical pathway. Li et al used disposable sensor

that was modified with single wall carbon nanotubes to electrochemically monitor

DNA hybridisation breast cancer disease [40]. Disposable electrochemical magne-to

immunosensor platform was also used by Jodra et al to monitor Ochratoxin A

contaminant in coffee samples [41]. In addition, Wang and co-researchers reported Pb

(II) concentration in blood down to about 0.10 µg/L using square-wave voltammetry

with a disposable screen printed electrode [42].

2.3 Fabrication of nanostructured substrates on conductive solid platforms for

surface enhanced Raman spectroscopic (SERS) sensing

2.3.1 Surface enhanced Raman spectroscopy

Surface enhanced Raman spectroscopy is an analytical technique with

widespread applications in catalysis [43], biomolecule detection [44], environmental

monitoring [45] agriculture [46-48], forensic [18, 49, 50] and food safety [51, 52]. This

is due to its sensitivity, non-destructive nature and the ability to directly screen aqueous

samples which are often problematic for other spectroscopic techniques such as infra-

red spectroscopy [53]. In addition, its multiplexing ability allows for the detection and

identification of multiple analytes in a given sample [54]. Due to the availability of

portable and handheld Raman spectrometers, SERS can be easily carried out in the

field with minimum sample pre-treatment [55-57].


In SERS the analyte molecules are adsorbed onto the rough nanostructured

metallic surface (usually a noble metal) where the inherently weak Raman scattering

from the analyte becomes significantly enhanced as explained below [58, 59].

Gold and silver are the most widely used metals in fabricating SERS substrates.

This is due to the presence of unbound electrons within their electronic structure which

supports surface plasmons such as surface plasmon polaritons (SPP) and localised

surface plasmons (LSP)[60-62]. It is the presence of these surface plasmons which

contribute to the enhancement effects observed in SERS. The SPP is a coherent

oscillation of electrons that propagates effectively as a longitudinal wave and occurs

between the surfaces of these coinage metals and a dielectric medium (Fig 2.1A) [63].

The LSP, which is the coherent oscillation of electrons within the vicinity of coinage

metal nanostructures (Fig 2.1B), can be produced quite easily using Au or Ag

nanostructures in the visible to near infra - red region where most Raman

measurements are undertaken to avoid fluorescence background [64].

Fig. 2.1 Schematic illustration of (A) surface plasmon polariton and (B) localised

surface plasmon.


Fig. 2.2 Schematic representation of SERS phenomenon for an analyte on Au

nanoparticles.

Metals such as Pd, In, Rh and Ru have been explored in the fabrication of SERS

substrates. However, the difficulties associated with their LSP excitation within the

visible region of the electromagnetic spectrum, make them less suitable for SERS

applications [65].

The enhancement effect of SERS is well-known to also originate from a

chemical effect, often referred to as a charge transfer (CT) mechanism and an

electromagnetic (EM) effect. The chemical effect results from electronic transitions

between analyte molecules and the surfaces of the metallic nanostructures onto which

the molecules are chemisorbed [66]. Electromagnetic enhancement, on the other hand,

occurs when the analyte orients itself within the LSP resonance. Upon light incidence,

the incoming monochromatic radiation creates a localised electromagnetic field which

extends up to ca. 20 nm from the metal surfaces. The Raman radiation of a molecule

within the LSPR (shown as B in Fig. 2. 2) experiences enhancement while those

outside the LSPR (A in Fig. 2.2) do not. A stronger enhancement is experienced when

the molecule is trapped within the gap between adjacent metal nanostructures, often


referred to as “hotspot”, where the LSPR of the nanostructures overlap (C in Fig

2.2)[18, 66]. Resonance enhancement can also occur when the wavelength of the

incident light is in resonance with the absorption wavelength of the analyte. A

combination of the CT, EM and resonance effects can produce an enhancement to the

Raman signal intensity up to 1014 [67, 68].

2.3.2 Fabrication of SERS substrates

Since Raman enhancement is greatly influenced by the choice of SERS

substrates, significant advances have been made on developing a variety of substrates

for SERS measurements. These substrates can generally be grouped into two

categories: (1) metal nanoparticles (MNP) in suspension and (2) metal nanostructures

on surfaces of solid platforms. The use of MNP in suspension for SERS applications

come with some advantages among which are the ease of their chemical synthesis,

without sophisticated instrumentation, the excellent Raman signal enhancement they

can generate and their ability in detecting single molecules [58]. However, they are

known to suffer some drawbacks in that, their enhancement factors are often difficult

to control. This, in part, is due to the difficulties in producing suspensions containing

nanoparticles of uniform/homogeneous sizes. Although this problem has been reduced

with recent development of methods that can produce suspensions with homogenous

nanoparticle sizes, identifying the precise location of “hotspots” in these suspensions

still remains a challenge [69]. In addition, the aggregation of MNPs are often required

for SERS observation [70]. This requirement is problematic as MNPs aggregation is

usually difficult to control. For instance, in some cases the aggregation of MNPs can

occur even before SERS spectra acquisition making data collection challenging when

using suspension based MNPs. A combination of these issues gives rise to difficulties

in signal reproducibility thereby, making reliable and reproducible Raman


measurements quite challenging. To minimise the aforementioned problems, metal

nanostructures on surfaces of solid platforms as SERS substrates have been developed.

These solid platforms have well-defined structures that provide stability to the SERS

substrate and improves / creates coupling of different SPR modes for better Raman

signal enhancement. Since the SERS active nanostructures are fixed on the surfaces of

1D, 2D and 3D solid platforms, the problem of uncontrolled nanoparticle aggregation

is essentially eliminated. Furthermore, this approach allow the control of the inter-

nanoparticle distances that are crucial in generating hotspots [71]. This is because of

the observed electric field enhancement when the inter nanoparticle distances are

within the nanometer range. There is an even sharper increase in this electric field

enhancement when the adjacent nanoparticles are at a distance <2 nm apart from each

other [72]. This was confirmed by Jiang et al [73] in their experiment using 60 nm

sized Ag nanoparticles separated by 9 nm, 3 nm and 1 nm. From their experiment, the

corresponding enhancement factor increased from 1.5 x 104 to 1.7 x 106 through to 5.5

x 109 for the 9 nm, 3 nm and 1 nm inter nanoparticle distances respectively. As a result,

numerous methods have emerged to fashion SERS active substrates with

nanostructures immobilised on solid platforms such as conductive carbon nanotubes

[74], polyaniline (PANI) materials [75], silicon wafers [76], carbon fibre cloth (CF)

[77] and indium tin oxide (ITO) [78, 79]. Examples of these methods are lithography

[80-82], dry reactive ion etching [83, 84] and atomic layer deposition [85] which have

been used extensively to build nanopatterned SERS substrates. However, the cost of

production and the instrumentation required for their fabrication are relatively

expensive and the throughput is usually low [86]. Unlike nonconductive materials, the

use of conductive solid supporting materials in the design of SERS substrates opens

up the possibility of electrophoretic and electrochemical deposition techniques which


are more cost-effective routes to homogeneous and reproducible surfaces with high

density of nanostructures [71]. With these methods, applied voltages are either used to

immobilise nanoparticles onto conductive solid platforms (by electrophoretic

deposition) [87, 88] or form nanostructures directly on such surfaces (by

electrochemical deposition) [89, 90] with good mechanical adhesion. In the case of

electrochemical deposition, nanostructures can be generated without capping agents.

This eliminates the problems of Raman fingerprint interferences which occurs for

SERS based applications using chemically synthesised nanoparticles in solution that

require capping agents to prevent severe nanoparticle aggregation [91]. In view of the

flexibility they present, conductive solid platforms, here referred to as electrodes, have

been used extensively as support materials in the fabrication of a variety of SERS

substrates.

2.3.3 Scope of this review

Different kinds of nanostructures have been developed using different

fabrication strategies towards the design of SERS active substrates and have seen a lot

of growth during the past few years (Fig. 2.3). Considering the wide range of research

reporting on the fabrication of SERS substrates using metal nanostructures on solid

platforms, it is impossible to review every contribution to this area. Hence, this review

is limited to the use of more commonly employed electrode materials such as indium

tin oxide, carbon fibers, silicon wafers, polyaniline fiber and carbon nanotubes as the

supporting solid platforms for fabricating SERS active substrate over the past decade.


Fig. 2.3 Number of publications per year from 2009 – 2018 period searched

through Web of Science using the keywords “SERS Substrate and fabrication” and

“SERS and nanostructures”

The impact that these electrodes have had on SERS substrate development as

well as the various methods of fabrication using these electrodes will be discussed.

Examples of the application of SERS substrates using these conductive support

materials in analytical science will also be detailed. Although there exist reviews

reporting on SERS [92, 93], SERS substrates [59, 94, 95] and their applications [96-

98], to the best of our knowledge, the types of solid platforms used as supporting

materials (particularly conductive solid platforms) and their impact on the SERS

substrates fabricated have not been reviewed.

2.3.4 Fabricating nanostructures on conductive solid platforms

Many supporting solid platforms have been used to develop SERS active

substrates that usually lack electrical conductivity such as quartz and paper [99, 100].

As a result, during the design of SERS active substrates, it is often difficult to introduce

fabrication processes that require the conductivity of supporting materials for the


development of nanostructures. Below are some examples of conductive solid

platforms and how they have been utilised as SERS substrates.

2.3.5 Indium tin oxide

ITO has frequently been used as a solid platform for SERS substrate

development because it is relatively inexpensive, optically transparent, its excellent

electrical conductivity and good heat tolerance [101]. For instance, taking advantage

of its electrical conductivity, Zhu et al [87] deposited a thin film of spherical gold

nanoparticles (GNPs) on ITO by electrophoresis. Fig. 2.4 shows the field emission

scanning electron micrograph of the gold nanoparticle film immobilised on an ITO.

The ITO served as both the cathode and anode using an inter-electrode distance of 3

mm and an applied voltage of 4.5 V. Random aggregation of the ≈40 to 47 nm sized

GNP sol (prepared by a seed mediated method) was prevented by the use of

cetyltrimethylammonium bromide (CTAB) as a capping agent which kept a net

positive charge on the GNPs. This net positive charge ensured mobility of the GNPs

under an applied external electric field thereby facilitating deposition onto the ITO

electrode. The as prepared SERS active substrate detected the dye rhodamine 6G

(R6G) at a concentration of 10-7 M. According to Ye et al, [89] flowerlike gold

nanostructures can be developed directly on an ITO by first coating a polydopamine

layer on a bare ITO surface. The dopamine layer facilitated rapid gold nucleation and

the abundant OH groups present in its structure acted as a capping agent which is

crucial for both GNP formation and for the control of GNP aggregation. The

polydopamine layer was made by first dipping ITO in a dopamine solution of Tris-

HCl. This approach allows for the deposition of high nanostructure density and hence

high hotspot density. This produces nanostructured substrate with high SERS

performance.


Fig. 2.4 Field emission scanning electron microscope (FSEM) images of GNPs

immobilised on ITO by electrophoresis at ≤ 2mins (a and b), 8 mins (c) and (d) High

resolution image at 8 mins. Red circles indicate sub-10-nm gaps between neighbouring

nanoparticles. From Reference [87]; Reprinted with permission of Springer.

To obtain a SERS active substrate, gold nanostructures have been

electrodeposited directly onto the polydopamine/ITO electrode via cyclic voltammetry

and by chronocoulometry. Using R6G as a probe molecule, a concentration of 10−12

M was detected showing its high sensitivity as a SERS substrate. This work indicated

that hierarchical nanostructures with a good surface coverage together with an

exceptional size and shape uniformity can also be achieved using the electrochemical

deposition technique. Bain and co-workers [78] reported the use of low precursor

metal salt concentrations with a complexing agent (such as sodium citrate) to

electrochemically generate hierarchical Ag nanostructures on ITO. A relative standard

deviation (RSD) of 28.8% in the nanostructure size distribution can be obtained with

this method. From their report, a SERS substrate fabricated in this manner can detect

R6G at a concentration as low as 10-10 M with a 14.9% RSD in their SERS signal

intensity. Another example involving nanostructure development by electrochemical


means is the highly dense silver nanoplate arrays synthesised by Liu et al [102] in 2010

using a seed-assisted electrochemical growth method (depicted in Fig. 2.5) on an ITO

electrode. These nanoplates were formed by spin coating the ITO with colloidal an Ag

solution to form Ag seeds. Subsequently, Ag nanoparticles (AgNP) were

electrodeposited on the ITO at room temperature at a low current density of 5 μA cm−2.

Under these conditions, nucleation of Ag nanoplates occurred preferentially on the Ag

seeds. A highly SERS active substrate with homogeneous SERS signal generation was

achieved from this approach with an enhancement factor (EF) of 2x105 using 4-

aminothiophenol as a demonstrator reagent.

Fig. 2.5 A schematic illustration of the preferential nucleation and oriented

growth for cross-linking Ag nanoplate arrays: (a) random-oriented Ag seeds laying on

the ITO substrate, and Ag nuclei are preferentially formed on some <110>-oriented

seeds (see arrow’s marks); (b) oriented growth of the nuclei along the fastest <110>

within (111) plane under a low deposition current density; (c) cross-linking Ag

nanoplate array structure is formed and standing on the substrate vertically. From

Reference [102]; Reprinted with permission of The Royal Society of Chemistry.

Silver micro-spheres consisting of numerous vertically aligned nanosheets have

been formed on an ITO electrode by electrodeposition in an aqueous solution of

AgNO3 (2 g/L) and citric acid (18 g/L) at a constant current of 0.17 mA for 60 mins


(Fig. 2.6) [86]. On this substrate, there was a sub-10 nm separation between adjacent

nanosheets with rough edges. Its usefulness as a SERS active substrate originates from

both the numerous hotspots found within these sub-10 nm gaps and the presence of

the rough edges. These nanostructures resulted from nucleation of Ag atoms followed

by their fusion into nanoparticle assembled micro-hemispheres. The fused

nanoparticles then formed layers of rough nanosheets aided by Oswald ripening

(which is the dissolution of small particles and their redeposition of the dissolved

species on the surfaces of larger particles). Reproducible Raman spectra for 10-15 M

R6G was observed on this substrate with an EF of 7x107 illustrating its high sensitivity.

Fig. 2.6 (a–c) Scanning electron microscope (SEM) images of Ag microhemispheres

at different magnification. The inset in (a) is the SEM side view of the

microhemispheres. (d and e) Cross-sectional SEM views of a microhemisphere. (f)

The size distribution of the Ag micro-hemispheres in (a). (g and h) Differently

magnified SEM images of an imperfect Ag microhemisphere. (i) TEM image of fan-

shaped pieces broken off from an imperfect Ag micro-hemisphere as shown in (g). The

inset in (i) is the SAED pattern from the black circle. (j) SERS spectra of 10−11 M R6G

from five randomly chosen individual micro-hemispheres. From Reference [86];

Reprinted with permission of The Royal Society of Chemistry.


Fig. 2.7 A schematic illustration of the seed mediated two step electrochemical

approach for deposition of AuNPs on ITO and its utilization for the detection of 4-

Mercaptobenzoic acid (p-MBA). From Reference [90]; Reprinted with permission of

The American Chemical Society.

Wang’s group reported a seed mediated two-step electrochemical approach to

deposit AuNPs on ITO in an electrolyte containing 0.1 mM HAuCl4 and 0.1 M NaClO4

(Fig. 2.7). [90] This approach generates monodispersed AuNPs on the electrode

surface which is vital for Raman signal reproducibility. Their method involved (i) A

nucleation step carried out by applying a potential step from +0.89 to -0.80 V (vs SCE)

for 10s to ensure nanostructures with a high density and monodispersity; and (ii) A

growth process using cyclic voltammetry at a potential range from +0.3 to -0.04 V (vs

SCE). Gold seeds with low particle size distribution (RSD of 24%) were produced in

the nucleation step and acted as sites for preferential growth of Au during the cyclic

voltammetry stage. This led to the generation of a SERS substrate with monodispersed

Au nanoparticles. An EF of 1.26x106 was obtained using 4-mercaptobenzioc acid as

an analyte with a SERS intensity deviation of only 9.2% for 30 spectra (obtained at

1074 cm-1). Another example of a two-step electrochemical approach on an ITO was

reported in 2015 where an EF of 5.9 × 108 was achieved with a limit of detection of

10-11 M for R6G [103]. The ITO electrode was coated with reduced graphene oxide–


Ag nanoparticle nanocomposite by a two-step chronoamperometric method. This

substrate was fabricated using an aqueous solution of diamminosilver ion (Ag

(NH3)2]+) and reduced graphene oxide with KNO3 as the supporting electrolyte. The

advantage of reduced graphene oxide in this approach is its high molecular absorption

capacity as well as being electron rich which greatly enhanced the charge transfer

effect in SERS measurements. Despite the many reports on ITO as a SERS support

substrate, the conductive layer can easily be damaged with little mechanical contact.

In addition, moulding them into different shapes and designs poses a challenge as

special tools are required for this because of their brittle nature. Table 2.1 below is a

list of some SERS applications using ITO-based substrates.

Table 2.1: A list of some SERS applications using ITO-based substrates

Material Fabrication method

Application Sensitivity References

Au nanodots on ITO glass

Electrodeposition HIV – 1 virus detection

35 fg/mL to 350 pg/mL

[104]

Ag nanostar pattern on ITO-glass

Electrodeposition Detection and characterization of breast cancer cells (SK-BR-3 and MCF-7)

1×105 cells/mL

[105]

Ag nanosheet-assembled micro-hemispheres on ITO-glass

Electrodeposition Polychlorinated biphenyls(PCB-1 and PCB-77) detection

3x10-7M (PCB-77) and 3x10-5(PCB-1)

[86]

Au NP on ITO-glass

Electrodeposition Detection of β-Amyloid

100 fg/mL [106]

Microfluidic chip with an Ag-Au nanocomposites modified ITO

Electrodeposition and galvanic displacement reaction

Melamine detection 0.1 nM [107]

AgNP bearing Au nanoplate on ITO

Immersion of ITO bearing Au nanoplate in AgNO3 solution

Detection of Biotin 1.0 nM [108]

ITO modified with anisotropic Ag nano-pine tree

Electrodeposition Myoglobin detection

10 ng/mL [109]


ITO modified with Ag NP

Electrodeposition Nitrate and Nitrite detection

1 ppm (nitrate) and 0.1 ppm (nitrite)

[110]

ITO modified with Au nano flowers (AuNF)

Functionalization of ITO glass with APTES followed by deposition of AuNF

CCl4-induced acute liver injury

N/A [111]

2.3.6 Polyaniline (PANI)

Polyaniline (PANI) is an inherently conductive polymer because of the

conjugated π electron system present in its structure. It has been utilised as a solid

supporting material in the design of SERS substrates due to its light weight, good

electrical conductivity, low cost, ease of synthesis and stability [112]. Unlike ITO, it

can easily be cut into different sizes and shapes due to its flexibility [113, 114]. The

abundance of reactive NH- groups in its polymer chain and the ability of these groups

to act as reducing agents promotes the formation and deposition of MNPs directly on

the surfaces of PANI, making it a good support material for SERS substrate

development. These properties were explored by Xu and co-workers [115] to

fabricate a SERS substrate via the direct formation of homogeneous 3D Ag

nanostructures on an Au coated PANI membrane. In the fabrication process, the Au

coated PANI membrane, which was obtained by immersing citric acid doped PANI

in an AuCl3 aqueous solution, was subsequently dipped into an aqueous solution of

AgNO3. This led to the formation of a continuous Ag thin film on the Au surface

followed by the growth of Ag nanospheres. The as prepared substrate, with

homogeneous nanostructures of high hotspot density (Fig. 2.8), produced a SERS

response with an average enhancement factor of 106-107 using mercaptobenzioc acid

as a demonstrator reagent. Li et al [116] also proposed a dual acid doping technique

for the fabrication of SERS active substrates from PANI. In their work, they used

ascorbic acid as a reducing agent together with succinic acid which controlled the


morphology of the Ag nanoparticles formed on the surface of PANI by regulating the

nanostructure evolution caused by ascorbic acid. This technique greatly enhanced the

growth rate of AgNPs and produced Ag nanostructures with numerous hotspots needed

for efficient SERS performance. Concentration as low as 10-10 M was detected for R6G

on this substrate.

Fig. 2.8. SEM images of (a) Au nanoparticles grown on a PANI membrane (doped by

citric acid) by immersing the PANI membrane in 10 mM AuCl3 aqueous solution for

10 s, and the Ag nanostructures produced by immersing the Au nanolayer-supported

PANI membrane in 50 mM AgNO3 aqueous solution for (b) 10 s, (c) 30 s, (d) 1 min,

(e) 10 min, and (f) 60 min. The scale bar is 500 nm. From Reference [115]; Reprinted

with permission of The American Chemical Society.

According to He et al [117], homogenous Ag nanostructures can be generated on

undoped PANI (Fig.2.9) within a minute to yield a highly sensitive SERS active

substrate. This can be achieved by introducing hydrazine and an organic acid in the

fabrication process. Hydrazine converts the emaraldine base form of PANI into the

leucoemaraldine form which is thermodynamically favourable for rapid reduction of


Ag+ ions. The organic acid, on the other hand, directs the growth of Ag nanostructures

ensuring homogeneity and wide coverage on the surfaces of PANI. Salicylic acid,

citric acid, succinic acid and lactic acid were individually used as the organic acid

component to grow nanostructure on PANI fibres by He et al [117] for comparison

purposes. The as prepared substrates were used for SERS detection of 4-

mercaptobenzoic acid. The substrates prepared with succinic and lactic acid yielded

the best SERS performance detecting 4-mercaptobenzioc acid at a concentration of 10-

8 M. Likewise, Yan et al [118] used three different AgNO3 solutions (prepared in

succinic acid, camphorsulfonic acid and lactic acid respectively) to deposited Ag

nanostructures each on three PANI fibres that has previously been doped. PANI was

first doped with the acid which was used to prepare the individual AgNO3 solution (i.e.

either succinic acid, lactic acid or camphorsulfonic acid). The lactic and succinic acid

solutions gave the best surface coverage and homogeneity (Fig. 2.10). This doping

process was crucial in achieving homogeneity and full coverage of nanostructures over

the PANI’s surface. This is because the nucleation of Ag seeds on PANI is greatly

improved by surface wettability provided by the doping process. According to this

research group, the surface wettability provided by only the acid used in the AgNO3

solution preparation was found to be insufficient in achieving full Ag nanostructure

coverage on the PANI membranes. A SERS signal response was obtained, at a

concentration as low as 10-12 M for 4-mercaptobenzioc acid, when the doped PANI

membranes were used with AgNO3 solution containing succinic acid in the substrate

fabrication process. Mondal et al [119] also fabricated a recyclable SERS substrate

using PANI fibres doped with benzene tetracarboxylic acid. The doped material acted

as a reducing agent, template and stabilizer. They were able to detect 4-

mercaptobenzoic acid and R6G by SERS in the nanomolar concentration range.


Fig. 2.9 SEM images of Ag nanostructures produced on hydrazine treated PANI

films at a reaction time of 30 s (a), 1 min (b), 2 min (c) and 5 min (d), with lactic acid

present and 30 s (e), 1 min (f), 2 min (g) and 5 min (h), with succinic acid present in

the AgNO3 solution. Scale bar: 3 mm. From Reference [117]; Reprinted with

permission of The Royal Society of Chemistry.

Fig. 2.10 SEM images of Ag nanostructures produced on the camphorsulfonic-

acid-doped PANI membranes, with (a, b) succinic acid, (c, d) lactic acid, and (e, f)

camphorsulfonic acid present in the AgNO3 solution. From Reference [118]; Reprinted

with permission of The American Chemical Society.


One drawback associated with the use of PANI is the aniline group in this

material. This group is Raman active and hence poses background interference

challenges, particularly, when the fabrication method is not tuned to properly cover

the entire polyaniline surface. Although PANI is electrically conductive, the use of

applied voltages for the development of highly dense nanostructures on their surfaces

towards SERS substrate design is still an underdeveloped area. in as much as PANI

has been used as a solid support material on which MNPs have been deposited for the

fabrication of SERS substrates, very little can be found in the literature on the use of

these SERS substrates for real life applications. However, solution based Ag@PANI

and Au@PANI have been used for the detection of Hg(II) ions at concentrations of 1

pM and 10 pM respectively [120, 121]. The detection mechanism was based on

nanoparticle aggregation triggered by the attachment of Hg(II) ions to the nitrogen

atoms of PANI.

2.3.7 Carbon based solid platforms

Carbon nanotubes (CNTs) are elongated fullerenes or rolled graphene cylinders.

They are potential surface plasmon enhancers and have attracted applications in the

fabrication of SERS substrates [122, 123]. Due to their high strength, good chemical

stability, electrical conductivity and the fact that they are nanoporous in nature, they

can be used as templates for the direct electrochemical deposition of nanostructures on

their surfaces with excellent control over nanoparticle size and density [123, 124].

Carbon fibres (CFs) have also been used in SERS substrate development due to their

flexibility, high conductivity, good corrosion resistance and large surface area. With

the emergence of CFs, the design of easily foldable and portable SERS substrates that

can be fashioned into different shapes to suite different applications [77]. Unlike PANI

and other organic based substrates, which may pose background interference problems


due to the presence of relatively high polarizable molecules with extended π–π systems

and electron-rich atoms, clean CNTs and CFs have the advantage of producing

insignificant background signal interferences [125]. Beqa et al [126] designed a SERS

substrate by decorating an aminothiophenol (ATP) functionalized CNT with popcorn

and rod shaped Au nanostructures separately and evaluated their SERS performance.

The popcorn shaped nanostructures yielded the best results due to the existence of

sharp edges and corners on the nanostructures. Whilst the central sphere of the popcorn

shaped Au nanostructures acted as electron reservoirs, their tips contributed positively

to the field enhancement by concentrating the electric field at their apexes. A 10-9 M

R6G concentration was detected and a SERS enhancement factor of 8.9 x 1011 was

observed on this substrate. Jiang et al [127] developed a nest-shaped CNT,

incorporated it in a silicon nanoporous pillar array and coated it with an Au film which

possessed nanoscale surface roughness. The nest-shaped CNT provided a large surface

area for efficient target molecule adsorption. This property, combined with the

nanoscale surface roughness of Au, leads to the SERS detection of R6G at a

concentration of 10-8 M. Bui and co-workers [128] also demonstrated the possibility

of producing patterned Au nanostructures on carbon nanotube solid platforms as

described in Fig. 2.11. The Au deposition was done electrochemically from a 1 mM

aqueous solution of HAuCl4 using chronocoulometry at a set voltage of 0.6 V vs

Ag/AgCl. A SERS enhancement factor of 3.0 x 104 was obtained using 10 mg/ mL

K3[Fe (CN)6].


Fig. 2.11 FE-SEM images of gold nanoparticles patterned onto Single walled carbon

nanotube (SWCNT) films by electrochemical deposition. The deposited charges for

(A), (B), and (C) were 1 mC, 10 mC, and 30 mC, respectively. The scale bars in the

left, middle and right columns are 100 mm, 10 mm and 1 mm, respectively. From


According to Zhao et al [77], R6G at a concentration of 10-14 M with good SERS

signal reproducibility can be realized when Ag nanoparticles are deposited on CFs. An

electroless plating method where Tollen’s reagent was used as the silver source and

glucose as the reducing agent was used. Duy et al [129] developed a nanostructured

CF substrate for both SERS and an electrochemical sensing by electrochemically

depositing Au nanodendrites on CFs from a solution containing HAuCl4, H2SO4, KI

and NH4Cl. Preferential nanodendrite formation was made possible by the introduction

of iodide which inhibited nanoparticle aggregation. 2-Naphthalenethiol was detected


at 1 nM by SERS (and a good substrate-to-substrate reproducibility with an RSD of

8.5% and an EF of 4x106) and 0.09 ppb Hg(II) by stripping voltammetry.

Noble metal-semiconductor nanocomposites such as Ag-decorated NiCo2O4

[130], Ag-decorated TiO2 [131, 132], Ag-decorated ZnO [133] and Au-decorated

SnO2 [134] have also been incorporated in the fabrication of SERS substrates. These

structures have the ability to enhance the CT mechanism of SERS via electron

promotion from the semiconductor to the MNPs. This increases the number of

electrons available for CT between adsorbed molecules and the MNPs [133, 135].

Atomic layer deposition [136], vapor-liquid-solid growth mechanism [137],

hydrothermal method [78] and electrodeposition have also been used to graft these

semiconductors onto CFs to ensure greater SERS activity. ZnO has seen much use in

SERS substrate fabrication because of its superior refractive index that promotes

strong light confinement which is essential for SERS activity [133, 135]. With respect

to this phenomenon, Wang et al [136] developed a SERS substrate by grafting ZnO

mesoporous nanosheets (ZnO-NS) on CFs using atomic layer deposition method (Fig.

2.12); the latter method involved immersion of the ZnO-seeded CFs in a precursor

solution (zinc nitrate and urea) and then placing it in an oven (at 90 °C) for subsequent

deposition of Zn4(CO3)(OH)6·H2O. The formed ZnO-NS were subsequently

decorated with AgNPs using an ion-sputtering technique. Good signal reproducibility

and sensitivity were achieved from this substrate detecting R6G at a concentration of

10−10 M. Table 2.2 below lists some SERS applications using carbon based SERS

substrates.


Fig. 2.12 ZnO-mesoporous-NSs grafted on CFs (a) Top-view SEM image. The lower-

left inset is an optical photo of a piece of ZnO-mesoporous-NSs@CFC. (b) Magnified

SEM observation on two adjacent CFs grafted with ZnO-mesoporous-NSs. (c) Close-

up SEM view of a few ZnO-mesoporous-NSs. (d) TEM image of a single ZnO-

mesoporous-NS and its corresponding selected area electron diffraction pattern. From


Table 2.2: Some selected SERS applications using Carbon-based substrates



Copper nanowires coated CF

Electrophoretic deposition

Sensing of designer drugs

10-10 – 10-12 M

[138]

CF bearing ZnO nanosheet coated with Ag nanoparticles

A combination of Atomic layer deposition, pyrolysis and ion-sputtering

Methylparathion(MP) and PCB-77

10−7 M for MP 5 × 10−6 M for PCB-77

[136]

CF with Ag coated NiCo2O4 nanorods and Ag coated SiO2 microspheres hybrid

A combination of Stober’s method, hydrothermal method and ion sputtering

Alpha fetoprotein

2.1 fg m/L [130]

Au nanoparticle coated CNT

Synthesis of CNT by chemical vapor deposition followed by the electrostatic attachment of Au nanoparticles

Glycine in solution and bio-molecules inside organelles

1 pM [139]

CNT/AuNP hybrid Chemical method Melamine in milk

1 nM [140]


2.3.8 Silicon wafers

Silicon wafers are another type of electrode that have seen a lot of use in SERS

substrate fabrication due to the excellent electronic and mechanical properties,

biocompatibility and surface tailorability of silicon [141]. Porous silicon wafers have

large surface areas which allow for the formation of nanoparticles inside their holes to

yield highly sensitive SERS active substrates [142]. Coinage MNPs can be grown in

situ on the surfaces of silicon wafers without the use of templates or surfactants. The

random aggregation of free metal NPs can be controlled by this route of synthesis

resulting in SERS substrates with excellent reproducibility [143, 144]. Shao et al [145]

demonstrated this by an in situ deposition of Cu nanoparticles (which are thought to

have limited SERS effect) on a Si wafer giving rise to an EF of 2.29x107 and an RSD

of < 20% using R6G. They reported that, the said method can be used to prevent

nanoparticle aggregation or growth that can result from laser irradiation during SERS

detection. This in situ growth of metal nanoparticles on surfaces of Si wafers can be

achieved by immersing porous silicon wafer into solutions containing the coinage

metal ion of interest. The metal ions, are reduced by Si-H bonds that can be introduced

on the wafer’s surface by a galvanic displacement reaction method. Giorgis et al [146],

Panarin et al [147] and Chursanova et al [142] have all successfully fabricated SERS

active substrates by immersing porous Si wafer into an AgNO3 solutions after forming

the Si-H bonds on the wafer’s surface by the galvanic displacement reaction method.

According to Jiang’s group [141], this method involves a hydrogen fluoride (HF)

etching technique, which can produce a highly sensitive and reproducible SERS active

substrate that is capable of yielding SERS enhancement factors of about 8.8x106 and

a RSD of 12.4%. As this process involves chemical etching, it is debatable whether

this truly is a galvanic displacement reaction. The simplicity of this method and its


ability to generate nanoparticles of high purity with good nanoparticle adhesion to

surfaces of Si wafers, motivated the Rajkumar group [148] to fabricate a SERS

substrate using this synthetic route. In their work, a discontinuous film of hierarchical

Ag nanostructures was formed on Si wafers which resulted in a substrate that could

detect R6G at a concentration of 10-16 M by SERS. Unlike glassy materials, such as

ITO, which are relatively fragile, nanoholes acting as sites for nanoparticle deposition

can be mechanically patterned in a compact Si wafer and used as a supporting material

for SERS active substrate fabrication without any breakage or deformity. For instance,

Alexander and co-workers created patterned nanohole arrays with 120 nm by 100 nm

(WxH) hole depth and a centre-to-centre spacing of 350 nm using a soft lithographic

technique, known as Pattern Replication in Nonwetting Templates (PRINT) [149].

Here, nanoclusters comprising of two 60 nm sized gold nanospheres were deposited

in these nanoholes by immersing the patterned Si wafer in an aqueous solution of

colloidal gold nanoparticles (Fig. 2.13).

Fig. 2.13 (a) Scanning electron micrograph of an array of nanoholes patterned into a

Si wafer after Cr deposition, removal of poly (lactic-co-glycolic acid) (PLGA) posts,

and reactive ion etching. (b) Aspect of whole wafer patterned with nanoholes. From

Reference [150]; Reprinted with permission of Wiley online library.


Before the deposition process, the Si wafers were made hydrophilic by plasma etching.

An EF range of 108–109 was recorded using thiophenol. Fang et al [151] also

demonstrated that, ordered arrays of silicon nanostructures can be fabricated from Si

wafers by UV photolithography. According to the authors, these nanostructured arrays

can be coated with a 30 nm thick layer of Ag followed by an Au layer (of 15 nm

thickness) using an electron beam evaporation system. The 15 nm Au layer was used

as a protective covering for the unstable Ag layer and to prevent peeling during sample

incubation periods. Although lithographic techniques generate patterned surfaces

which are essential for reproducibility in SERS, it is limited by the difficulties that

come with fabricating arrays that have interparticle separation less than 10 nm [69].

For this reason, this technique produces substrates with nanoparticle separations

limited to 10 nm - 20 nm which is larger than that theoretically predicted for giant

electric field enhancements [152, 153]. Semiconductor materials such as SiO2 and

ZnO2 can be grown into nanorods and nanopillars on Si wafers and used as platforms

for designing 3D nanostructured SERS active substrates to achieve substrates of

similar patterned surfaces. Hau et al [154] illustrated this by growing ≈140 nm length

SiO2 nanorods on Si wafers via a glancing angle deposition technique using an E beam

evaporation system. AuNPs were then sputtered on these rods forming a SERS active

substrate. The potential to reuse this substrate was shown through multiple detection

of monochlorobiphenyl congeners by washing the loaded substrate after each use with

acetone. The mechanical strength of a Si wafer and strong mechanical adhesion

between silicon based materials and metal nanostructures helped in maintaining the

substrate’s surface morphology after the repeated washing protocols. Vertically

aligned SiO2 nanowires can also be grown on Si wafers in a patterned manner by

chemical etching and the oxide assisted growth method. This is possible by initially


using a UV light to selectively expose the surface of a Si wafer covered with a

photoresist and a photomask. A metal nanoparticle layer can then be chemically

deposited on the exposed layer to act as a catalyst for Si nanowire growth by either

chemical etching or oxide assisted growth [155]. Tao et al [156] took advantage of the

ease of modifying the surface texture of Si wafers and fabricated a SERS active

substrate by first, texturizing the silicon wafer surface before hydrothermally growing

ZnO nanorods (ZnO NRs) on the texturised surface. The ZnO NRs were then decorated

with Ag nanoparticles by sputtering (Fig. 2.14).

Fig. 2.14 Schematic procedure to fabricate the 3D SERS substrate and the related SEM

images, (a) Top view of texturised Si and side view (the inset) (b) ZnO NRs on

texturised Si, (c) plasmonic structures of ZnO NRs with Ag decoration. From

Reference [156]; Reprinted with permission of Elsevier.

Texturising the surface triggered the growth of ZnO NRs at a high density, about 1.7

times that grown on a non-texturised flat surface. This high density is vital for

extensive hotspot formation. Furthermore, Si wafers can be designed into a regular

network of structured arrays on which a network of periodic spherical nanoparticle

arrays can formed. According to Zhang’s [157] research group, this structured arrays

with regular network can be formed on a Si wafer by a monolayer colloidal template-

induced solution-dipping approach. This can then be followed by the growth of


spherical nanoparticle arrays with tuneable gaps between adjacent nanoparticles using

a sputtering deposition technique. Fig. 2.15 shows the substrate fabrication process.

Zhang et al used this approach and subsequently coated the spherical nanoparticle

arrays with a 30 nm layer of Au to generate a highly sensitive and reproducible SERS

active substrate (Fig. 2.16). A detection limit as low as 10 −12 M for R6G can be

achieved on this substrate. This was reported to be due to the hydrophobicity of the

fabricated substrate which promoted rapid concentration of analyte solutions onto its

surface after surface modification with a perfluorodecanethiol compound. Another

property of Si wafers which makes it attractive as a supporting solid platform for SERS

substrate fabrication is its ability to generate surface plasmon polaritons (SPP). This

plasmon (i.e. SPP), when coupled with surface plasmon resonance (SPR), produces a

large Raman signal enhancement.

Fig. 2.15 Fabrication strategy for periodic spherical nanoparticle arrays. a) Monolayer

PSs were formed on silicon wafer by self-assembling process. b) The monolayer PSs

were heated on electric-plate at 120 °C for 25 s. c) 0.5 M Fe (NO 3) 3 with addition of

20 × 10 −3 M Triton X-100 was dropped onto the surface of monolayer PSs. d) After

drying at 110 °C for 30 min, the samples were annealing at 400 °C for 2 h to remove

the template of PSs, and regular network-structured arrays with prism-like protrusions

among three neighbouring holes were formed. e) After magnetron sputtering

deposition at 50 W for certain time, hexagonal periodic spherical nanoparticle arrays


were formed. From Reference [158]; Reprinted with permission of Wiley online

library.

Fig. 2.16 FE-SEM images of periodic spherical nanoparticle arrays using regular

network-structured arrays as sputtering deposition template (b) is expanded image of

(a) Scale bars: 500 nm. From Reference 154; Reprinted with permission of Wiley

online library.

Du et al [159] showed this by coating Ag nanoparticles on a Si wafer substrate which

was previously patterned with an array of SiO2 cuboids. A Raman enhancement of

≈2x109 was attainable when these two surface plasmons were coupled on the prepared

substrate. Some applications of SERS technique involving Si wafer-based substrates

are indicated in Table 2.3 below:

Table 2.3: Some selected SERS applications using Si wafer-based substrates



Si wafer with Ag nanodendrites

Electroless deposition

For PCB77 detection 10−10 M [160]

AgNPs bearing Si nanocones on Si wafer

Plasma etching and ion sputtering

Detection of dimethyl phthalate

10-7 M [161]

Si wafer with Si nanopillars bearing AgNPs

Electroless deposition

Detection of melamine 10-5 M [162]

Au nano rods on Si wafer

Seeded growth method followed by drop casting

Cannabinol detection 10-6 M [163]


Au nanoparticles on a thiol bearing Si wafer

Thiolation of Si wafer surface and subsequent immersion into AuNPs

Detection of Ricin 47.5 ng /mL [164]

Although there are different varieties of solid platforms on which

nanostructures have either been developed in situ or immobilised form nanoparticle

solutions to produce SERS substrates with homogenous morphologies, high density of

hotspots, good signal reproducibility and sensitivity, the added advantage of

electrodes, particularly their conductivity, have not been fully explored. Electrodes

such as Si wafers, CNTs and PANI are all electrically conductive and can support

electrophoretic and electrochemical deposition methods. However, these methods

have not been fully explored and there is room for improvement of these methods with

conductive materials. The electrophoretic and electrochemical techniques are cost-

effective, rapid, simple and robust methods for the generation of SERS active

substrates with a high density of uniformly distributed nanostructures, differently

shaped nanostructures and substrates possessing numerous hotspots needed for

excellent SERS performance. The development of SERS and electrochemical-based

dual sensors from conductive nanostructured materials have also not been fully

explored. The application of SPR-SPP coupling and semiconductor materials towards

the improvement on SERS sensitivity also needs more exploration.

2.4 Detection of toxicants by SERS

To detect environmental toxicants that have a small Raman cross-section or low

affinity to coinage metal nanoparticle by SERS, modification of the metal nanoparticle

surface is needed. Molecules with high Raman cross-section, good affinity for metal

nanoparticles and ability to recognize analytes of interest are used to modify the


nanoparticle surfaces of the SERS substrates. Such molecules are often called surface

recognition molecules. The observable changes that occur in the Raman fingerprint of

a surface recognition molecule are often used for detection and identification of

toxicants [165, 166]. Examples of such recognition molecules are DNA/RNA,

oligonucleotides, antibodies, alkylated-thiols and macrocyclic molecules [167, 168].

With the aid of recognition molecules such as viologen, thiolated cyclodextrin

and an alginate gel network, detection of different organic pollutants by SERS is

achievable. For instance, pyrene has been detected at 1 nM by a viologen modified

colloidal AgNP substrate. Chrysene, benzo[a]pyrene, pyrene and triphenylene have

also been detected by AuNPs modified with alginate gel network with detection limits

of 100 nM, 0.365 nM, 10 nM and 1000 nM respectively [169]. Additionally,

microtoxins such as ricins have been detected by SERS with a 4 μg/mL detection limit

on an Ag dendrites substrate modified with recombinant protein G, coupled to super-

paramagnetic beads [170]. 3, 3, 4, 4-Tetrachlorobiphenyl has also been detected by Su

et al using a label-free detection approach with DNA aptamer-modified Ag-nanorod

arrays. (The Ag-NR array substrate was prepared by depositing Ag nanoparticles on

an anodic aluminium oxide template by top-view ion-sputtering before functionalizing

with DNA aptamer as a recognition molecule.) This 3, 3, 4, 4-Tetrachlorobiphenyl was

sensed at a detection limit of 3.3 × 10−8 M [171]. Using a Fe3O4@C@Ag composite

as a SERS substrate, pentachlorophenol, diethylhexyl phthalate and trinitrotoluene

were detected by An et al [172]. The substrate was labelled with 4-aminobenzenethiol.

By using a direct SERS detection approach, a detection limit of 1 pM was achieved

for all the three organic toxicants. Other organic pollutants have been studied and

detected at different limits of detections using SERS (Table 2.4).


Table 2.4: Detection of some organic toxicants by SERS

Organic toxicant Substrate Limit of detection

Drawback References

Methyl parathion Mono-6-thio-β- cyclodextrin coated Au NPs

1pM Substrate with poor reproducibility

[173]

Dipterex Citrate-capped AgNPs modified with Acetylthio-choline

0.18 ng m/L Substrate with poor stability and uniformity

[174]

Tetrachlorobiphenyl

Decanethiol modified AgNPs

50pM Solution based hence difficult to use on the field

[175]

Carbaryl, phosmet, azinphos-methyl

Q-SERS™ G1 4.51 ppm, 6.51 ppm, 6.66 ppm

[11]

Pentachlorophenol Cys-Modified Ag/Cu Substrate

0.2 μM. Poor sensitivity [176]

Pyrene and anthracene

AuNPs modified with calixarene

0.5 nM Poor selectivity [177]

Aldrin, dieldrin, lindane, and α - endosulfan

Metal NP modified with aliphatic dithiol

0.1nM

Substrate with poor reproducibility

[178]

Ricin Ag dendrites modified with a 5 – thiol DNA aptamer

50 ng /mL (in orange juice) and 100 ng /mL (in milk)

A relatively expensive SERS substrate

[179]

Metal ions also have a small Raman scattering cross-section and, in most cases,

lack vibrational modes. Therefore, they are not easily recognized by SERS and hence

it is difficult to directly identify their specific Raman fingerprints in environmental

applications. Detection of metal ions by SERS is consequently done indirectly via the

observable changes that occurs in the Raman fingerprint of a surface recognition

molecule upon exposure to metal ions [165, 166]. Example of such molecules used for

the indirect detection and identification of metal ions is trithiocyanuric acid. It has high

selectivity for Cd(II) ions in water due to its strong affinity towards Cd(II) through one

of its ring amine and one thiol groups. They are chemisorbed onto SERS substrates

through bond formation between metallic NPs and the remaining thiol group and ring

amine. Using this approach, LODs and LOQs at nano concentrations can be obtained


for Cd(II) ions. This was demonstrated by Chen et al when they used trithiocyanuric

acid together with AuNP colloids as SERS substrates. LOQ and LOD of 2.9 and 8.7

nM, respectively, were reported [180]. However, the colloidal substrate used in their

work makes the reproducibility of this sensor difficult. Although citrates have been

used as capping agents, its ability to act as a recognition molecule for metal ions in

SERS has been reported by Frost et al who detected Pb(II) ions at 25 ng/L

concentrations with the citrate group acting as a recognition molecule and capping

agent at the same time. However, the selectivity of citrate was observed to be very

poor as it showed affinity for other divalent metal ions such as Hg and Cd [181].

Mercaptoisonicotinic acid is another recognition molecule that has demonstrated

selective recognition for Hg and Pb. This has been reported by Tan et al [182] using

2 - mercaptoisonicotinic acid which was adsorbed on AuNPs colloids to detect Hg and

Pb ions at concentrations of 0.3 nM and 0.1 nM, respectively. The possibility of

variable selectivity using 2 - mercaptoisonicotinic acid with masking agents was also

reported by Tan et al. This was shown when other metal ions were masked with

Na2S2O3 and L-cysteine for the selective detection of Pb(II) ions in water. Bismuthiol

II [183] and 1,4-diethynylbenzene [184] have also shown high sensitivity (with 30 nM

and 0.8 nM detection limits respectively) and selectivity for the indirect detection of

Hg(II) ions over other environmentally relevant metal ions. Cysteine is also known to

be a recognition molecule for Cu and Hg. The specific recognition of Cu and Hg by

cysteine is due to the presence of glycine in the cysteine moiety, which selectively

binds with Cu(II) and Hg(II) ions. The attachment to metallic NP surfaces and bringing

Cu and Hg ions into the vicinity of the LSPR using cysteine is through its thiol end.

According to Li et al, when cysteine is used together with 3,5-dimethoxy-4-(60-

azobenzotria- zolyl) phenol (as a Raman active reporter), detection limits of 10 pM


and 1 pM for Cu (II) and Hg(II) ions, respectively can be achieved in water [185].

Although this recognition layer produces good sensitivity and some degree of

selectivity, the colloidal NP SERS substrates used have poor reproducibility and

stability. Handling and transportation are also difficult, making its field application

unattractive.

In addition to their Raman activity, crown ethers have been used as recognition

molecules for capturing metal ions due to their high reactive properties and their ability

to discriminate between different metal ions. Their chemical performance is highly

influenced by the chelating ring size, macrocyclic rigidity and number and type of

donor atoms. Careful tuning of these factors is known to provide substantial degree of

metal ion selectivity [186]. Velu et al modified the surface of AuNPs with a 1-aza-15-

crown-5 ether acridinedione to detect Ca(II) and Mg(II) ions in solution [187].

Alizadeh et al also detected Pb(II) ions by colorimetry using AuNPs that were

modified by monoazacrown ether-terminated alkanethiolates [188]. This assay showed

high selectivity towards the Pb(II) ion. Similarly, Mariappan et al [189] and Lin et al

[190] used crown ethers and crown ether derivatives of different crown sizes to host

different guest metal ions. While Mariappan et al used thio-18-crown-5, seleno-18-

crown-5, telluro-18-crown-5 for the selective detection of Pb(II) ions, AuNPs

modified with crown-tagged alkanethiols was used for K+ detection in water by Lin et

al. The possible recognition of Zn(II) ions by crown ethers has also been proven by

the use of crown ether derivatives such as 5,6,14,15-dibenzo-1,4-dioxa-8,12-

diazacyclopentadeca-5,14-diene [191]. Likewise, Hamidinia et al reported the

potential use of cyclohexane carboxylic acid-capped 15-crown-5 for the treatment of

Pb and Cd intoxication [192]. Aminodibenzo-18-crown-6 and its derivatives have been

used for the detection of Hg(II) ions in water due to its ability to selectively recognize


the mercuric ion even in the presence of other environmentally important metal ions.

For instance Patil et al reported the ability of synthesised enamine derivatives of

dibenzo-18-crown-6 to detect Hg(II) ions in water [193] while 4, 4-bis-(carboxyl

phenylazo)-dibenzo-18-crown-6 has also shown high recognition for Hg(II) ions in

water. These derivatives showed selectivity towards Hg(II) ions in the presence of

other environmentally relevant ions such as K+, Mg2+, Ca2+, Ba2+, Na+, Ni2+, Cd2+, Ag+,

Cu2+, Pb2+, Al3+, Co3+, Fe3+, Eu3+ and Dy3+ with a detection limit of 2.9×10−8 M using

colorimetry [194]. Most of these studies with crown ethers were conducted using UV

visible and fluorescence spectroscopy. The use of these techniques with colloidal

AuNPs by these researchers was possible since the plasmonic band of Au is in the

visible region of the electromagnetic spectrum. A distinct and observable colour

change of the colloidal AuNPs occurs when the localised surface plasmon resonance

band location changes due to the interaction between the metal ions and the recognition

molecule. However, the poor sensitivity of UV visible spectroscopy as well as the

background, spectral and chemical interference involved in the use of UV visible and

fluorescence spectroscopy present some limitations. In addition, they are both

destructive techniques [195, 196]. Although all the above recognition molecules and

their accompanied detection techniques can potentially be used for monitoring and

controlling toxicants, they either have poor selectivity or contain substrates with poor

reproducibility and low stability. The difficulty associated with the handling of some

of these substrates makes quick field detection of these toxicants difficult.

2.5 Implications from review

The literature review indicated that different analytical techniques have been

used extensively to detect and quantify a variety of toxicants successfully in different

matrices. Among these were AAS, fluorescence spectroscopy, INAA, UV visible


spectroscopy, HPLC and hyphenated techniques such as ICP-MS, LC–MS/MS,

HPLC-MS/MS, HPLC-DAD, LC-ESI-MS/MS and GC-APLI-MS. Despite the success

rate in their applications, these techniques were demonstrated to have several

drawbacks. Among these are their sophisticated and bulky nature, making them

difficult. Among these are the sophisticated and bulky nature of some, making them

difficult to operate and not attractive for field applications. Those that are less

sophisticated and portable may either be relatively insensitive or less selective. In some

cases, they suffer from background and spectral interferences.

Electrochemical and SERS techniques can be used for field analysis due to the

availability of easy-to-operate handheld devices. Their selectivity can be enhanced

either by surface functionalisation or a pre-analysis extraction/clean up protocol.

Although the electrochemical technique can detect analytes at relatively high

concentrations compared to SERS detection, their use for identification and

characterization of target analytes is sometimes challenging. The SERS technique on

the other hand, can be used for the identification and characterisation of target analytes

even at ultra-trace concentrations though spectra reproducibility can be a major

challenge.

Therefore, there is a need for an analytical technique that combines SERS and

electrochemical techniques to detect a wide range of toxicant concentrations. In

addition, the technique should be highly reproducible, easy- to-handle, stable, field

deployable, rapid, sensitive and selective.

As a result, this thesis is designed to address this gap in knowledge in the field.

The ensuing chapters describe the specific experiments and results obtained in the

quest to fabricate suitable substrates for rapid, sensitive, selective, portable, field


deployable and cost-effective SERS and electrochemical detection of some commonly

encountered toxicants.

Chapter 3: Fabrication of crown ether functionalised Au nanostructured substrate for trace detection and quantification of Hg (II) ion 47

Chapter 3: Fabrication of crown ether functionalised Au

nanostructured substrate for trace detection and

quantification of Hg (II) ion

This chapter is made up of the published journal article below:

D. K. Sarfo, A. Sivanesan, E. L. Izake and G. A. Ayoko, Rapid detection of mercury

contamination in water by surface enhanced Raman spectroscopy, RSC Advances,

2017, 7, 21567-21575

48 Chapter 3: Fabrication of crown ether functionalised Au nanostructured substrate for trace detection and quantification of Hg (II) ion

STATEMENT OF CONTRIBUTION OF CO-AUTHORS

The authors listed below have certified* that:

1. they meet the criteria for authorship in that they have participated in the conception, execution, or interpretation, of at least that part of the publication in their field of expertise;

2. they take public responsibility for their part of the publication, except for the responsible author who accepts overall responsibility for the publication;

3. there are no other authors of the publication according to these criteria;

4. potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or publisher of journals or other publications, and (c) the head of the responsible academic unit, and

5. they agree to the use of the publication in the student’s thesis and its publication on the QUT ePrints database consistent with any limitations set by publisher requirements.

In the case of this chapter: D. K. Sarfo, A. Sivanesan, E. L. Izake and G. A. Ayoko, Rapid detection of mercury contamination in water by surface enhanced Raman spectroscopy, RSC Advances, 2017, 7, 21567-21575

Contributor Statement of contribution*

D. K. Sarfo Conducted experiments, collected and analysed data and wrote the manuscript

A. Sivanesan Supervised and aided experimental work and manuscript editing

E. L. Izake Supervision of research and major manuscript editing

G. A. Ayoko Supervision of research and major manuscript editing

Principal Supervisor Confirmation

I have sighted email or other correspondence from all Co-authors confirming their certifying authorship.

------------------------------------ ------------------------------------ ------------------------------------Name Signature Date


Preface

From the literature reviewed in the previous chapter, it was evident that an

analytical technique that is sensitive, selective, cost-effective and field deployable is

vital for the detection and monitoring of toxicants. Due to the availability of handheld

Raman and electrochemical devices, it was realised that, if SERS and electrochemistry

can be used as complementary techniques, not only will it be possible to detect

toxicants within a wide range of concentrations, but, field analysis can also be

achieved. Hence, this chapter had the objective of developing a nanosensor platform

together with a requisite recognition molecule that is suitable for the rapid detection of

mercury contamination in water by surface enhanced Raman spectroscopy. To this

end, a nanostructured platform was designed by electrochemically depositing Au

nanostructures onto a gold disc. The surface of the designed substrate was then

functionalised with a thiolated aminodibenzo-18-crown-6 derivative for selective

detection and quantification of the notorious toxicant, Hg(II) ions. The possibility of

using this substrate for toxicant analysis by electrochemistry was also demonstrated.


Abstract

Mercury (Hg) is a potent neurotoxin in fish, wildlife, and humans. The

detection of Hg(II) ions in water therefore requires accurate, ultra-sensitive, rapid and

cost effective analytical methods. We present a novel nanosensor for the field detection

of Hg(II) in water by surface enhanced Raman spectroscopy (SERS). In the new SERS

nanosensor, aminodibenzo-18-crown-6 (ADB18C6) was coupled with

mercaptopropionic acid and the resultant crown ether derivative (TCE) was self-

assembled as a recognition surface layer for Hg(II) onto the surface of a nanostructured

gold substrate. The coordination of Hg(II) to the oxygen atoms of TCE led to the

spontaneous binding of the metal ion into the cavity of the crown ether layer. This

caused the intensity of the Raman band at 1501 cm-1 for the crown ether to increase

linearly with the concentration of the Hg(II) in the range of 1 x 10-11 M to 1 x 10-6 M.

Complexation between TCE and Hg(II) was further confirmed by UV-visible

spectrometry, spectrofluorimetry and electrochemistry. The method was successfully

applied to the determination of Hg(II) in tap water using a handheld Raman

spectrometer and it demonstrated high selectivity towards Hg(II) in the presence of

Pb(II) and Cd(II). This eliminated the need for extensive sample preparation and

extraction procedures prior to the analysis. The lower limit of Hg(II) quantification by

the new SERS nanosensor and the limit of detection were 1000 fold below the EPA

and WHO defined levels for Hg(II) ions in water.

3.1 Introduction

Mercury is a heavy transition-metal that is widely released into the

environment from anthropogenic sources (e.g. mining, solid waste incineration etc)

and natural activities (e.g. volcanic and oceanic emissions). Even at very low

concentrations, Hg(II) has highly toxic and deleterious effects on both the environment


and humans [183]. Exposure to Hg (II) can lead to brain damage, heart failure,

anaemia, kidney damage and nervous system disorders. Because of its abundance,

stability and good water solubility, Hg(II) accumulates in water and, subsequently, in

humans as well as other living organisms through the intake of Hg(II) contaminated

water [14, 182]. The maximum allowable concentration of Hg (II) in drinking water

has been indicated by the U.S. Environmental Protection Agency (USEPA) as 2 μg/L

(10 nM). Therefore, rapid detection and removal of Hg (II) is vital for its monitoring,

control and remediation.

Consequently, various analytical techniques have evolved over the past decade

for the detection and quantification of Hg (II) in water. Among these are inductively

coupled plasma mass spectrometry (ICP-MS)[197], colorimetry and

chemiluminescence [198], atomic absorption spectroscopy and X-ray fluorescence

spectrometry [199], high performance liquid chromatography [200], neutron

activation analysis(NAA) [201] and enzyme linked immune sorbent assay (ELISA)

[202]. However, most of these analytical methods require time-consuming sample

preparation procedures, expensive instrumental infrastructure and, are therefore, not

suitable for remote or on-site applications.

SERS is an ultra-sensitive analytical method that can be utilized for the rapid

field detection of Hg (II) in environmental matrices with minimum or no sample pre-

treatment [97, 203]. Many examples of SERS detection of Hg (II) in water were

demonstrated recently in literature [204-206]. In these examples various approaches

were adopted for the metal ion detection, such as the use of Raman reporters or the

utilization of the thymidine-Hg(II)- thymidine coordination chemistry [97].

Generally, metal ions are known to have small Raman scattering cross-section

with no vibrational modes making their direct detection by SERS difficult. Therefore,


SERS detection of Hg (II) usually adopts an indirect approach. In this approach, a

Raman reporter is used to detect the presence of Hg (II) in the sample. The SERS

signals of the Raman reporter is monitored and the changes in the SERS spectrum of

the reporter is attributed to its interaction with the mercuric ions [97]. Among the

Raman active molecules that can be utilized for the SERS detection of Hg (II) are

crown ethers. In addition to their Raman activity, crown ethers are excellent ligands

that can form complexes with metal ions. The ability of crown ethers to form

coordination complexes with different metal ions is highly influenced by the chelating

ring size, macrocycle rigidity and number and type of donor atoms present[207]. In

addition, the geometric shape, charge density and ionic radius of the metal ions also

affect its selective reactivity towards specific crown ethers [208, 209]. Careful tuning

of these factors can lead to the formation of a highly selective complex between a

given crown ether and a specific metal ion [186]. Many researchers have utilized

crown ether derivatives of different crown sizes to host different guest metal ions [188,

191, 192]. The selective complexation of Hg (II) over a wide range of environmentally

relevant metal ions by dibenzo 18-crown-6 azo dye derivatives using UV–visible and

fluorescence spectrometry has been reported in the literature [194, 210, 211]. In this

work, a different mode of detection (i.e. SERS) with high sensitivity and a new

aminodibenzo-18-crown-6 (ADB18C6) derivative was used. ADB18C6 was coupled

with mercaptopropionic acid (MPA) to form a thiolated crown ether derivative (TCE)

that was utilized as a recognition molecule for the selective sensing of Hg (II) in water

by SERS. The advantage of TCE is its ability to form a self-assembled monolayer by

firmly attaching itself unto a SERS substrate through the strong Au-S bond.

To improve upon the reproducibility of SERS measurements, the firm

attachment of the recognition molecule to the surface of a SERS substrate is important.


Several approaches have been demonstrated for the attachment of recognition

molecules onto SERS substrates. For instance, alkane thiol compounds can be used as

a linker to attach recognition molecules to the nanostructures of a gold SERS substrate

[20, 212, 213]. In this scenario, the thiol group of the linker molecule interact with the

gold surface via stable Au-S bond and the other end of the linker molecule may carry

an amine group that forms an amide linkage with an active carboxyl group on the

recognition molecule [18, 20]. In this work, MPA was utilized as a linker between a

nanostructured gold substrate and the ADB18C6 recognition molecule. The carboxylic

functional group of MPA was attached to the primary amine end of ADB18C6 through

EDC coupling reaction [214]. The gold nanostructures were electrochemically

prepared and deposited onto the surface of the solid gold substrate. This approach for

preparing the nanostructured gold substrate is relatively fast, does not involve the use

of capping agents (which may cause spectral interferences) and eliminates the problem

of uncontrolled nanoparticle aggregation. To demonstrate the potential of the new

nanosensor for real time analysis, a handheld Raman spectrometer was utilized for the

SERS detection of Hg (II) contamination in water samples at the ultra-trace level down

to 0.51pM.

3.2 Experimental

3.2.1 Chemicals and reagents

All reagents and solvents were of analytical grade and used without further

purification. Concentrated sulfuric acid (98%), hydrogen peroxide solution (30%),

mercaptopropionic acid (MPA), 4-amino-dibenzo- 18-crown-6 (ADB18C6), , Gold

(III) chloride (HAuCl4), phosphate buffered saline (PBS), N-hydroxysuccinimide

(NHS), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC),

Dimethyl sulphoxide (DMSO), Pb(NO3)2, Cd(NO3)2, and Hg(NO3)2 were all purchased


from Sigma-Aldrich (USA). All solutions were prepared using ultra-pure water

(18.2MΩ.cm@25 °C, Milli-Q).

3.2.2 UV–visible and fluorescence studies

To investigate the complex formation between ADB18C6 and Hg(II), 1mL of

1mM ADB18C6 (in DMSO: H2O, 1:1 v/v) solution was mixed with 150 µl of 10µM

HCl. This was followed by the addition of 1 ml of the Hg(II) aqueous solutions in the

concentration range of 20 mM, 40 mM, 60 mM and 80 mM. The mixtures were

transferred into cuvettes and their UV absorptions were measured in the wavelength

range 250 nm to 500 nm. To the same mixtures, fluorescence emissions between 280

nm and 800 nm were measured at an excitation wavelength of 281nm. To investigate

the effect of interfering ions on the UV and fluorescence measurements of Hg(II), the

above procedures were repeated after the addition of 1 mL of 80 mM Cd(II) and 80

mM Pb(II) to the ADB18C6/HCl/ Hg(II) mixtures.

3.2.3 ADB18C6 coupling to MPA

EDC (0.2 M) and NHS (0.1 M) were prepared in PBS (10 mM, pH 7.0) and

mixed together (50:50, v/v). One ml of MPA (0.4 mM in PBS, pH 7) was then added

to 1ml of EDC/NHS mixture. The reaction mixture was allowed to stand for 15

minutes to ensure the complete activation of the COOH terminal group of MPA. To

the MPA/EDC/NHS mixture, 1ml of ADB18C6 (1mM in DMSO) was added to form

an amide bond between the amine group of the crown ether and the activated COOH

group of the MPA. The MPA-coupled ADB18C6 was labelled “TCE” and used

directly as a recognition molecule for the detection of Hg (II). The scheme for the TCE

ligand formation is shown in Appendix A1 (A1).


3.2.4 Electrochemical deposition of gold nanostructures on a gold substrate

The surface of the gold substrates were polished with 0.5, 0.3 and 0.05 µm

grain sized alumina slurry respectively. The substrates were then rinsed with water and

sonicated in Millipore water for about 15 minutes to remove all the physically

adsorbed alumina slurry. The polished substrates were then soaked in piranha solution

(3 parts of 98% sulfuric acid and 1 part of 30% hydrogen peroxide solution) for 10

minutes and then rinsed with Millipore water. HAuCl4 solution (4 mM in 0.1 M

HClO4) was used for the electrochemical deposition of gold nanostructures onto the

surface of the gold substrate using the method described by Sivanesan et al [215] after

a minor modification where the deposition time was increased from 400s to 900s. This

was done to increase the number of gold nanostructures and hence the number of

hotspots available for SERS enhancement. Finally, the substrates were treated with

oxygen plasma for 15 minutes to remove any organic molecule that may become

adsorbed to the nanostructured gold surfaces (A 2). The prepared substrates were

characterized and used as a SERS active substrate for Raman measurement of Hg(II).

3.2.5 Surface functionalization of the nanostructured gold substrate with crown

ethers

The nanostructured gold substrates were functionalized with crown ethers by

their incubation in 500 µL of ADB18C6 and TCE solutions overnight. The substrates

were then removed from solution and washed with PBS. It was again washed with

ultra-pure water to remove all unbounded crown ether and other unreactive molecules

from the surface. To prevent the non-specific binding of foreign molecules to the bare

area of the nanostructured gold surface, the functionalized substrates were dipped into

butanethiol (10-7 M aqueous solution). This procedure was carried out for 2 hours in

order to allow the butanethiol molecules to backfill the bare gold sites on the substrate


surface via stable Au-S bonds. The functionalized and backfilled nanostructured gold

substrates were then washed several times to remove the excess butanethiol from their

surfaces.

3.2.6 Cyclic voltammetry measurements of the complex formation between Hg

(II) and tADB18C6

To confirm the complex formation between the TCE surface layer of the

functionalized nanostructured substrates and Hg (II) ions by electrochemistry, the bare

nanostructured gold substrate and the TCE -functionalized substrate were first

screened by cyclic voltammetry (CV). The voltammograms were acquired in 100mM

PBS solution as an electrolyte. The CV scans (6 cycles) were carried out between -

0.5 to 0.6V at a scan rate of 0.1 V/s. Ag/AgCl was used as the reference electrode and

a platinum wire as an auxiliary electrode. After the first set of CV measurements, Hg

(II) standard solutions at 1µM concentration were loaded onto TCE-functionalized

substrates and allowed to stand for 15 min. The CV measurements were then repeated

and the oxidation and reduction peaks at 0.40V and 0.35V, respectively, of the Hg (II)

ion were monitored after washing severally with milli-Q water.

Scheme 3.1: A schematic representation for the modification of nanostructured gold

substrate by TCE and subsequent binding of Hg (II)


3.2.7 SERS detection of Hg (II) by the functionalized nanostructured gold

substrates

The TCE-functionalized and ADB18C6-functionalized gold substrates were

used for the detection of Hg(II) (A 3). Prior to this, the background spectra of the bare

AuNS coated gold substrate was acquired (A 4). These substrates were screened by

Raman spectroscopy to acquire their native SERS spectra. The spectrum for MPA was

also recorded (A 5). It is noteworthy that these SERS spectra were acquired using a

handheld Raman spectrometer with the “reference spectrum” and “clean peaks” modes

enabled. With the “reference spectrum” enabled, ambient light and other

environmental background as well as any fixed pattern noise is excluded. The “clean

peaks” mode is an in built beta correction algorithm that does background correction

and removes the effect of fluorescence (A 4). These spectra were also collected in the

raster orbital scanning mode (over a wavelength range of 400 cm-1 to 2000 cm-1),

which rapidly scans a tightly focused beam in an orbital pattern, allowing lower

average power to produce high-integrity data from a larger area of the sample without

sample damage. An excitation wavelength of 785 nm laser line was used for the SERS

measurements. The laser power at the sample was 10 mW while the scan time was set

at 2 accumulations and 3 seconds integration time. After these sets of SERS

measurements, 1mL aliquots of Hg(II) standard solutions, in the concentration range

1µM to 0.1pM, were loaded onto the functionalized gold substrates for 5 min to allow

for the complex formation between the crown ether surface layer of the substrate and

Hg(II) ions. The SERS spectra of the ADB18C6- functionalized and the TCE-

functionalized substrates were then repeated after the complex formation (Scheme

3.1). A calibration curve was plotted between the SERS signal intensity at 1501 cm-1

and the concentrations of the Hg(II) standard solutions.


3.2.8 Determination of Detection limit and lower limit of quantification

In order to determine the detection and quantification limits of Hg(II) ions by

SERS measurement, seven 0.1pM Hg(II) bound TCE nanosensors were screened with

the handheld Raman spectrometer and the concentration was determined (A 6). The

LOD and LOQ were calculated as the standard deviation (σ) x t95% and σ x10

respectively (Where t is the threshold value of student t-distribution with a degree of

freedom (n - 1) at 95% confidence interval)[216].

3.2.9 Selectivity of tADB18C6 towards Hg (II)

The selectivity of TCE crown ether towards Hg (II) was tested using 1 mL

aliquots of 1µM Pb(II), Cd(II) and their mixed ion ( Pb(II) and Cd(II)) standard

solutions. These solutions were loaded onto individual TCE -functionalized substrates

and allowed to stand for 5 mins and the SERS spectra were acquired with the handheld

Raman spectrometer as previously described. The spectra of the functionalized

substrates before and after interaction with the metal ions were compared (A 7).

3.2.10 Determination of Hg (II) in tap water by tADB18C6-functinalized gold

substrate and handheld Raman spectrometer

To utilize the TCE -functinalized gold substrate as a new nanosensor for Hg (II)

contamination studies in the environment, 1mL of tap water was collected and spiked

with 25µL of 1µM Hg (II). The spiked water sample was then loaded onto the TCE-

functinalized gold substrate and allowed to stand for 15 mins. SERS measurements

were carried out using the handheld Raman device (n=3). The concentrations were

calculated from the calibration curve and the percentage recovery determined from the

relation:


% Recovery =𝐶𝐶𝐶𝐶𝐶𝐶(𝑥𝑥) − 𝐶𝐶𝐶𝐶𝐶𝐶(𝑏𝑏)

𝐶𝐶𝐶𝐶𝐶𝐶(𝑠𝑠) 𝑋𝑋 100

Where CHg(x), CHg(b) and CHg(s) represent the concentration of: Hg (II) as

calculated from the SERS calibration curve, blank sample and the spiked Hg (II)

standard respectively.

3.2.11 Instrumentation and characterization

The nanostructured gold substrate was characterized by Scanning Electron

Microscopy (SEM) to determine the geometry, size and distribution of the developed

gold nanostructures. The UV absorption spectra were recorded by Cary 60 UV–visible

spectrophotometer (Agilent Technologies, USA). Fluorescence Cary eclipse

spectrophotometer was used for fluorescence measurements. SERS measurements

were carried out using the handheld ID Raman Mini2 (ocean optics, USA). A μAutolab

potentiostat (Metrohm Autolab) with a custom made three-electrode cell setup was

also used for all electrochemical measurements.

3.3 Results and Discussion

3.3.1 UV–visible and fluorescence studies on Hg (II)

The absorption spectra of ADB18C6 in the absence and presence of Hg (II) are

shown in Fig. 3.1. ADB18C6 showed a shoulder band at 275nm and absorption bands

at 280 nm and 295 nm respectively (Fig. 3.1A). The absorption band at 295 nm can be

attributed to the n→π* transitions between the oxygen atoms electron lone pairs and

the crown ether ring of ADB18C6. The band at 280 nm can be attributed to π

→π*transitions within the unsaturated aromatic benzene rings on the crown ether

molecule. The shoulder at 275 nm may also be due to n→π transitions between the

amino group and the benzene ring of the aniline moiety of the crown ether system


[217, 218]. Upon the addition of different concentrations of Hg (II) ions, the absorption

band at 295 nm decreased and was completely quenched at 80 µM of Hg (II)

concentration (Fig. 3.1B spectra a - d).

Fig. 3.1, UV-visible spectra of (A) ADB18C6 and (B) ADB18C6 in different

concentrations of Hg (II): (a) 20 (b) 40, (c) 60 and (d) 80 µM.

The decrease in the 295nm band intensity indicated that a host-guest interaction

between Hg (II) and 18-Crown-6 where the Hg (II) metal ions are chelated by the

oxygen atoms of the crown ether [214, 219]. This leads to the formation of a stable

complex between ADB18C6 and Hg (II). A closer look at the spectra (a-d in Fig. 3.1B)

reveals an isosbestic point at 287nm which is attributed to the presence of both the Hg

(II) – ADB18C6 complex and the free ADB18C6 in solution[220]. The fluorescence

spectra of ADB18C6 in the absence and presence of Hg (II) are likewise shown in Fig.

3.2. As indicated by the figure, emission bands at 311 nm, 360 nm, 563 and 612 nm

were observed in the native spectra of ADB18C6. After the addition of different

concentrations of Hg (II), the band at 311 nm increased while the intensity of the band

at 360 nm quenched gradually (spectra a – e). Moreover, the addition of Hg (II) led to

an increase in the intensity of the emission bands at 563 nm and 612 nm (Fig. 3.2).


The quenching of the band at 360 nm and the increased intensity of the 311 nm, 563

nm and 612 nm can be attributed to the formation of the Hg (II) - ADB18C6 complex.

Fig 3.2 Fluorescence spectra of ADB18C6 in different concentrations of Hg (II): (a)

0, (b) 20, (c) 40, (d) 60 and (e) 80 µM.

Fig. 3.3 (A) UV Absorption spectra and (B) Fluorescence emission spectra of

ADB18C6 crown ether after reaction with Hg (II), Cd (II) and Pb (II) ions respectively.


3.3.2 Selectivity of ADB18C6 crown ether towards Hg (II)

The selectivity of ADB18C6 towards Hg(II) over 19 other metal ions ( i.e. K+,

Li+, Na+, Ag+, Ca2+, Mg2+, Ba2+, Sr2+, Ni2+, Co2+, Cd2+, Pb2+, Cu2+, Fe3+, Al3+, La3+,

Eu3+ and Dy3+has been reported in literature [194, 210] . In this work, Cadmium (II)

and Pb(II) ions were chosen for the selectivity studies using UV visible and

fluorescence spectroscopy because of the similar chemical properties between Cd (II),

Pb(II) and Hg(II) and the fact that these toxic heavy metals are among the commonly

known environmental pollutants. The UV absorption (Fig. 3.3A) and fluorescence

emission spectroscopy (Fig. 3.3B) showed no observable changes in the absorption

and fluorescence spectra of ADB18C6 after the addition of Cd(II) and Pb(II). These

results confirmed the selective complex formation between ADB18C6 and Hg(II).

3.3.3 Thiolation of ADB18C6 with MPA

To utilize ADB18C6 for the nanosensing of Hg (II) by SERS, it is necessary

to immobilize the crown ether onto the surface of a SERS substrate where it acts as a

recognition layer for the mercuric ions. For this purpose, the carboxylic acid group of

the alkane thiol (i.e. MPA) was coupled to the crown ether using the EDC/NHS

coupling reaction to produce the thiolated crown ether “TCE”. Upon interaction with

gold surface, the free thiol (SH) terminal group of TCE forms a stable Au-S bond with

the gold nanostructures to assemble the crown ether molecules in a highly ordered

monolayer with a common orientation. The assembled crown ether was subsequently

used to bind Hg (II) ions.

3.3.4 Study of complex formation between Hg (II) and TCE by electrochemistry

To confirm the complex formation between the crown ether and Hg (II) ions

on the functionalized nanostructured surface, the TCE-functionalized gold substrate


was screened by cyclic voltammetry (CV). The cyclic voltammogram of the bare

nanostructured gold substrate (Bare Au), the TCE-functionalized gold substrate

(Au/TCE) and the TCE-functionalized gold substrate after binding with Hg (II) are

shown in Fig. 3.4. As indicated by the figure, there was no observable redox peak(s)

in the voltammogram of the bare nanostructured gold substrate. However, two well-

defined redox peaks appeared in the voltammogram of TCE-functionalized gold

substrate at 0.20V (oxidation peak) and 0.14V (reduction peak) respectively. New

oxidation and reduction peaks at 0.40V and 0.35V, respectively, appeared in the

voltammogram of TCE-functionalized gold substrate after its interaction with Hg (II)

ions (Au/ TCE /Hg). The emergence of these two peaks indicated the binding of Hg

(II) to the host crown ether [221-224] and they may be attributed to the inclusion of

Hg (II) in the cavity of 18-crown-6 moiety of TCE and the accompanied corresponding

changes to the redox potential of the substrate [225, 226].

Fig. 3.4 Cyclic Voltammograms of bare nanostructured gold substrate (Bare Au),

TCE-functionalized gold substrate (Au/TCE) and TCE-functionalized gold substrate

after complex formation with Hg (II) [(Au/ TCE /Hg)]


3.3.5 SERS nanosensing of Hg (II) by the tADB18C6-functionalized gold

substrate

Some research groups [203, 227-229] have reported SERS studies that utilizes

colloidal nanoparticles for the detection of mercury contamination in various

environmental matrices. While some of these SERS sensors feature high selectivity,

the colloidal nanoparticles are quiet unstable since they are prone to uncontrolled

aggregation and, in effect, create non-reproducibility challenges in their SERS

measurements. Unlike colloidal substrates, solid SERS-active substrates offer the

advantages of easy handling, long term stability and adaptability to in-field screening

[230, 231]. We recently developed a solid nanostructured gold substrate for highly

reproducible SERS measurements. The substrate was manufactured by

electrochemical deposition of gold nanostructures onto a gold surface to deliver high

density of hot spots and high SERS enhancement factor[215, 230]. In the present work,

we functionalized this substrate with ADB18C6 and TCE and was used as a

nanosensor for the detection of Hg (II). The SERS spectra for both ADB18C6 and

TCE after their immobilization onto the nanostructured gold substrate (Fig. 3.5) were

recorded with the handheld ID Raman mini 2 and the corresponding band assignments

given in Table 3.1. Since the handheld device has the ability to perform fluorescence

and background interference corrections, the spectra is reported as raw data. This saves

time, giving it advantage over a bench top spectrometer.


Fig. 3.5 SERS spectra of ADB18C6 and TCE on nanostructured gold substrate

Table 3.1: SERS wavenumbers/cm of ADB18C6 and TCE and their band assignments

Raman shift/cm Band assignment References ADB18C6 TCE ADB18C6 TCE 496 596 ∂ (C-O-C),sym Au-S [168] 734 Cph-H, out of plane

bending [232]

813 ѵ (C-O-C),sym [168] 938 Cph-H, out of plane

bending [232]

1137 ѵ (C-O-C),asym [168, 232] 1327 CH2 - wag,

C-C-H deformation [168, 232,

233] 1440 1437 H-C-H deformation √ [168] 1501 1492 Cph-O str., Cph-H √ [232] 1599 1602 C=C str. ( aromatic) √ [234]

When ADB18C6 is adsorbed onto the substrate, Au-N and Au-O bonds are formed

between the substrate’s gold nanostructures and the amine and oxygen groups of

ADB18C6 respectively. As a result, we propose that the crown ether moiety assumed

a horizontal position to the gold surface. The appearance of the out-of-plane

vibrational modes at 734 and 938 cm-1 indicated that, these modes of vibration adopted

a perpendicular orientation to the gold surface while the crown ether moiety lies

horizontal to this nanostructured gold surface [235, 236]. In this orientation, since the

ADB18C6 molecule lies close to the nanostructured gold substrate’s surface, it

experiences a strong localized surface plasmon resonance effect (LSPR). The close


proximity of the ADB18C6 molecule to the LSPR and the fact that its scattering tensor

components for the out-of-plane vibration modes lie perpendicularly to the gold

surface, led to a strong Raman intensity enhancement observed in the spectra of

ADB18C6 [235, 236].

The Raman spectra of TCE has relatively low intensities compared to the spectra of

ADB18C6 (Fig. 3.5). These changes may be attributed to a non-horizontal orientation

of TCE onto the substrate’s surface. Thiolated ADB18C6 forms a strong Au-S bond

between its MPA moiety and the nanostructures of the gold substrate. Therefore, TCE

may assume a more vertical position on the substrate’s surface when compared to that

of ADB18C6 [213, 237, 238]. The vertical orientation of TCE causes the crown ether

structure to lie further away from the effective range of the LSPR. Since the LPSR

effect decays exponentially with the distance from the gold surface [18], a significant

reduction in the Raman signal intensity was observed for TCE. The Raman bands at

1501 cm-1 experienced a strong reduction in its intensity as well as a bathochromic

shift to 1492 cm-1 [235, 239]. In addition, the low intensities of the bands at 1492 cm-

1 and 1599 cm-1 suggested that the phenyl rings of the crown ether structure are not

directly adsorbed onto the substrate surface and, therefore, their interaction with the

LSPR is weak [20]. The vertical orientation of TCE also causes the out-of-plane

vibrational modes at 734 cm-1 and 938 cm-1 to lie parallel to the surface of the substrate

[236, 238]. Hence, these vibrational modes become Raman inactive leading to a

quenching of the 734 cm-1 and 938 cm-1 bands in the Raman spectrum of TCE.

The TCE -functionalization gold substrate was utilized for the SERS detection

of Hg (II) ions. Upon complexation of Hg (II) ions with the TCE surface layer of the

functionalized substrate, the Raman spectrum of TCE experienced significant changes

(Fig. 3.6). As illustrated by Fig 3.6, the bands at 1327 cm-1 and 1501 cm-1 and the H-


C-H deformation band at 1454 cm-1 reappeared. These changes may be attributed to

the structural deformation of TCE upon its complexation with Hg (II) [232, 240]. In

addition, the affinity of Hg (II) to gold may cause the mercuric ions to move close to

the substrates surface. This will force the TCE to move to the proximity of the

substrates surface in order to chelate the mercuric ions that lie at the gold interface.

These combined effects cause the crown ether structure to align itself within the LSPR

of the SERS substrate [233]. This in turn, led to the strong enhancement of the Raman

spectrum of TCE -Hg (II) complex compared to that of TCE alone.

Fig. 3.6 SERS spectra of TCE and TCE -Hg (II) complex

Fig. 3.7 A linear correlation plot of the Raman intensity (at 1501) versus the logarithm

of Hg(II) concentration and corresponding SERS spectra(Insect) from (a) 1x10-11 M

to (f) 1x10-6 M


3.3.6 SERS Quantification of Hg(II) in the environment by TCE -functionalized

gold nanosensor

The TCE-functionalized gold nanosensor was utilized for SERS quantification

of Hg(II) in aqueous solutions. For the quantification of Hg(II) by SERS, the intensity

of the Raman band at 1501 cm-1 was recorded at different concentrations of Hg(II).

The Raman signal intensity at 1501 cm-1 was found to increase with the concentration

of Hg(II) (Fig. 3.7 Insect). A linear relationship was obtained between the SERS signal

intensity and the corresponding log concentration of Hg(II) (Fig. 3.7). Close

correlation (R2 = 0.9909) was found over the Hg (II) concentration range of 1µM to

0.1pM. The LOD and LOQ of the new nanosensing method were obtained at a standard

deviation (σ) of 0.21pM from seven measurements and estimated to be 5.1 x 10-13 M

(0.51pM) and 2.07 x 10-12M (2.07pM) respectively. The LOD and LOQ values of the

SERS nanosensor are 1000 fold lower than the recommended EPA and WHO limits

of Hg (II) in water (i.e. 10nM and 30nM respectively) [97, 241]. Other SERS sensors

for Hg (II) have been demonstrated in the literature. For example, Shaban et al [242]

developed a SERS sensor for Hg (II) by functionalizing porous anodic alumina

membrane with CoFe2O4 nanoparticles and growing carbon nanofibers on the inner

walls of the functionalized membrane to capture the target analyte. The method

development of the senor was complex and the detection limit for Hg (II) was only 1

µg per L (1 ppb). Ren et al [228] synthesised and functionalized Ag nanoparticles with

crystal violet, as a Raman reporter, and utilized them to determine Hg (II) down to 90

pM. The disadvantage of this method is that, the Raman detection of Hg (II) was

carried out directly within the Ag colloids. This mode of detection is well known to

suffer from non-reproducible SERS measurements due to the inability to control the

formation of hot spots[243, 244]. Wang et al [245]used gold nanoparticles that were


functionalized with anti Hg antibody and a Raman reporter to detect Hg (II) by SERS

based competitive immunoassay. The method was selective and sensitive down to 0.4

pM. However, this method is disadvantaged by the use of expensive antibody and

Raman reporter as well as the limited shelf life of the functionalized nanoparticles due

to the potential loss of the antibody activity. By contrast, the developed sensor in this

work addresses these challenges in that, besides the ease of using this sensor in the

field, its LOD is relatively lower making it more sensitive than those for some other

methods reported previously (Table 3.2). In addition, it is more stable than the

antibody-functionalized counterpart, due to the chemical nature of the used crown

ether and its immobilization on the nanostructured substrate via stable Au-S bonds.

The TCE-functionalized gold nanosensor was utilized for the detection of Hg (II) in

tap water. The TAP water was contaminated with Hg (II) (2.5x10-8 M), loaded onto

the TCE -functionalized gold nanosensor and screened by a handheld Raman

spectrometer. This concentration was chosen because it represents a case of Hg(II)

Table 3.2: Comparison of proposed Hg(II) sensing method with some reported

methods

Sensing material Analytical technique

Medium LOD References

Dibenzo-18-crown-6 azo dye derivative

Spectrofluorometry water 1.25x10-8M [210]

Dibenzo-18-crown-6 azo dye derivative

UV - visible spectroscopy

water 2.90x10-8M [194]

Microporous poly(2-mercaptobenzothiazole)

film

Electrochemistry water 1.0x10-10M [246]

Thiolated Dibenzo-18-crown-6 derivative

SERS water 5.1x10-13M This work


contamination in water that can cause toxicity (since it is above the EPA and WHO

defined levels for Hg(II) ions in water). The mean prediction concentration of Hg (II)

was 1.67x10-8 M (n=3) with a % recovery of 93.33 ± 16.17. The results obtained for

the spiked water sample indicated the potential of this nanosensor for the fast, sensitive

and selective field analysis of mercury contamination in water.

3.4 Conclusion

In this work, a new field-deployable crown ether-based SERS sensor for

detecting Hg (II) ions in water has been demonstrated. The new nanosensor showed

high sensitivity and selectivity towards Hg (II). The LOD (0.51pM) and LOQ

(2.07pM) of this new mercury nanosensor were 1000 fold below the EPA and WHO

defined levels for Hg (II) ions in water. The TCE-functionalized nanostructured gold

substrate provided a convenient sensing platform for the field detection of Hg (II) in

water by surface enhanced Raman spectroscopy. In view of these advantages, this

nanosensor provides a promising rapid and easy assay that can be used for the field

detection of Hg (II) in water. Investigations on the fabrication of more sensitive, rapid,

easy-to-use, selective and field deployable nanosensors for environmental toxicants

are in progress in our laboratory.

3.5 Acknowledgements

We thank the Ghana Atomic Energy Commission (GAEC) for the study leave

granted and the Queensland University of Technology (QUT) for their support through

the QUT Postgraduate Research Award (QUTPRA) and the QUT International HDR

Tuition Fee Sponsorship to DKS. The authors also acknowledge the staff of Central

Analytical Research Facility (CARF) of QUT, Natalia Danilova and Llew Rintoul

(PhD) for the support. Access to CARF was supported by the generous funding from

the Science and Engineering Faculty of QUT.

Chapter 4: A reusable Au nanostructured substrate for selective detection and quantification of Pb(II) ions by SERS 71

Chapter 4: A reusable Au nanostructured substrate for selective

detection and quantification of Pb(II) ions by SERS

This chapter is made up of the published journal article below:

D. K. Sarfo, E. L. Izake, A. P. O’Mullane and G. A. Ayoko, Molecular recognition

and detection of Pb (II) ions in water by aminobenzo-18-crown-6 immobilised onto a

nanostructured SERS substrate, Sensors and Actuators B: Chemical, 2018, 255, 1945-

1952

72 Chapter 4: A reusable Au nanostructured substrate for selective detection and quantification of Pb(II) ions by SERS








In the case of this chapter: D. K. Sarfo, E. L. Izake, A. P. O’Mullane and G. A. Ayoko, Molecular recognition and detection of Pb (II) ions in water by aminobenzo-18-crown-6 immobilised onto a nanostructured SERS substrate, Sensors and Actuators B: Chemical, 2018, 255, 1945-1952




A. P. O’Mullane Supervision of research and major manuscript editing






Preface

From the previous chapter, an Au nanostructured gold disc was fabricated. The

possibility of detecting toxicants at ultra-trace levels and doing so selectively using the

fabricated substrate was demonstrated with Hg(II) ions. To make the application of

this Au nanostructured substrate cost-effective and user-friendly, it was the objective

of the current chapter to find a way of using the substrate for repetitive measurements.

In addition, the prospect of amending the nanostructured substrate surface for the

detection of other toxicants besides Hg(II) ions was investigated. To achieve these

objectives, another type of crown ether, aminobenzo-18-crown-6, was identified and

used as a recognition molecule together with the nanostructured substrate to selectively

detect and quantify Pb(II) ions. This was done by developing a three-step method

involving an initial formation of a Pb(II)-crown ether complex, followed by

immobilization of the complex onto the nanostructured substrate and finally, a

detection and quantification step. The method demonstrated good selectivity and

sensitivity by detecting Pb(II) ions in the presence of other competing toxic and

potentially toxic metal ions at the pico- molar concentration level. An electrochemical

method was also developed to clean the surface of the Au nanostructured substrate

after its use for further analysis. Applying this method, the possibility of reusing the

substrate was shown by successfully cleaning the Pb(II) – crown ether complex from

the substrate surface and using it for repetitive measurements.


Abstract

In this work, we propose a new sensitive, selective and portable surface

enhanced Raman spectroscopy (SERS) methodology for the rapid on site detection of

Pb(II) pollution in water. The new method utilises aminobenzo-18-crown-6 (AB18C6)

as a selective recognition molecule to form a spontaneous complex with Pb(II) ions.

The formed AB18C6-Pb(II) complex was rapidly immobilised onto a nanostructured

gold substrate via Au-N bond formation and reproducibly screened by SERS using a

handheld Raman device. For the SERS measurements, a substrate was fabricated by

electrochemical deposition of gold nanostructures onto a flat gold disc, creating

multiple hotspots for ultrasensitive SERS measurements. The limit of quantification

(LOQ) for Pb(II) ions by the SERS method was 2.20 pM. The limit of detection (LOD)

was 0.69 pM which is five orders of magnitude lower than the maximum Pb(II) level

of 72 nM allowed by the US Environmental Protection Agency. The high sensitivity

of the SERS substrate is attributed to the coupling between the Surface Plasmon

Polariton (SPP) of its gold surface, the localised Surface Plasmon Resonance (SPR) of

the gold nanostructures and the Raman radiation from the immobilised AB18C6-Pb(II)

complex. The new SERS detection method was successfully applied for the selective

and rapid screening of Pb(II) ion contamination in water proving its practical

application for environmental analysis.

Keywords: SERS Nanosensor, Nanostructured gold substrate, Field deployable

Selective Pb(II) detector

4.1 Introduction

There is a continuous search for rapid, selective, and sensitive techniques for the

in-field screening of Pb(II) ions in the environment [247]. The toxicity of Pb(II) ranges

from chronic inflammation of the kidney and heart to inhibited brain development and


poor nerve conduction [248]. Though traditional methods such as Inductively

Coupled Plasma Mass Spectrometry (ICP-MS), Atomic Absorption Spectrometry

(AAS) and Inductively Coupled Plasma Atomic Emission spectroscopy (ICP-AES)

are useful for the detection of these toxins, they are usually not suitable for on-site

applications since they require sophisticated instruments with significant footprints

[249]. Other detection methods such as fluorescence spectroscopy [250], UV visible

spectroscopy [188] and dynamic light scattering [251] have been proposed in the

literature for the detection of Pb(II) ions. However these methods suffer from

disadvantages such as poor selectivity, interferences from spectral, chemical and

background, low sensitivity at trace concentrations and lack of optimisation for the in-

field screening of Pb(II) ions [195, 196].

Surface enhanced Raman spectroscopy (SERS) is a very sensitive mode of Raman

spectroscopy where an enhanced electromagnetic field near the surface of a metal

nanostructure causes significant amplification of the inherently weak Raman signal of

analytes by several orders of magnitude [252]. This significant enhancement of the

Raman signal allows for the detection of analytes at ultra-low concentrations down to

the single molecule level. In addition, SERS can be used for the multiplexed analysis

of multiple analytes in a sample [54]. Furthermore, with the emergence of commercial

handheld Raman spectrometers and sensitive SERS substrates, in-field screening of

environmental pollutants has been made easier [55]. Hence, SERS based nanosensors

have attracted much attention over the past decade.

Metal ions are generally known to have a small Raman scattering cross-section

and, in most cases, lack vibrational modes. Therefore, the direct detection of heavy

metal ions by SERS is challenging and the use of Raman active recognition molecules

that can bind these metal ions to the surfaces of SERS substrates is required [165, 166].


For this reason, SERS detection of Pb(II) ions by recognition molecules such as nucleic

acid sequences [253], 4-(2-pyridylazo)resorcinol [254] and citrates [181] have been

demonstrated in the literature where observable changes in the Raman fingerprint of

these recognition molecules were used to indicate the presence of Pb(II) ions in

samples. However, some of these recognition molecules may have poor selectivity

towards the metal ion and their immobilisation onto the surface of SERS substrates is

challenging.

Crown ethers and their benzo derivatives are Raman active ionophores that

spontaneously form stable complexes with metal ions and, in many instances, do so

selectively due to their unique coordination chemistry with the ion of interest [219].

For example, we recently demonstrated the use of aminodibenzo-18-crown-6

(ADB18C6) for the detection of Hg(II) ions in water [255]. The availability of electron

donor atoms in their molecular structure facilitates their immobilisation on noble metal

substrates such as gold via Au-O, Au-N, and Au-S bond formation [255].

The in-field SERS detection of metal ions requires substrates that can produce high

SERS enhancement factors and be easily manufactured at low cost with a potential to

reuse the substrate for multiple sample tests. We previously demonstrated a

nanostructured gold disc electrode, that was fabricated by a cost-effective and

relatively simple electrochemical method, providing very high SERS enhancement

factors due to the presence of multiple hotspots on its surface [215, 255]. The substrate

was also characterised by high SERS signal reproducibility due to the very low relative

standard deviation in SERS measurements that were obtained. Therefore, this substrate

was utilised in combination with aminobenzo-18-crown-6 (AB18C6) for the SERS

detection of Pb(II) ions. In this work, we aim to develop a simple and ultra-sensitive

method for the rapid detection of Pb(II) ions by SERS for the in-field environmental


monitoring of Pb(II) contamination. Significantly, this new SERS detection method

presented in this work offers high selectivity towards Pb(II) ions over other metal ions.

In addition, besides the ease of transporting this SERS substrate, it can be easily

recycled by a simple and rapid electrochemical cleaning procedure and used for

repeated SERS measurements. This new method can therefore be used for on-site

detection of Pb(II) contamination with a handheld Raman device. To demonstrate the

potential of the new method for real time analysis, it was utilised for the determination

of Pb(II) in drinking water at ultra-trace concentration levels down to 0.97 pM which

is significantly lower than the maximum allowable concentration (72 nM) for Pb(II)

ions in drinking water [256].

4.2 Materials and methods

4.2.1 Chemicals and reagents

All reagents and solvents were of analytical grade and used without further

purification. Sulfuric acid (98%), potassium hydroxide, hydrogen peroxide solution

(30%), 4-amino-benzo - 18-crown-6 (AB18C6), gold (III) chloride (HAuCl4),

dimethyl sulphoxide (DMSO), Pb(NO3)2, Cd(NO3)2, NaNO3, Hg(NO3)2,

Ca(NO3)2.4H2O, CuSO4, BaO, KCl, LiCl, NiCl2.6H2O and CoCl2.6 H2O, were all

purchased from Sigma-Aldrich (USA). All solutions were prepared using ultra-pure

deionised water (18.2MΩ.cm@25°C, Milli-Q)

4.2.2 Instrumentation

The nanostructured gold substrate was characterised by Scanning Electron

Microscopy (SEM) to determine the geometry, size and distribution of the developed

gold nanostructures. UV absorption spectra were recorded using a Cary 60 UV–visible

spectrophotometer (Agilent Technologies, USA). Fluorescence measurements were


carried out using a Fluorescence Cary eclipse spectrophotometer. All electrochemical

studies were conducted using a μAutolab potentiostat (Metrohm Autolab) with a

custom made three-electrode cell setup. SERS measurements were carried out using a

handheld ID Raman Mini2 (ocean optics, USA).

4.2.3 Study of AB18C6-Pb (II) complex formation by fluorescence spectroscopy

The complex formation between AB18C6 and Pb (II) was investigated by

fluorescence spectroscopy. For the fluorescence measurements, 1 mL of Pb (II)

aqueous solutions in the concentration range of 1 μM to 800 μM was added to 1 mL

of 1 mM AB18C6 (in DMSO: H2O, 1:1 v/v) solution. The pH of the formed AB18C6-

Pb(II) was 4.5. Aliquots of the formed complex, at different Pb concentrations, were

transferred into fluorescence-free quartz cuvettes and their fluorescence emission was

measured between 296 and 800 nm using an excitation wavelength of 295 nm. The Kf

value for the complexation reaction was calculated using the expression given by Shah

et al [257].

4.2.4 Study of AB18C6 selectivity towards Pb(II) by UV-Vis spectroscopy

To determine the selectivity of AB18C6 towards Pb (II) over other

environmentally relevant metal ions, 1 mL aliquots of 1 mM Pb (II), Cd(II), Cu(II), Ca

(II), K, Ba (II), Li, Ni (II), Co (II), Na and Hg (II) were added to 1 mL of 1 mM

AB18C6 solution (in DMSO: H2O, 1:1 v/v) and the UV absorption spectra of the

prepared mixtures were acquired within the wavelength range 200 to 600 nm.

4.2.5 Electrochemical deposition of gold nanostructures on gold substrate

A gold disc electrode cut into dimensions with geometric area of 8 mm was used

as a substrate to electrochemically deposit gold nanostructures on its surface. The

surface of the gold substrate was polished with 0.5, 0.3 and 0.05 µm grain sized


alumina slurry respectively, rinsed with water and sonicated in deionised water for 15

minutes to remove all the physically adsorbed alumina slurry. The polished substrate

was then soaked in piranha solution [(H2SO4 (98%): H2O2 (30%), 3:1 v/v] for 10

minutes and rinsed with deionised water. HAuCl4 solution (4 mM in 0.1 M HClO4)

was used for the electrochemical deposition of gold nanostructures using the method

described in our previous studies [255] to achieve a high number of deposited

nanostructures per surface area. After electrochemical deposition, the substrate was

treated with oxygen plasma for 15 minutes to remove adsorbed organic contaminants.

The nanostructured gold substrate was characterised and used for the SERS detection

of Pb(II) ions.

4.2.6 SERS detection of Pb (II) using AB18C6 crown ether and nanostructured

gold substrate

2 mL of 5 µM AB18C6 (in DMSO: deionised water 1:1 v/v) were mixed with 2

mL of 5 µM Pb(II) aqueous solution, to spontaneously form a complex between the

lead ion and the crown ether. 1 mL of the formed complex was loaded onto the gold

nanostructured substrates and incubated overnight to form a self-assembled monolayer

of the crown ether-Pb(II) complex on the nanostructured substrate. The substrate was

then rinsed with deionised water and screened by SERS to acquire the spectrum of the

AB18C6-Pb(II) complex. The AB18C6-Pb(II) complex formation and its

immobilisation onto the gold nanostructured substrate for SERS screening by

handheld Raman spectrometer is depicted in scheme 4.1.

For comparison purposes, 1 mL of the crown ether only was loaded onto another gold

nanostructured substrate, incubated overnight, rinsed with deionised water and

screened to acquire the Raman spectrum of the crown ether alone.


The SERS measurements were carried out by a handheld Raman spectrometer

at an excitation wavelength of 785 nm, and a laser power of 5 mW. The sample

interrogation time was 2 seconds at 7 accumulations. The spectra were collected in the

raster orbital scanning mode over a wavelength range of 400 cm-1 to 2000 cm-1. In this

mode, spectra from a large area of the sample is acquired at low average power while

the integrity of the sample is preserved. In addition, the Raman spectra were collected

with the “reference spectrum” and the “clean peaks” modes enabled. These scanning

modes remove ambient light, and fluorescence background using an automated built-

in correction algorithm.

Scheme 4.1. Schematic illustration of the formation of AB18C6-Pb(II) complex and

its immobilisation onto the nanostructured gold substrate for the detection of Pb(II)

ions by SERS.

4.2.6 SERS quantification of Pb(II) in water

For the quantitative analysis of Pb(II) by SERS, crown ether-Pb(II) complexes at

various Pb(II) concentrations were prepared by mixing 2 mL of Pb(II) standard

solution (from 1 µM to 1 pM) with 2 mL of the crown ether solution. Aliquots of the

formed complex were loaded onto the nanostructured substrates to form a Au-N bond

between the AB18C6-Pb(II) complex and the gold nanostructured substrate. The


substrates were then rinsed with deionised water and screened by SERS. A calibration

curve was plotted of the SERS signal intensity at 820 cm-1 versus concentrations of

Pb(II) ions.

The detection limit and limit of quantification for Pb(II) ions by SERS were

estimated by loading the gold nanostructured substrate with Pb(II)-AB18C6 (1 pM)

complex and screening with the handheld Raman spectrometer (n = 7). After

determining the concentration, the LOD and LOQ were calculated as the standard

deviation (δ) x t99% and δ x10 respectively (where t is the threshold value of student t-

distribution with a degree of freedom (n - 1) at 99% confidence interval) [216, 258].

To detect Pb (II) contamination in the environment, 1 mL of drinking water

containing 0.12 µM Pb(II) was mixed with 1 mL of AB18C6 solution to form a Pb(II)-

AB18C6 complex. The resulting complex was then loaded onto the gold

nanostructured substrate and allowed to stand for an hour. SERS measurements were

carried out using the handheld Raman device (n=3). The concentration of Pb (II) in

water was calculated from the calibration curve and the percent recovery determined

using the following equation:

% Recovery =C𝑃𝑃𝑏𝑏(𝑥𝑥) − C𝑃𝑃𝑏𝑏(𝑏𝑏)

C𝑃𝑃𝑏𝑏(𝑠𝑠) 𝑋𝑋 100

Where CPb(x) is concentration of spiked Pb(II) in the water (calculated from the SERS

calibration curve); CPb(b) represents the concentration of Pb(II) in blank water (before

spiking) and CPb(s) is the known concentration of the spiked Pb (II) standard solution.

The drinking water containing 0.12µM Pb(II) was also analysed using ICP-MS. SERS

spectra for AB18C6 in mixed metal ion water solution [containing 1µM each of Cd(II),

Cu(II), Ca(II), K, Ba(II), Li, Ni(II), Co(II), Na and Hg(II)] was acquired before and

after adding 1µM Pb(II) ions [Appendix B1(BI)].


4.2.7 Regeneration of the nanostructured gold substrate

1 nM AB18C6-Pb(II) complex were prepared, loaded onto a nanostructured gold

substrate and screened by SERS. To regenerate the substrate surface and re-use it for

SERS screening of Pb(II) ions, the AB18C6-Pb(II) complex was electrochemically

desorbed from the substrate. For this purpose, the substrate bearing the AB18C6-Pb(II)

complex was connected as the working electrode in an electrochemical cell. The

electrochemical desorption process was carried out in 0.1M KOH using Ag/AgCl (KCl

sat.) as a reference electrode and a platinum wire as an auxiliary electrode. Repetitive

scans (20 cycles) were carried out between 0.10 and -1.40 V at a scan rate of 0.1 V/s.

Afterwards, the substrates were screened with the handheld Raman device to ascertain

the presence or absence of the AB18C6-Pb(II) complex on the substrate’s surface.

After regeneration, a new aliquot of the AB18C6-Pb(II) complex was loaded onto the

substrate and re-screened by the handheld Raman device.

4.3 Results and discussion

4.3.1 SERS Substrate characterisation and Properties

The morphology and size of the gold nanostructure on the gold substrate were

studied by SEM at different magnifications (Fig. 4.1a). The SEM measurements

showed a high density of spherical gold nanostructures (average size 10 and 100 nm)

that are distributed all over the gold disc [215]. Therefore, the nanostructured gold disc

has a high coverage of hot spots where the Raman spectrum of the analyte experiences

significant electromagnetic field enhancement. This enhancement is due to the

coupling between the surface Plasmon Resonance (SPR) of the nanostructures, the

Surface Plasmon Polariton (SPP) of the underlying gold and the Raman scattering of

adsorbed molecules and leads to the ultra-sensitive SERS detection of analytes [159,

215, 259] (Fig. 4.1b).


Fig. 4.1. (A) SEM images of gold nanostructures electrochemically deposited onto a

flat solid Au material at different magnifications (B) Coupling of the SPP (from Au

solid base material) and SPR (from gold nanostructures) for high SERS signals

4.3.2 AB18C6 and Pb (II) ion binding studies

Crown ethers and their derivatives have been reported to form complexes with

heavy and transition metal cations through ion-dipole interaction with metal ions [187,

260, 261]. The benzo-18-crown-6 and its derivatives have been reported to selectively

form stable complexes with Pb(II) ions [219, 262, 263]. This is attributed, in part, to

the similarity of the host cavity size of the 18-crown-6 crown ether (2.2–3.2 Ao) and

the diameter of the Pb(II) ions (2.4 Ao), which allows a stable complex to be formed

[209]. In this work, the complex formation between AB18C6 and Pb(II) ions was

studied by spectrofluorimetry. Fig. 4.2 shows the fluorescence spectra of AB18C6 in

the presence and absence of Pb(II). In the absence of Pb(II), a strong band at 360 nm

and a relatively weaker band at 582 nm were observed. The band at 360 nm was

quenched while the band at 582 nm increased concomitantly with increasing Pb(II)

concentration from 1 µM to 0.8 mM. This change in the crown ether spectra with the

change of the Pb(II) ion concentration indicates complex formation between the crown

ether and the metal ion. The calculated Kf value for this reaction was 1x105. This is


similar to that reported by Rounaghi et al [264] using 18-Crown-6 to form a complex

with pb(II).

Fig. 4.2. Fluorescence spectra of AB18C6-Pb(II) complex at different concentrations

of Pb (II) ions.

4.3.3 Selectivity and interference studies

The selectivity of AB18C6 towards Pb(II) ions was investigated by reacting the

crown ether with Pb(II) ions and other environmentally relevant ions, K(I), Li(I),

Na(I), Mg(II), Ba(II), Ca(II), Cd(II), Mn(II), Co(II), Cu(II), Hg(II), and Ni(II). The

reaction mixtures were then screened by UV-Vis spectroscopy to monitor any changes

in the UV-visible spectrum of AB18C6. The crown ether showed a characteristic

absorption band at 295 nm (Fig. 4.3), which is attributed to the n→π* transitions

between the electron lone pairs of the oxygen atoms and the amino benzene moiety of

the crown ether [217, 218]. As shown by Fig. 4.3, the crown ether absorption band at

295 nm was quenched with the emergence of another band at 282 nm only in the

presence of the Pb(II) ions. This change in the crown ether UV-visible spectrum is

attributed to the formation of an ABC186-Pb(II) complex where the metal ion acts as

a Lewis acid to withdraw the electron density from the oxygen atoms of the crown


ether and cause the n→π* transitions within the formed complex to occur at higher

energy [233]. The other metal ions had no significant effect on the crown ether

absorption due to their inability to form a stable complex with AB18C6.

Fig. 4.3. UV-visible absorption spectra of mono and divalent ions after interaction with

AB18C6

4.3.4 Detection of Pb (II) by SERS

To test the feasibility of the nanostructured gold substrate as a SERS sensor for

Pb(II) ions, equal volumes of equimolar solutions of the crown ether and Pb(II) were

mixed to form a complex in solution. The Pb(II)-AB18C6 complex was loaded on the

gold nanostructured substrate to form a self-assembled monolayer where the amino

group of the crown ether binds to the gold nanostructures of the substrate. The SERS

spectrum of the AB18C6-Pb(II) complex was acquired and compared to that of the

bare substrate (Fig. 4.4). The Raman spectrum of the AB18C6 crown ether showed

vibration modes at 589 cm-1, 628 cm-1, 670 cm-1, 720 cm-1, 740 cm-1, 785 cm-1, 910

cm-1, 985 cm-1, 1011 cm-1,1047 cm-1, 1124 cm-1, 1185 cm-1, 1271 cm-1, 1349 cm-1 1427

cm-1, 1575 cm-1, 1584 cm-1 and 1602 cm-1. The band assignments for these vibration

modes are given in table 4.1. As indicated by Fig. 4.4 and Table 4.1, when AB18C6 is

reacted with the Pb(II) solution, many of the crown ether vibration modes shift to


higher wavenumbers. In addition new bands at 820 cm-1, 958 cm-1, 1149 cm-1, 1284

cm-1, 1440 cm-1, 1458 cm-1, 1483 cm-1 appear. The band at 820 cm-1 can be attributed

to the complex formation between the crown ether and the divalent lead ion [265]. The

bands at 958 cm-1, 1149 cm-1, 1284 cm-1, 1440 cm-1, 1458 cm-1, 1483 cm-1 may be

attributed to conformational changes of the crown ether moiety upon binding with the

lead ion [266].

Fig. 4.4 A: SERS spectra of (i) nanostructured gold substrate, (ii) AB18C6 crown ether

and B: (iii) AB18C6-Pb(II) complex


Table 4.1: SERS wavenumbers/cm of Pb(II)-AB18C6 complex and AB18C6 and their band assignments

Raman shift/cm Band assignment References AB18C6 Pb(II)-

AB18C6 AB18C6 Pb(II)-AB18C6

589 589 C-C-O deformation C-O-C bending Ring deformation

C-C-O deformation C-O-C bending

[267]

628 640 Ring deformation Ring deformation [268, 269] 670 687 ring deformation ring deformation [268, 270] 720 - ring deformation , CH wag [268, 269] 740 737 CH bend CH bend [269, 270] 788 - CH, ring breathing [268, 269] - 820 CH2 rock, crown

ether-lead complex [265]

910 910 CH bend, C-C- stretch, C-O stretch

CH bend, C-C- stretch, C-O stretch

[265, 271]

- 958 CH2 rock, C-C, C-O vibrations in D3d conformer of the crown ring

[272]

985 - CH2 rock, C-O vibrations in Ci conformer of the crown ring

[265, 272, 273]

1011 1002 Ring breathing Ring breathing [270] 1047 1052 C-C, C-O vibrations in Ci

conformer of the crown ring C-C, C-O vibrations in Ci conformer of the crown ring

[272, 273]

1124 - C-C, C-O vibrations of the crown ring

[265, 272, 273]

- 1149 CH2 rock vibration in D3d conformer of the crown ring

[272]

1185 1204 CH2 wagging, CH bend CH2 wagging, CH bend

[168, 270]

1271 1274 CH2 twist , Ci, D3d conformers of the crown ring

CH2 twist , Ci, D3d conformers of the crown ring

[272]

1284 CH2 twist , CH2 deformation in the D3d conformers of the crown ring

[265, 272, 273]

1343, 1377 1353 CH bend and ring deformation

CH bend and ring deformation

[268-270]

1427 - CH2 wagging and CH2 scissoring vibrations in Ci conformer of the crown ring

[272]

- 1440, 1458, 1483

CH2 deformation in D3d conformer of the crown ring

[265, 273]

1584 C-C [271] 1602 1607 C-C, ring stretch C-C,ring deformation

and ring stretch [268, 270, 271]


4.3.5 Reproducibility of the nanostructured gold substrate in the SERS detection

of Pb(II)

To confirm the reproducibility of the nanostructured gold substrate for the Raman

detection of Pb(II) ions, 100 µL aliquots of 1 pM AB18C6-Pb (II) complex were

loaded on 6 independent nanostructured gold discs and screened by SERS. As

indicated by figure B 2, matching Raman spectra with similar intensities were acquired

by the handheld Raman spectrometer, thus confirming the reproducible SERS

enhancement of the electrochemically prepared nanostructured gold substrates.

4.3.6 Recycling the nanostructured SERS substrate

In order to regenerate the nanostructured gold substrate for repeated SERS

detection of Pb(II) ions, the deposited AB18C6-P(II) complex was desorbed from the

substrate’s surface by using cyclic voltammetry. Fig. 4.5 shows a clear cathodic

desorption peak of the AB18C6-P(II) complex from the substrate surface at – 0.27 V.

This peak at -0.27 V disappears after 20 cycles of cathodic desorption which indicates

the complete removal of the adsorbed AB18C6-Pb(II) complex.

Fig. 4.5. Cyclic voltammograms between 0.10 and -1.40 V at a scan rate of 0.1 V/s for

10 µM AB18C6-Pb(II) functionalised substrate.


In order to confirm the removal of the AB18C6-Pb(II) complex, the substrate

was screened by SERS before and after the electrochemical desorption process. The

SERS spectra before and after desorption are shown in Fig. 4.6 and clearly indicate the

disappearance of the AB18C6-Pb(II) complex Raman fingerprint. When a new aliquot

of the complex was deposited on the substrate after its regeneration, the Raman

fingerprint of the AB18C6-Pb(II) complex re-emerged (Fig. 4.6c) confirming that the

electrochemical treatment did not compromise the SERS activity of the nanostructured

substrate.

Fig. 4.6 (a) Raman spectrum of the AB18C6-Pb(II) complex on the nanostructured

gold substrate before electrochemical desorption, (b) Raman spectrum of the substrate

after desorption, (c) Re-emergence of the AB18C6-Pb(II) complex Raman fingerprint

after depositing a new aliquot of the complex.

4.3.7 Quantitative SERS detection of Pb (II)

For quantitative detection of Pb(II) ions by SERS, the intensity of the AB18C6-

Pb(II) complex Raman band at 820 cm-1 was recorded and found to be directly


proportional to the concentration of Pb(II) ions (Fig. 4.7). A linear relationship was

obtained between the SERS signal intensity and the corresponding log concentration

of Pb(II) ions over the concentration range from 1x10-12 M to 1x10-6 M with a

correlation coefficient (R2) of 0.9742. The LOQ and LOD of Pb(II) ions by SERS were

found to be 2.20 x 10-12 and 6.90 x 10-13 respectively (n=7), which are lower than the

maximum allowable concentration (72 nM) for Pb(II) in drinking water as defined by

the United States Environmental Protection Agency [256]. In addition, this new SERS

detection platform for Pb(II) ions by AB18C6 and the gold nanostructured substrate

showed significantly improved sensitivity when compared to other Pb (II) detection

methods that utilise 18 crown 6 derivatives (Table 4.2).

Fig. 4.7. A plot of the Raman intensity (at 820 cm-1) versus the logarithm of Pb(II)

concentration (1x10-12 M to 1x10-6 M) and the corresponding SERS spectra from 750

to 900 cm-1(Inset).


Table 4.2 Comparison of the proposed Pb(II) sensing method with some reported

methods

Sensing material Analytical technique

Medium LOD References

4-aminobenzo-18-crown-6

UV - visible spectroscopy

water 5.0x10-8 M [262]

Dicyclohexano-18-crown 6-ether

Electrothermal atomic absorption

spectrometry

water 1.0x10-11 M [274]

Benzo-18-crown-6 Electrochemistry water 1.0x10-9 M [219]

4-aminobenzo-18-crown-6

SERS water 6.9x10-13 M This work

4.3.8 Environmental application

To demonstrate the potential application of the new SERS detection method for

the environmental analysis of Pb(II) ions, it was used to screen a drinking water sample

that was spiked with 1.2x10-7 M Pb(II). Another aliquot of the same spiked water

sample was re-screened by ICP-MS for cross validation. The amount detected and the

% recovery of each method are shown in Table 4.3. Although the % recoveries

obtained by both methods are within the usually acceptable limit of 85-115%, the %

recovery by the ICP-MS analysis was higher than that of the SERS analysis, the SERS

detection is comparatively rapid and easily applied to in-field screening of lead

contamination.

Table: 4.3 Recovery studies with spiked drinking water (n = 3)

Amount added (M) Amount detected (M) % Recoveries 1.2x10-7 SERS ICPMS SERS ICP-MS

1.06 x 10-7 1.37 x 10-7 88.83 ± 9.19 114.17 ± 1.97


4.5 Conclusions

In summary, we demonstrated a new SERS sensor for the selective detection of

Pb(II) ions. The new method utilises aminobenzo-18-crown-6 for the molecular

recognition of Pb(II) via the formation of a coordination complex with the metal ion.

The formed complex is immobilised onto a highly sensitive nanostructured gold

substrate, through Au-N bond formation, for the indirect SERS detection of Pb(II) ions

by a handheld Raman spectrometer. The new sensor showed excellent selectivity

towards Pb(II) ions as well as low LOD and LOQ values of 0.69 pM and 2.20 pM

respectively and was used successfully to determine the lead ion content of drinking

water. Therefore, this SERS nanosensor has excellent potential for the rapid on-site

detection of Pb(II) ions in the environment.


The authors thank Queensland University of Technology (QUT) for the award

of QUT Postgraduate Research Award (QUTPRA) and QUT International HDR

Tuition Fee Sponsorship to DSK and the Ghana Atomic Energy Commission (GAEC)

for granting him study leave. The authors also acknowledge the staff of Central

Analytical Research Facility (CARF) of QUT. Access to CARF was supported by the

generous funding from the Science and Engineering Faculty of QUT.

Chapter 5: Fabrication of disposable and dual function substrates for SERS and electrochemical detection of toxicants 93

Chapter 5: Fabrication of disposable and dual function substrates

for SERS and electrochemical detection of toxicants

This chapter is made up of the manuscript below:

D. K. Sarfo, E. L. Izake, A. P. O’Mullane, T. Wang, H. Wang, T. Tesfamichael and

G. A. Ayoko, Fabrication of dual function disposable substrates for

spectroelectrochemical nanosensing, Sensors and Actuators B: Chemical, 2019, 287,

9-17.

94 Chapter 5: Fabrication of disposable and dual function substrates for SERS and electrochemical detection of toxicants








In the case of this chapter: D. K. Sarfo, E. L. Izake, A. P. O’Mullane, T. Wang, H. Wang, T. Tesfamichael and G. A. Ayoko, Fabrication of dual function disposable substrates for spectroelectrochemical nanosensing, Sensors and Actuators B: Chemical, 2019, 287, 9-17.




A. P. O’Mullane Supervision of research and major manuscript editing

Teng Wang Assisted in the design of ZnO nanowires and manuscript editing

Hongxia Wang Editing of manuscript

Tuquabo Tesfamichael Assisted in E beam coating of plane gold experimental design and manuscript editing






Preface

To reduce the cost in the fabrication of the substrate, disposable substrates with

both plasmonics and conductive properties were prepared using indium tin oxide (ITO)

and carbon fibre as support materials. The fabricated substrate can act as a dual sensor

for both SERS and electrochemical detection. It was the objective of this part of the

thesis to design cheaper and disposable substrates with enhanced sensitivities. A SPP-

SPR coupling similar to that observed on the substrate in the previous chapters was

adopted on an ITO material by introducing a thin, planar gold film on its surface before

electrochemically depositing Au nanostructures. This produced multiplicative

electromagnetic enhancement effect in SERS. Highly dense ZnO nanowires were

grown on a carbon fibre material upon which Au nanostructures were deposited.

Charge transfer from the plasmonic Au nanostructures to adsorbed analytes was

enhanced because the ZnO nanowires increased the number of available electrons. The

ZnO nanowires also provided a large surface area for deposition of Au nanostructures

at high density and hence, generating many hotspots. A combination of these effects

from the ZnO nanowires enhanced the sensitivity of the nanostructured carbon fibre

substrate. Ultra-trace detection of melamine by SERS was achieved using these

nanostructured substrates. The compatibility of the developed substrate with

electrochemical technique has been demonstrated in this chapter.


Abstract

In this work, we demonstrate the fabrication of disposable and field deployable

nanostructured conductive substrates for dual detection by Surface Enhanced Raman

Scattering (SERS) and electrochemistry. Using a one-step potentiostatic process, gold

nanostructures were electrodeposited on three substrates: bare indium tin oxide (ITO)

electrode, ITO coated with plane gold and carbon fibre (CF) covered with ZnO

nanowires (ZnO NWs). Their sensitivities were enhanced by incorporating the plane

gold layer and ZnO NWs. The intensity of SERS signals produced on the

nanostructured ITO substrates with 0.1µM quinolinethiol were of the order:

nanostructured gold-coated ITO > nanostructured bare ITO. The higher SERS signal

on the nanostructured gold-coated ITO was attributed to the coupling between the

surface plasmon polariton provided by the gold under layer and the surface plasmon

resonance of the Au nanostructures. The ZnO NWs on the carbon fibre provided

additional surface area for electrodeposition of gold nanostructures at high density.

This led to multiple hotspots formation yielding high SERS signal intensity relative to

that on a nanostructured bare carbon fibre. The nanostructured substrates,

demonstrated good SERS signal reproducibility with relative standard deviation of

5.19%, 3.28% and 4.53% for Au/ITO, Au/Au-ITO and Au/ZnO-CF respectively. To

demonstrate the potential application of these substrates and estimate their

sensitivities, they were used to detect melamine by SERS at 1 pM (for Au

nanostructures on bare ITO), 1 fM (for Au nanostructured gold-coated ITO), and 0.1

nM (for Au nanostructures on ZnO NWs-coated CF) concentrations with LOD of

0.118 pM, 0.189 fM and 57.4 pM respectively. Taking advantage of the conductive

properties of gold nanostructured ITOs, electrochemical detection of 0.1 µM melamine


(with an LOD of 0.05 µM) was also demonstrated. Hence, these substrates are

potentially useful for SERS and electrochemical-based detection of organic toxicants.

5.1 Introduction

The utilization of Surface Enhanced Raman Scattering (SERS) spectroscopy

for the detection and monitoring of toxicants continues to attract strong attention. This

is due to favoured features of SERS such as high sensitivity, ability to directly screen

aqueous samples, and its capacity to provide the characteristic fingerprints of a wide

range of compounds [53].

The high sensitivity of SERS arises mainly from an electromagnetic effect (EM)

that is caused by the interaction of the Raman photons with the surface plasmon

resonance (SPR) of the metallic substrate [275]. However, when metallic

nanostructures are deposited on the flat surface of a noble metal, the coupling between

the SPR of the nanostructures and the surface plasmon polariton (SPP) of the flat noble

metal produces an additional multiplicative electromagnetic effect that causes further

enhancement to the SERS signal [61, 276, 277]. Another factor that is known to

contribute to SERS enhancement is the chemical effect caused by the formation of a

charge transfer (CT) complex between an analyte and the metallic substrate [278]. To

maximize the CT contribution in SERS, researchers have explored the inclusion of

semiconductor materials such as TiO2 [132], ZnO [133] and SnO2 [134] with

plasmonic noble metals to form noble metal-semiconductor nanocomposites. Among

these, ZnO has been the most widely used due to its superior refractive index which

promotes strong light confinements and its ability to transfer charge to other metallic

nanostructures, thus enhancing the subsequent transfer of charges from electron-rich

metallic nanostructures to analyte molecules that are adsorbed on their surfaces [133,


135]. This then increases the CT effect and further enhances the SERS signal of

adsorbed analytes.

Colloidal metal nanoparticles have been used as 3 D SERS substrates to detect

numerous compounds [279-281] due to their high sensitivity for target analytes.

However, the uncontrolled aggregation of the metallic nanoparticles greatly affects

SERS signal reproducibility and intensity [282]. Therefore, methods such as

lithography [80, 82], reactive ion etching [83, 84] and atomic layer deposition [85]

have been used to generate highly patterned and reproducible 2D SERS substrates [76,

77, 79]. However, the associated costs of production are usually high and the

processing rates low [86]. The development of SERS substrates based on Au thin film

nanostructures have been reviewed elsewhere in the literature [283].

Carbon Fibre (CF) and Indium tin oxide (ITO) substrates have been used as solid

support materials for the direct deposition and growth of metallic nanostructures (e.g.

gold, silver) because they are relatively inexpensive, electrically conductive and have

good heat tolerance [101, 129]. In addition to its low cost, CF has a high surface area

for the deposition of nanostructures, good resistance to corrosion, high flexibility [77]

and high porosity which allow for the facile absorption of analytes [129].

Electrochemical deposition methods have been used to develop metallic

nanostructures on CF and ITO substrates for SERS applications. Wang et al [90] used

a two-step electrochemical method to deposit gold nanostructures onto an ITO

electrode in which a single potential pulse was first utilised to form gold seeds on the

ITO’s surface. This was subsequently followed by a growth stage using cyclic

voltammetry.

In addition to their SERS utility, gold/silver nanostructured 2D conductive

substrates can be used for the sensitive and cost effective electrochemical detection of


toxicants [29, 34, 284]. In these measurements, the metallic nanostructures on the

surface of the electrode enhance the mass transport of the analyte and, therefore,

influence high signal-to-noise ratio within the detected electrochemical response [34,

284]. To improve their selectivity, nanostructured electrodes can be functionalized

with target-specific molecules for the recognition of a target analyte [284]. For

example, Pham and co-workers [129] developed Au nanodendrite on a bare CF

substrate and used the nanostructured substrate for both SERS and electrochemical

detection.

Our research group previously developed a conductive SERS substrate with

excellent reproducibility and sensitivity by electrochemically depositing spherical

gold nanostructures onto a gold disc electrode [91, 215, 285]. In the present work, we

develop disposable SERS substrates via a single-step chronoamperometric method for

the deposition of Au nanostructures onto a bare ITO electrode, ITO electrode coated

with plane gold and CF coated with ZnO nanowires (ZnO NWs). We explore the

contribution of SPR-SPP coupling to the electromagnetic field enhancement and the

contribution of ZnO nanowires to the CT enhancement in SERS. We also demonstrate

the application of the developed substrates as dual nanosensors for combined SERS

and electrochemical detection of melamine. The dual nanosensing of melamine was

sensitive (0.1 µM and 1fM by cyclic voltammetry and SERS respectively), simple and

cost-effective. The developed sensors have strong potential for the detection of many

analytes by SERS and/or electrochemistry.

5.2 Experimental

5.2.1 Chemicals and materials

Gold (III) chloride (HAuCl4), sodium hydroxide (NaOH), 2-quinolinethiol,

melamine, acetone, ethanol, diethanolamine (DEA), hexamethylene tetraamine


(HMT), ethylene glycol monomethyl ether [CH3O-CH2-CH2OH], zinc nitrate

(Zn(NO3)2·6H2O), Zinc acetate dihydrate [Zn(CH3COO)2·2H2O] and perchloric acid

(HClO4) were purchased from Sigma-Aldrich (USA). All reagents were of analytical

grade and used without further purification. Indium Tin Oxide (ITO) coated glass

substrates (7 x 50 x 0.7 mm, resistance = 4-8 Ω) were purchased from Delta

Technologies Ltd, U.S.A. Bare carbon fibre (CF) substrate was provided by

ElectroChem Inc. (U.S.A). Deionised water (18.2MΩ.cm@25 °C) was used in all

experiments.

5.2.2 Pre-treatment of ITO and CF substrates

The ITO and carbon fibre (CF) substrates were cut into 1.7 mm x 0.7 mm

rectangles and sequentially sonicated in acetone, ethanol, and ultra-pure water for 10

minutes. The washed substrates were dried under a stream of nitrogen gas.

5.2.3 Deposition of thin gold film on ITO

A 100 nm thick film of Au was deposited from a high purity (99.9%) gold pellet

target onto ITO substrates using a PVD 75 Kurt J. Lesker electron beam evaporator

(Kurt J. Lesker Co., USA). Before deposition, the chamber was evacuated to a base

pressure less than 1 x 10-6 Torr. The deposition was conducted using an acceleration

voltage of 10 kV and the beam current was adjusted accordingly to a deposition rate

of 1Å/sec. The film was monitored with a quartz crystal thickness monitor. The

substrates were placed perpendicular to the evaporation source at a distance of ≈ 40

cm and continuously rotated at 10 rpm in order to ensure a uniform and homogenous

Au film coating.


5.2.4 Development of ZnO NWs on CF

ZnO NWs were fabricated using a modified method based on our previous

report. [286, 287] Briefly, equimolar amounts of 0.1 M zinc acetate dihydrate and

diethanolamine were dissolved in ethylene glycol monomethyl ether at 60°C under

continuous magnetic stirring for 2 hours. The solution was then cooled and aged for at

least 48 h before being used for seed deposition of ZnO. The CF was dipped into the

precursor solution, rinsed by ethanol and subsequently heated for 10 min at 150°C.

The dip-dry procedure was repeated 3 times to ensure full coverage of the ZnO

precursor on the CF. The CF substrate was then annealed at 350°C for 30 min to obtain

the ZnO seed layer. To grow ZnO NWs forest on the ZnO seeded CF substrate, a

hydrothermal reaction was utilised. An aqueous solution containing 0.04 M zinc nitrate

and 0.04 M hexamethylene tetraamine was used as the precursor solution and added

to the ZnO seeded CF substrate in a Teflon lined stainless-steel autoclave. The

autoclave was then sealed and placed into an oven for 6 h at 95°C to grow the ZnO

NW. The final product was thoroughly rinsed sequentially with deionised water,

absolute ethyl alcohol, and finally dried in an oven overnight at 60°C.

5.2.5 Electrochemical deposition of gold nanostructures on ITO and CF

substrates

Chronoamperometry was used for the electrochemical deposition of gold

nanostructures on bare ITO, gold coated ITO, bare CF and ZnO-coated CF. The

deposition was performed in a one-compartment cell using a μAutolab potentiostat

(Metrohm, Switzerland). A standard three-electrode cell setup was established using

ITO or CF substrates as working electrodes, Ag/AgCl in saturated KCl as a reference

electrode and platinum wire as the counter electrode. The deposition of gold

nanostructures were carried out using a 10 mL solution that contained 0.1 M NaClO4


and 4 mM HAuCl4 (the pH adjusted to 7 with 0.1M NaOH). After an equilibration

time of 5 s, a potential of -80 mV was applied for 400 s, 600 s and 900 s to investigate

the effect of deposition time on the development of the gold nanostructures on the

substrates. After deposition, the nanostructured substrates were removed from

solution, washed thoroughly with deionised water and left to dry at room temperature.

The substrates were placed under oxygen plasma for 15 minutes to remove any organic

molecule that may have adsorbed onto the nanostructured gold surfaces.

5.2.6 Characterisation of the developed substrates

The gold nanostructures developed on the substrates were characterised by

Scanning Electron Microscopy (SEM) (MIRA 3 TESCAN, Brno, Czech Republic) to

determine their geometry, size and distribution. The chemical composition of the ZnO

NWs on CF substrate was confirmed by energy-dispersive X-ray spectroscopy (EDX).

5.2.7 SERS detection of quinolinethiol and melamine

For SERS measurements using the developed substrates, quinolinethiol was used

as a Raman probe. The gold nanostructured ITOs and CFs were immersed in 0.1µM

quinolinethiol solution for 2 h, rinsed with deionised water, dried under a stream of

nitrogen and screened to acquire the Raman spectrum of the Raman probe.

To detect melamine by SERS, 1.26 mg of the toxicant was dissolved in 1 mL of

tap water and concentrations ranging from 0.1 µM to 1fM were subsequently prepared

by serial dilution. The detection limit (LOD) approach was used to estimate the

substrates’ sensitivities to SERS measurements. This was done by loading melamine

onto the Au nanostructured bare ITO (at 1 pM, n=7), gold coated ITO (at 1 fM, n=7)

and ZnO-coated CF (at 0.1 nM, n=7) followed by SERS spectra acquisition. After

determining the concentration using the calibration curves in Appendix C1[C 1 (a-c)],


the LODs were calculated as the standard deviation (δ) x t99% (where t is the threshold

value of student t-distribution with a degree of freedom (n - 1) at 99% confidence

interval) [216, 258].

To detect the melamine in a complex food matrix, it was extracted from milk

samples spiked with 0.1mM melamine using the procedure in C 2. The extracted

melamine samples were deposited onto the nanostructured ITO substrates and

incubated for 1 hour. The substrates were then rinsed with deionised water and left to

dry prior to the SERS measurements.

All SERS measurements were carried out using a handheld ID Raman Mini2

(ocean optics, USA). The laser excitation was done with a 785nm laser line at a laser

power of 10mW. Five accumulations were taken per measurement with an overall

sample interrogation time of 2 s. The spectra were collected using the raster orbital

scanning (ROS) mode over a wavelength range of 400-1800 cm-1. The algorithm for

the instrument’s software (OceanView Spectroscopy 1.5.07) was operated to

automatically average and correct the acquired spectra for background noise and

florescence.

5.2.8 Electrochemical detection of melamine

To detect melamine using electrochemistry, 0.1 µM aqueous solution of the

toxicant was prepared in tap water and screened on the nanostructured ITO electrodes

by cyclic voltammetry (CV) using the μAutolab potentiostat. The ITO substrates were

used as working electrodes, Ag/AgCl in saturated KCl as a reference electrode and a

platinum wire as the counter electrode. The potential was scanned from 0.6 V to -0.6

V at a scan rate of 30 mV/s using 0.1 M KOH as an electrolyte. The LOD for melamine

detection was calculated from the expression previously described in this manuscript

using the calibration curve from C 1(d).


5.3 Results and discussion

5.3.1 Characterization of fabricated substrates

The morphology of the deposited gold nanostructures on bare ITO and gold-

coated ITO substrates, at a constant potential and different deposition times are shown

in Fig. 5.1. As indicted in Fig. 5.1a, quasi spherical Au nanostructures with an average

size of ≈ 70 nm were formed on the bare ITO substrate after 400 s. This was due to the

reduction of the gold chloride and subsequent deposition of Au0 atoms onto the bare

ITO surface. The deposited Au0 atoms fuse and grow to form the 70 nm Au

nanostructures [86, 288, 289]. By increasing the deposition time from 400 s to 600 s

and finally to 900 s, the formed quasi spherical Au nanostructures continue to grow

and form flower-like clusters that are separated by smaller spaces compared to those

formed using 400 s deposition time (Fig. 5.1b-c) [89, 290]. The observed colour

change of the nanostructured ITO substrates (Fig. 5.1 inset) confirms the growth of the

Au nanostructures with increasing deposition time. Therefore, the size and

morphology of the deposited Au nanostructures can be controlled by controlling the

deposition time.

Similar morphological changes were seen when gold nanostructures were

deposited onto the gold coated ITO substrate. However, compared to that on bare ITO

substrate, higher density of Au nanostructures were deposited onto the gold coated

ITO substrate at the same deposition times (as shown in Fig. 5.1). This may be

attributed to the 100 nm gold film on the ITO surface. Unlike the bare ITO (where the

initially deposited Au nanostructures at 400 s acted as nucleation sites for further

deposition), the thin gold layer underneath provided Au nucleation sites that acted as

nanoelectrodes for the ongoing deposition and growth of Au nanostructures over the

entire surface (Fig. 5.1d-f)[255].


Fig. 5.1 SEM images of electrodeposited Au nanostructures on bare ITO at (a) 400s

(b) 600s and (c) 900s and on gold-coated ITO at (d) 400s (e) 600s (f) 900s respectively.

The ease and hence rapid formation of Au nanostructures on the gold coated ITO

relative to the bare ITO was also evidenced by the positive shift in the reduction peak

potential from 0.51V on the nanostructured bare ITO to 0.61 V on the nanostructured

gold-coated ITO (C 3) when CV scans were performed on these two substrates (from

2.0V to -0.2V at 30mV/s using 4 mM HAuCl4 in 0.1 M NaClO4) [291].

Fig. 5.2, depicts the morphological changes to the surface of bare CF due to the

growth of ZnO NWs (average height ~ 550 nm, C 4), and the subsequent deposition

of gold nanostructure. As indicated in the figure, the ZnO NWs were uniformly and

densely grown onto the surface of the CF substrate and adopted an upward orientation

(Fig. 5.2b). The ZnO NWs forest provided additional surface area for the deposition

and growth of gold nanostructures (Fig. 5.2d) compared to the bare CF (Fig. 5.2c).


Fig. 5.2 SEM images of (a) bare CF (b) ZnO NWs-coated CF (c) electrodeposited Au

nanostructures on bare CF (d) electrodeposited Au nanostructures on ZnO NWs-

coated CF (e) Energy Dispersive X-ray spectrum (EDX) of gold nanostructured ZnO

NWs-coated CF.

The performance of the developed nanostructured electrodes as SERS

substrates were assessed by depositing 0.1 µM QT Raman probe onto their surfaces.

The acquired SERS spectra of QT are depicted in Figures 5.3 and 5.4 respectively. As

indicated by the figures, the SERS spectra were in good agreement with the reference

spectrum of QT (C 5). In addition, the QT signal intensity increased with increasing

electrodeposition time on both the Au nanostructured bare ITO (Au/ITO) and Au

nanostructured gold coated ITO (Au/Au-ITO) substrates (Fig. 5.3a, b). This can be

ascribed to the increase in the coverage and size of nanostructures with increasing

deposition time. The deposition of closely packed Au nanostructures onto the substrate

surface led to strong plasmon coupling between the adjacent Au nanostructures and

the formation of large excess of hotspots for high SERS signal intensity [87, 292].


Compared to the Raman signal intensity of QT on Au/ITO prepared at the same

deposition time of 900 s, Au/Au-ITO gave the highest Raman signal intensity. This

result indicates the contribution of the gold underlying film towards the sensitivity of

the SERS measurements in the Au/Au-ITO substrate. The large increase in the signal

intensity can be attributed to the coupling between the SPP of the 100 nm gold

underlayer and the SPR of the electrodeposited nanostructures [61, 92, 276].

Fig. 5.3 SERS spectra of 0.1µM quinolinethiol on (a) Au/ITO and (b) Au/Au-ITO.

Fig. 5.4 (a) SERS spectra of 0.1 µM QT on Au/ZnO-CF at different deposition times

(brown spectrum = clean Au/ZnO-CF) (b) SERS spectra of 0.1 µM QT on (i) Au/ZnO-

CF substrate (red line) and (ii) Au/CF substrate (green line) at 600 s deposition time.


A combination of the SPR-SPR coupling between the closely packed gold

nanostructures on the surface of the gold coated ITO and the SPR-SPP coupling

between the gold nanostructures and the thin gold film, give rise to the formation of

highly efficient hot spots at the nanostructures-gold film interface and, in effect,

increases the SERS signal enhancement for the analyte molecules that are adsorbed at

this interface [276] . The relative variation in the SERS spectra of QT on Au/ITO and

Au/Au-ITO (Fig. 5.3a and b) may be attributed to the different orientations that the

QT molecule assume when adsorbed onto the two substrates with different surface

morphologies [293].

Fig. 5.4a shows the Raman spectra of 0.1 µM QT deposited onto the Au

nanostructured ZnO-coated CFs (Au/ZnO-CF) when screened by a handheld Raman

spectrometer. The acquired spectra were in good agreement with that of QT standard

(C 5). In addition, the SERS signal intensity increased with increasing

electrodeposition time up to 600 s. To confirm this observation, gold nanostructured

were directly deposited onto CF (deposition time = 600 s) to develop Au/CF substrate

and utilised for SERS detection of 0.1 µM QT. The spectrum collected by the Au/CF

was compared to that obtained by Au/ZnO-CF substrate (prepared using deposition

time of 600 s, Fig. 5.4b). As shown in Fig. 5.4b-ii, a lower SERS signal was obtained

from Au/CF as compared to that obtained by the Au/ZnO-CF (Fig. 5.4b-i).

This result can be attributed to the high density of ZnO NWs on the CF substrate

which offered additional electroactive surface area for the deposition of more gold

nanostructures compared to the bare CF. As a result, this allows for the adsorption of

more analyte molecules on the Au/ZnO-CF than those on the Au/CF of similar

dimensions [282, 294]. In addition, the forest-like morphology of the Au/ZnO-CF

substrate allows for the creation of numerous hot spots on its surface that are not


available on the surface of the Au-CF substrate where the Au nanostructures are

randomly distributed (Fig. 5.2c, d; C 4, Fig. 5.5).

Fig. 5.5 Schematic diagram showing enhanced Raman scattering resulting from

plasmon coupling between neighbouring Au nanostructures and Au- ZnO junctions.

These abundant hotspots on the Au/ZnO-CF substrate experience strong SPP

coupling that lead to strong signal enhancement [133]. In addition, it has been reported

that, the ZnO NWs at the Au nanostructure-nanowire interface enhance light scattering

from the Au nanostructures, and, therefore, contribute to the overall field enhancement

effect at the interface [133, 282, 295].

Another factor that may explain the higher QT signal intensity on Au/ZnO-CF,

is the contribution of the ZnO NWs towards the formation of charge transfer complex

between the QT molecules and the substrate (i.e. the chemical effect in SERS) [102].

The charge transfer mechanism (CT) in SERS emanates from electron transfer

between the Fermi level of Au nanoparticles and the LUMO energy level of adsorbed

molecules [135, 296]. The electron-rich ZnO NWs on the CF surface promotes

additional electrons into the Fermi level of the Au nanoparticles where they become

more readily available to be transferred between the metal and the absorbed QT


molecules [297]. Hence, the CT mechanism is relatively enhanced by Au/ZnO-CF

when compared to Au/CF without ZnO NWs.

For comparison purposes, gold nanostructures were deposited onto Au/ZnO-CF

for 900 s and the substrate was used to detect 0.1 µM QT. The SERS signal intensity

was found to drop back when compared to that obtained by a substrate prepared using

600 s deposition time (Fig. 5.4a, black line = 900s and red line = 600 s). This

phenomenon was also observed by Wang et al [136]. The observation may be

attributed to the collapse of the ZnO NWs under the effect of the increasing weight of

the deposited gold nanostructure. Therefore, the SPR coupling between the gold

nanostructures on the collapsed ZnO NWs diminishes, leading to the reduced SERS

signal intensity.

Despite the role of ZnO NWs in promoting field enhancement at the Au-ZnO

NWs interface and the chemical effect in the SERS measurement of QT, the signal

intensity of the Raman probe on Au/ZnO-CF was lower than that on Au/Au-ITO at the

same deposition time (600 s) (red lines in Fig. 5.4a and Fig. 5.3b). This may be as a

result of the different modes of electromagnetic enhancement experienced in these

substrates. Both substrates experience SPR coupling between their gold

nanostructures. However, Au/Au-ITO experience an additional strong electromagnetic

enhancement caused by the SPR-SPP interaction between the Au nanostructures and

the underlying gold film [92]. It is known that the contribution of the electromagnetic

effect in SERS is higher by orders of magnitude than the chemical effect [92].

Therefore, the additional electromagnetic effect delivered by the SPR-SPP coupling in

the gold-coated ITO outweighs the additional chemical effect from the ZnO NWs on

Au/ZnO-CF.


The enhancement factor (EF) was calculated by monitoring the signal intensity of the

QT vibration band at 1361 cm-1 and using the equation reported by Yang et al [298]

(The concentrations of QT used for SERS and direct Raman measurements were

0.1µM and 0.1 M respectively.) The EF values were 3 x 107, 4.5 x 107 and 3.6 x 108

for Au/ZnO-CF, Au/ITO and Au/Au-ITO substrates respectively. These EF values

compare well with those obtained for similar substrates by other researchers [129,

299]. Although Kalachyova et al and Rodríguez-Lorenzo et al used colloidal

nanoparticles and a thiol self-assembly monolayer chemistry method to design SERS

substrates that provide EFs of 1011 and 1012 respectively [300], the use of colloidal

substrates is challenging due to uncontrolled aggregation of the nanoparticles. In

addition, while the approach used in the current work is applicable to the analyses of

both thiolated and non-thiolated molecules, it might be challenging to extent the

application of the method reported by Rodríguez-Lorenzo et al to non-thiolated

molecules [300].

5.3.2 Reproducibility of the SERS measurements on the nanostructured ITOs

and CFs

To demonstrate the signal reproducibility in the Raman spectra acquired by the

nanostructured ITO substrates as well as the nanostructured ZnO NWs-coated CF,

several measurements were carried out using the QT probe. The spectra were acquired

from ten random spots on the substrate’s surfaces (C 6). The relative standard

deviation (RSD) in the SERS measurements were found to be 5.19%, 3.28% and

4.53% for Au/ITO, Au/Au-ITO and Au/ZnO-CF respectively. The low RSD values

indicated good reproducibility of the SERS signals by the developed substrates.

5.3.3 SERS nanosensing of melamine


Melamine is a known organic toxicant [301] that forms insoluble cyanurate

crystals in the kidney and causes kidney malfunction/renal failure in humans [302,

303]. Consequently, the maximum allowable melamine content is 0.5 mg/kg (3.9 µM)

in powdered baby formula and 2.5 mg/kg (19.8 µM) for other foods[43, 304]. As a

proof-of-concept, the developed substrates were utilised for qualitative screening of

melamine in water at 1 pM, 1fM and 0.1 nM concentrations on Au/ITO, Au/Au-ITO

and Au/ZnO-CF substrates respectively, where Au/Au-ITO showed the highest signal

intensity of the measured analyte (Fig. 5.6a-c). To assess the sensitivities of the

fabricated substrates, their LODs were determined from the melamine measurements

and were found to be in the decreasing order of 0.189 fM > 0.118 pM > 57.4 pM for

the Au/Au-ITO, Au/ITO and Au/ZnO-CF respectively.

Fig. 5.6 SERS spectra of melamine in water on (a) Au/ITO (b) Au/Au-ITO (c)

Au/ZnO-CF and SERS spectra of melamine extracted from milk on (d) Au/Au-ITO.

The black spectra represent the Raman spectra of blank water (a –c) and milk (d)

samples. The red spectrum (d) is for melamine standard at the same extraction

conditions


The detection LODs obtained for melamine on the developed substrates satisfy the

current limit of detection set by food and health authorities (2.5 mg/kg). The identity

of melamine in the water sample was confirmed by mass spectrometry (C 7).

The reproducibility of the SERS screening of melamine on the three substrates

was assessed by monitoring the melamine Raman band at 1370 cm-1 (C 8) in repeated

measurements (n = 7). The RSD values were 4.55%, 4.22% and 4.77% for Au/ITO,

Au/Au-ITO and Au/ZnO-CF substrates respectively, confirming the reproducibility of

the measurements. Fig. 5.6 (d) shows the SERS spectra of melamine after extraction

from a spiked milk sample using the Au/Au-ITO substrate. The extraction process was

used to reduce the effect of matrix on spectra acquisition as SERS is known to be a

non-selective technique. This demonstrate the potential use of the substrate for the

analysis of real world samples in complex matrices after an appropriate

extraction/purification step.

5.3.4 Electrochemical detection of melamine

The utilization of nanostructured CF for electrochemical detection of analytes

has been demonstrated in the literature [129]. Therefore, we focused our efforts on

exploring the potential of the nanostructured ITOs as dual nanostructured sensors for

the detection of toxicants by Raman and electrochemistry. The electrochemical

performance of Au/ITO and Au/Au-ITO were assessed by cyclic voltammetry. The

voltammograms obtained for Au/ITO, and Au/Au-ITO, in the presence and absence of

0.1 µM melamine are shown in Fig. 5.7(a-b). In the presence of melamine, a new

reduction band appeared at -0.16 V on both substrates. In addition, there was an

observed positive shift in the reduction peak from 0.14 V to 0.19 V on Au/ITO (Fig.

5.7a) and from 0.14 V to 0.17 V on Au/Au-ITO (Fig. 5.7b).


Fig. 5.7 Voltammograms of (a) bare ITO (i), Au/ITO (ii), Au/ITO with 0.1 µM

Melamine (iii) and (b) gold coated ITO (i), Au/Au-ITO (ii), Au/Au-ITO with 0.1 µM

melamine (iii). CV scans were carried out in 0.1M KOH.

A similar positive shift in reduction peak was observed for melamine by Li et al [305]

on a glassy carbon electrode decorated with multiwalled carbon nanotubes.

The reduction peak current at 0.17 V was found to have significantly larger

magnitude in the case of Au/Au-ITO. This indicates the higher sensitivity of Au/Au-

ITO substrate over Au/ITO substrate for electrochemical measurements. This result is

in agreement with our observation from the SERS measurements on Au/ITO and

Au/Au-ITO substrates (Fig. 5.6a-b). The ability of the two nanostructured ITO

substrates to detect melamine at concentration below its allowed limit in food indicates

the potential use of these substrates for electrochemical detection of a wide range of

organic toxicants.

Using the Au/Au-ITO, the intensity of the peak current at -0.16 V was plotted

as a function of the logarithm of melamine concentration, as shown in C 1d. A linear

relationship was obtained over the melamine concentration range from 0.1 µM to 0.01

M with a correlation of determination (R2) of 0.9727 (n = 7). The calculated LOD for

melamine detection on the Au/Au-ITO was 0.05 µM (at 0.1 µM, n=7). Reproducibility

of the electrochemical measurements were also assessed from repeated measurements


of melamine (at 0.1 µM, n=7) using the Au/Au-ITO substrate and the peak current at

-0.16 V. An RSD value of 8.89% was obtained confirming the reproducibility of these

measurements.

The conductive and plasmonic properties of the manufactured substrates allow

for their use as spectro-electrochemical dual sensors for the determination of analytes.

Routine screening of high concentrations of analytes can be carried out by the

relatively simple and inexpensive electrochemical mode while the SERS screening

mode would be advantageous for molecular structure identification of analytes and

their quantification at ultra-trace concentrations. The low fabrication cost of the

substrates promotes their potential use for cost-effective analysis of many analytes.

Table 1 depicts the detection mode, SERS sensitivity and fabrication cost of the new

substrates.

Table 5.1: A comparison of the three fabricated substrates

Substrate Detection modes

SERS enhancement factor

Fabrication cost/substrate (AUD)$

Nanostructured ZnO NWs-coated CF

SERS 3.0 x 107

≈0.80

Nanostructured bare ITO

SERS & Electrochemistry

4.5 x 107 ≈2.00

Nanostructured gold-coated ITO

SERS & Electrochemistry

3.0 x 108 ≈6.00

5.4 Conclusions

In conclusion, highly sensitive, disposable, field deployable and relatively cheap

nanostructured substrates were developed. Their potential use for SERS and

electrochemical dual sensing were demonstrated. The presence of the thin gold layer

and ZnO NWs on the surfaces of ITO and CF substrates was the key to the high SERS

sensitivity. A clean, simple and rapid single-step chronoamperometry method was


developed for the cost-effective production of Au nanostructured ITO and CF dual

nanosensors. This method lead to full coverage of Au nanostructures over the entire

surface of the substrates which is crucial for SERS sensitivity and signal

reproducibility. Nanostructured bare ITO (Au/ITO), nanostructured gold-coated ITO

(Au/Au-ITO) and nanostructured ZnO NWs-coated CF (Au/ZnO-CF) substrates were

used for the SERS detection of melamine with LODs of 0.118 pM, 0.189 fM and 57.4

pM respectively. The RSD value for the SERS detection of melamine by Au/ITO,

Au/Au-ITO and Au/ZnO-CF substrates were as low as 4.55 %, 4.22 % and 4.77 %

respectively. The developed substrates pave the way for potential detection of a wide

range of organic toxicants on the field and in the laboratory as well as the possible

validation of SERS measurement with electrochemical techniques and vice versa. The

developed substrates can also be used for the SERS/electrochemical determination of

target analytes in real world samples after their extraction from complex matrices.


We thank the Ghana Atomic Energy Commission (GAEC) for the study leave

and Queensland University of Technology (QUT) for the QUT Postgraduate Research

Award (QUTPRA) and International HDR Tuition Fee Sponsorship granted to DKS.

The authors also acknowledge the staff of Central Analytical Research Facility

(CARF) of QUT, Fawad Ali and Christopher East (PhD) for their assistance. Access

to CARF was supported by the generous funding from the Science and Engineering

Faculty of QUT.

Chapter 6: Summary, Conclusions and Recommendations for future work 117

Chapter 6: Summary, Conclusions and Recommendations for

future work

6.1 Summary

A cost-effective and easy-to-use analytical technique with short sample

preparation and analysis time is indispensable in the monitoring and control of

toxicants. Such a technique should be field deployable, with the ability to detect

toxicants within a wide concentration range. In view of this, fabrication of field

deployable, disposable and reusable nanostructured substrates with surfaces that can

easily be functionalised so as to selectively detect toxicants in various matrices at trace

and high concentrations is of immense value. Although some existing techniques do

demonstrate good analytical performances, they are too bulky and are not field

deployable. Some of these techniques also suffer from background and matrix

interferences, making them less selective towards target analytes. To address these

limitations of present methods, easy-to-use disposable and reusable nanostructured

substrates for the detection of various toxicants within a wide range of concentration

with the relatively cheap SERS and electrochemical techniques have been

demonstrated in this thesis. While the electrochemical technique can detect toxicants

at relatively high concentrations, the SERS technique can be used for ultra-trace

detection and identification. The technique presented in this study has better analytical

detection performance, selectivity and short sample analysis time compared to some

existing techniques. The substrates were fabricated by electrochemically depositing

highly dense Au nanostructures on the following conductive surfaces: gold disc, bare

ITO, ITO coated with a thin layer of plane gold and carbon fibre bearing ZnO

nanowires. The one step electrochemical nanostructure fabrication process presented

118 Chapter 6: Summary, Conclusions and Recommendations for future work

in this study does not require the use of templates, surfactants and capping agents.

Hence it is easier, faster, cost-effective, reduces the problem of background

interferences and has high throughput.

Most existing nanostructured substrates rely on the electromagnetic and charge

transfer effect for their SERS sensitivity. In this study, a way for enhancing these

effects and hence, increasing the sensitivity of SERS measurements has been shown,

via the use of a gold disc as well as the incorporation of a thin gold layer and ZnO

nanowires on the surfaces of ITO and carbon fibre. The gold disc and the thin gold

layer (on the ITO surface) generated SPP that coupled with the SPR of the Au

nanostructures to increase the electromagnetic effect. The charge transfer effect was

likewise enhanced by the ZnO nanowires as they promote additional electrons into the

Fermi level of the Au nanoparticles where they become more readily available for

transfer between the metal and absorbed molecules.

After developing these substrates with improved SERS and electrochemical

sensitivities, their potential use for real world applications were demonstrated. The

AuNS gold disc was used to selectively detect Hg(II) ions in water after

functionalizing the surface with a thiolated aminodibenzo-18-crown-6 (ADB18C6)

derivative through a self-assembled monolayer formation. The thiolated derivative

was formed by coupling the amino group of the crown ether to the carboxylic group

of mercaptopropionic acid via the EDC coupling reaction. The Hg(II) ions were

detected at a LOQ and LOD of 2.07 pM and 0.51 pM respectively by SERS. Using

this same functionalized substrate, its electrochemical application was demonstrated

by qualitatively detecting 1 µM Hg(II) ions by cyclic voltammetry. The substrate was

again used to selectively detect Pb(II) ions in water against other coexisting metal ions.

In this case, aminobenzo-18-crown-6 was utilized as the molecular recognition of Pb


(II) via the formation of a coordination complex with the metal ion. The formed

complex was immobilised onto the AuNS gold disc, through Au-N bond formation,

for the indirect SERS detection of Pb(II) ions at a LOD and LOQ of 0.69 pM and 2.20

pM respectively. The possibility of reusing the AuNS substrate was also demonstrated

by detecting Pb(II) ions in water repeatedly on this substrate. The corresponding

disposable AuNS bare ITO, ITO coated with plane gold and carbon fibre with ZnO

nanowires were used to qualitatively detect melamine at 1 pM, 1 fM, and 0.1 nM

respectively. The substrates displayed good signal reproducibility with a relative

standard deviation of 4.55 % (AuNS bare ITO), 4.22 % (AuNS ITO coated with plane

gold) and 4.77 % (AuNS carbon fibre with ZnO nanowires). In addition, 0.1 µM

melamine was detected by cyclic voltammetry on the two gold nanostructured ITO

substrates to demonstrate their electrochemical detection capabilities. Melamine

detection at 1 mM was also demonstrated after extraction from milk samples. The

performance of the substrates reported in this thesis, for the analysis of toxicants,

compared well with other related works previously reported in literature.

6.2 Conclusions

The objectives of this thesis were achieved in that disposable and reusable

nanosensing platforms suitable for both SERS and electrochemical detection of

toxicants have been developed and characterized. The potential use of the AuNS

platforms fabricated in this thesis to selectively detect various toxicant at ultra-trace

concentrations using a handheld Raman spectrometer after functionalizing their

surfaces with suitable recognition molecules, was likewise demonstrated. The ease of

use and relatively shorter analysis times associated with the detection techniques

demonstrated in this study, and the ability to use the fabricated nanostructured

substrate together with handheld devices makes them good candidates for field


analysis of various toxicants. The surfaces of the substrates presented in this study can

easily be tailored towards the detection of different toxicants in various matrices such

as food, biological fluids and the environment. The outcome of this thesis is

potentially useful to various groups in research and academia as well as law

enforcement bodies for the analysis of different toxicants.

6.3 Recommendations for future work

The fabrication and potential use of a reusable AuNS gold disc have been

presented in this thesis work. Although the AuNS gold disc was used for both

qualitative and quantitative SERS analysis of Hg(II) and Pb(II) ions in water at ultra-

trace levels, further studies needs to be carried out on the application of this substrate

for the detection of these toxic metal ions in blood and other body fluids. This is due

to the possible entry of Hg(II) and Pb(II) ions into humans (via the food chain) when

they escape detection in environmental matrices, such as water, air and soil.

After presenting the fabrication process for disposable gold nanostructured

bare ITO, ITO coated with a thin planar gold layer and carbon fibre cloth bearing ZnO

nanowires respectively, qualitative detection of melamine was used as a proof of

concept to show their potential use for the detection of organic toxicants by both SERS

and electrochemistry. The application of these substrates for quantitative SERS

analysis of other toxicants needs further studies. The surfaces of these substrates can

be modified with an appropriate recognition molecule for the selective detection of

target analytes in complex matrices. As a result, further studies are recommended in

this area to improve upon the selective detection of analytes using these substrates.

Though the potential use of the substrates presented in this thesis for

electrochemical analysis of toxicants have been shown, it was only demonstrated

qualitatively. Hence, optimization of the reusable AuNS gold disc as well as the


disposable gold nanostructured bare ITO, ITO coated with a thin planar gold layer and

carbon fibre cloth bearing ZnO nanowires for quantitative electrochemical analyses of

toxicants is recommended.

The contribution of SPP-SPR coupling and nanowires/nanorods (vertically

oriented on surfaces solid support platforms) to the SERS signal enhancement have

been demonstrated in this work. However, the combined effect of these two concepts

towards SERS signal enhancement was not investigated. Growing of

nanowires/nanorods on the gold disc and ITO coated with a thin layer of plane gold

before electrochemical deposition of AuNS on these materials needs to be explored.

This can lead to possible multiplicative enhancement effect on the SERS signals and

hence, the sensitivities obtained from these substrates.

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142 Appendices

Appendices

Appendix A (Chapter 3)

A 1: Attachment of MPA to the ADB18C6 molecule by the EDC/NHS coupling reaction (R1 = CH2CH3, R2 = (CH2)3N+H (CH3)2Cl-)

A 2: SEM images AuNS coated gold substrate

Appendices 143

A 3: SERS spectra of 1µMHg-ADB18C6 Complex

A 4: SERS spectra of unmodified AuNS coated gold substrate after 15 minutes oxygen plasma cleaning

144 Appendices

A 5: SERS spectra of 0.4mM Mercaptopropionic acid

A 6: Raman spectra of 0.1pM Hg(II) in thiolated 4-amino-dibenzo- 18-crown-6 taken from 7 different substrates.

Appendices 145

A 7: SERS spectra of tADB18C6 and Cd(II), Pb(II), Cd(II) and Pb(II) and Hg(II) in tADB18C6 respectively

A 8: A plot of Raman intensity@1501 verses Hg concentration (M)

146 Appendices

Appendix B (Chapter 4)

B 1: SERS spectra of AB18C6 in mixed metal ion solution (yellow line) and in mixed metal ion solution containing Pb(II) ions (red line)

B 2: SERS spectra of 1pM Pb (II)-AB18C6 complex on solid gold SERS substrate (n=7)

Appendices 147

B 3: SERS spectra of the bare nanostructured gold substrate (red line) and drinking water spiked with K (I), Li(I), Na(I), Mg(II), Ba(II), Ca(II),

Cd(II), Mn(II), Co(II), Cu(II), Hg(II), Pb(II) and Ni(II) ions (blue line)

B 4: Fluorescence spectrum of (A) water and (B) 0.1mM Pb(II) in water

148 Appendices

Appendix C ( Chapter 5)

C 1. A plot of the Raman intensity (at 1370 cm-1) versus the logarithm of melamine concentration on (a) Au/ITO (1x10-12 M to 1x10-6 M) (b) Au/Au-ITO (1x10-15 M to 1x10-9 M) and (c) Au/ZnO-CF(1x10-10 M to 1x10-5 M) and (d) A plot of the peak current (at -0.16 V) versus logarithm of melamine concentration on Au/Au-ITO (1x10-

7 M to 1x10-2 M) C 2. Extraction of melamine from spiked milk sample A 900 µL milk sample was spiked with 100µL of 1mM melamine standard solution and shaken for 3 minutes. The resultant solution was centrifuge for 6 minutes at 17500 rpm after adding 5 µL of 1M HCl (aq) solution (pH of solution = 4). Subsequently, the supernatant solution was carefully collected into a glass vial and its pH adjusted to 7 with 4 µL of 1M NaOH (aq) solution. The final solution was again centrifuged for 6 minutes at 17500 rpm after the addition of 4 mL acetonitrile. The supernatant solution was carefully collected into a glass vail and used for SERS measurements.

Appendices 149

C 3. CV on nanostructured ITO and nanostructured gold coated ITO at 900 s deposition time using 4mM HAuCl4 in 0.1M NaClO4 as electrolyte

C 4. SEM images of ZnO NWs-coated CF with estimated length of ZnO NWs

150 Appendices

C 5. Normal Raman spectra of standard QT powder

C 6. SERS mapping spectra of 0.1µM quinolinethiol collected from 10 random spots on (a) Au/ITO (b) Au/Au-ITO and (c) Au/ZnO-CF

Appendices 151

C 7. Collision-induced dissociation mass spectrum (MS/MS) of melamine-containing samples (0.1 µM melamine), exhibiting product ions at m/z 85.1 and 68.0. Analysis was done in 50 % acetonitrile and 0.1 % formic acid. (The mass spectrum was acquired using a Thermo Fisher Scientific Orbitrap Elite mass spectrometer at a mass resolution of 240,000 (FWHM at m/z 400).)

C 8. SERS spectra of melamine collected on seven (a) Au/ITOs (b) Au/Au-ITOs (d) Au/ZnO-CFs substrates.

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