Orientated agsio2 core shell nanocubes as dual functional plasmonic substrates for biomarker detection

A dual-functional plasmonic substrate with a unique design that allows both the sensitive detection of photoluminescence PL via metal-enhanced photoluminescence MEPL and the specific Ra

National Taiwan University of Science and Technology

Department of Chemical Engineering

Ph.D Dissertation

Student ID: D10206814

Orientated Ag@SiO2 Core-shell Nanocubes as Dual-functional

Plasmonic Substrates for Biomarker Detection

Graduate Student : Nguyen Minh Kha

December, 2016

Abstract

The progress in fundamental biological studies of disease has been revealing a variety

of new biomarkers that are difficult to be detected by traditional diagnostic tools Interestingly,

plasmonics is an ultrasensitive optical technology applicable in medical diagnostics and

biological imaging sensors A dual-functional plasmonic substrate with a unique design that

allows both the sensitive detection of photoluminescence (PL) via metal-enhanced

photoluminescence (MEPL) and the specific Raman fingerprint via surface-enhanced Raman

scattering (SERS) is highly desirable to improve accuracy and sensitivity in detection

This dissertation describes the role of coupling agents, spacer effects, and importance

characters of various platforms for the optimum bifunctional SERS-MEPL based on Ag@SiO2

core-shell nanocube(s) [NC(s)] for urinary biomarkers detection In this work, the Ag NCs

were synthesized by the polyol method and modified, firstly with different coupling agents,

such as 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-aminopropyltrimethoxysilane

(APTMS), and secondly with tetraethylorthosilicate (TEOS) The presence of coupling agents

greatly modified the Ag NC cores and manipulated the thickness and uniformity of the silica

shells Ultrathin silica-coated Ag NCs (with a ~1.5 nm silica layer) found to have a SERS

intensity 3 fold higher than synthesized Ag NCs In comparison with MPTMS, it was found

that APTMS modified Ag@SiO2 NCs improved significant SERS and MEPL enhancements

Moreover, a ‘dual functionality’ represented by the simultaneous strengthening of SERS and

MEPL signals can be achieved by mixing Ag@SiO2 NCs, with a silica shell thickness of ~1.5

nm and ~4.4 nm This approach allows both the Ag@SiO2 NCs SERS and MEPL sensitivities

to be maintained at ~90% after 12 weeks of storage

Additionally, it is known that interactions between substrate and plasmonic

nanostructures can influence the performance of plasmonic biosensing Therefore, in the final

approach, a substrate characterized with low refractive index and roughness was first fabricated

by creating flower-like alumina on etched Al foil (f-Al2O3/e-Al) The Ag@SiO2 NCs assemble

in the edge-edge configuration when they were deposited on this substrate It is to note that the

surface roughness of f-Al2O3/e-Al provides a pathway for the coupling of incident light to

surface plasmon The Ag@SiO2/f-Al2O3/e-Al substrate exhibits a coupling efficiency of laser

light sources into surface plasmon hotspots for both SERS and MEPL Moreover, the shelf life

of this substrate is significantly improved due to reduced oxygen diffusion rate by ultrathin

silica spacer and flower-like Al2O3 dielectric layer Creatinine (CR) and flavin adenine

dinucleotide (FAD) are biomolecules present in human blood and urine With the advanced

label-free SERS and MEPL techniques, these biomarkers in urine are detected, and it allows

cheap, non-invasive and yet sensitive analysis

The approaches explored in this dissertation could be developed as a powerful encoding

tool for high-throughput bio-analysis

Keywords: SERS; photoluminescence; Ag@SiO 2 nanocubes; ultrathin shell; flower-like alumina; plasmonic coupling; edge-edge orientation; stability; dual functionality; Rhodamine 6G; urine; biomarker detection

Acknowledgement

First and foremost, I would like to express my deep gratitude to my advisor, Prof

Bing-Joe Hwang, for giving me an opportunity to work in his group, and exploring the wonderful

plasmonic nanoscale world He has taught me not only knowledge in research but also behavior

in a new environment The completion of this dissertation cannot be possible without his

intellectual support I could not imagine having a better advisor and mentor for my Ph.D study

I would like to thank my sincere gratitude to all members at the Nano-electrochemistry

Laboratory, Department of Chemical Engineering, National Taiwan University of Science and

Technology (NTUST), who assisted me not only my study but also on many occasions Special

thanks to Prof Wei-Nien Su who essentially inspired me to do my best in performing my

experiment as well as in writing papers Thank you for your appreciated support, valuable

encouragement and considerable recommendations Additionally, I wish to express my

appreciation to Dr Ching-Hsiang Chen, Dr Chun-Jern Pan, Dr John Rick, Dr Meng-Che Tsai,

and Dr Alok Mani Tripathi for giving their time and effort in evaluating my work I have

benefited from their constructive comments on my report

My sincere thanks also goes to Prof Hongjie Dai at Stanford University for supporting

and suggesting the work to improve my knowledge in the field of biochemistry

I am thankful to the Department of Chemical Engineering and Department of Materials

Science and Engineering at NTUST for their help and research facilities Also, many thanks to

the Faculty of Chemical Engineering of Ho Chi Minh University of Technology (HCMUT),

Vietnam, and Office of International Affairs at NTUST for all the best things that they have

offered me during my study and research

I would like to acknowledge National Taiwan University of Science and Technology

for giving me the chance to improve my knowledge in the graduate school and the scholarship

during my Ph.D life Also, gratefully acknowledge financial support from the Ministry of

Science and Technology (MOST), and the Top University Projects of Ministry of Education

(MOE)

It is my pleasure to thanks my friends at Vietnamese Student Association who made

my study and stay at NTUST more colorful

Last but not least, I would like to thank my family with love and gratitude for supporting

me spiritually throughout studying Ph.D and my life in general

Table of Contents

Abstract i

Acknowledgement iii

Table of Contents v

List of Figures ix

List of Tables xvii

List of Acronyms xix

Chapter 1 Introduction 1

1.1 Overview about plasmonic technologies 1

1.2 Statement of the problem 6

1.3 Objective of the study 9

1.4 Significance of the study 10

1.5 Structure of the dissertation 11

Chapter 2 Literature Review 13

2.1 A brief overview of SERS and MEPL 13

2.1.1 Light scattering and optical properties of metals 13

2.1.2 Normal Raman and surface-enhanced Raman scattering 17

2.1.3 Photoluminescence and metal-enhanced photoluminescence 24

2.2 The major concerns related to SERS and MEPL enhancements 29

2.2.1 Type of metal 31

2.2.2 Size effects 34

2.2.3 Shape effects 35

2.2.4 Interacting objects, gaps, and coupled plasmon resonances 36

2.3 The research on SERS-MEPL bifunctional mode 37

2.3.1 Solutions for the integrated SERS and MEPL 37

2.3.2 Applications of SERS and MEPL 39

2.4 Controlling the fabrication of nanostructures for plasmonic applications 42

2.4.1 General nanofabrication techniques 42

2.4.2 Preparation of Ag nanostructures 43

2.4.3 Surface coating for protection of nanostructures 48

Chapter 3 Materials and Methods 51

3.1 Materials 51

3.2 Fabrication of the plasmonic substrates 51

3.2.1 Synthesis of Ag NCs 51

3.2.2 Synthesis of Ag@SiO2 core-shell NCs 53

3.2.3 Fabrication of Ag@SiO2/f-Al2O3/e-Al and Ag@SiO2/Al2O3/Al substrates 53

3.2.4 Preparation of artificial urine 54

3.2.5 Preparation of SERS and MEPL measurements 55

3.3 Characterization and measurements 55

3.3.1 General characterization 55

3.3.2 SERS and MEPL measurements 56

3.3.3 Calculation enhancement factor of the substrate 57

Chapter 4 SERS and MEPL of Ag@SiO 2 Nanocubes with Ultrathin Silica Shells 59

4.1 Introduction 59

4.2 Results and discussion 61

4.2.1 Synthesis of Ag NCs 61

4.2.2 Ultra-coating silica shell for Ag NCs cores 66

4.2.3 The SERS and MEPL of the Ag@SiO2 NCs vs Ag NCs 70

4.3 Summary 78

Chapter 5 Sensitivity and Stability of Bifunctional SERS-MEPL from the Mixture of Ag@SiO 2 Nanocubes 81

5.1 Introduction 81

5.2 Results and Discussion 83

5.2.1 Characterization of the Ag NCs and Ag@SiO2 NCs 83

5.2.2 Bifunctional SERS-MEPL activity of Ag@SiO2 NCs 88

5.2.3 The stability for dual SERS-MEPL activity 94

5.2.4 Direct label-free SERS-MEPL detection of the mixture of creatinine and flavin adenine dinucleotide 97

5.3 Summary 99

Chapter 6 A Plasmonic Coupling Substrate Based on Ag@SiO 2 /f-Al 2 O 3 /e-Al for Sensitive Detection of Biomarkers in Urine 101

6.1 Introduction 101

6.2 Results and discussion 103

6.2.1 Fabrication of the substrates and their characterization 103

6.2.2 SERS and MEPL activities of the substrates 109

6.2.3 Urinary biomarkers detection 115

6.3 Summary 118

Chapter 7 Conclusion and Future Perspectives 119

7.1 Conclusion 119

7.2 Future perspectives 122

References 123

List of Figures

Figure 1.1 Models of healthcare delivery in chronic disease management.[2] Where, primary

care physician (PCP); integrated practice unit (IPU) 1

Figure 1.2 The outcomes from the diagnostic process.[6] 2

Figure 1.3 Plasmonic-based technologies for versatile biosensor applications.[10] 3

Figure 1.4 A schematic illustration of the averaged EFs obtained in the visible region from

different types of SERS substrates: Au, Ag and Cu, transition metals, and core-shell

(transition metals @ noble metals) NPs.[24] 5

Figure 1.5 Poynting vector or energy flow (lines) around a subwavelength metallic colloid

illuminated at the plasmon wavelength (left) and at a wavelength longer than the

plasmon wavelength (right) The vertical lines on the left indicate the diameter of

the cross sections for absorption.[56] 8

Figure 2.1 Schematic diagram representing the SPP at a metal dielectric interface.[20] 15

Figure 2.2 Schematic diagram representing the LSP.[20] The equilibrium distribution of electron

cloud in a metal NP is modified in the presence of an external E-field 16

Figure 2.3 Coupled surface plasmon modes (a) propagating along a thin metallic film and (b)

formed by the Bragg scattering on periodically modulated surface.[81] 16

Figure 2.4 The optical path schematic of a Raman system 18

Figure 2.5 Energy-level diagram for Rayleigh, Raman and resonance-Raman scattering S0 and

S1 are the singlet electronic ground and first excited states 19

Figure 2.6 Raman peak shift ranges for organic bonds.[86] 20

Figure 2.7 Jablonski energy-level diagram for absorption and PL Quenching represents a

non-radiative decay process 25

Figure 2.8 Free-space emission.[36] 26

Figure 2.9 Simplified Jablonski diagram for a fluorophore in absence of (top) and in presence

of (bottom) a metal.[111] 27

Figure 2.10 Plasmon controlled emission.[36] 28

Figure 2.11 Schematic representation of the relative intensities of fluorescence and SERS for

molecules at different distances from a metal surface.[119] 30

Figure 2.12 A simplified periodic table of the elements The part marked in black are

‘free-electron metals’ and those marked in grey are transition metals 31Figure 2.13 Plot of the (A) real, εr , and (B) imaginary, εi, components of the dielectric function

of Ag, Au, and Si as a function of wavelength.[124] 32

Figure 2.14 Dielectric constants of Ag and Pt.[94] 33

Figure 2.15 Quality factor of the LSPR for a metal/air interface The shaded area represents the

region of interest for many plasmonic applications.[17] 34

Figure 2.16 Extinction (black), absorption (red), and scattering (blue) spectra calculated for Ag

nanoparticles of different shapes: (A) a sphere, (B) a cube, (C) a tetrahedron, and

(D) an octahedron.[129] 35

Figure 2.17 (a) Gap width dependence of the SERS intensity normalized by extinction values

at the incident laser wavelength and Raman scattering wavelength; and (b)-(d) The

gap width dependence of the averaged enhancement factor by multiplying FDTD

intensity maps monitored at the incident laser wavelength by the map monitored at

the Raman scattering wavelength [132] 36

Figure 2.18 The synthesis procedures of AuNRs functionalized for both SERS and MEPL

DSPE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG = poly(ethylene glycol), and OTMS = octadecyltrimethoxysilane.[137] 38

Figure 2.19 MEPL, SERS and bright field images of the precipitates acquired from: (A)-(C)

the goat anti-human IgG immobilized nanoprobes and magnetic nanobeads (MBs)

mixed with human, mouse or bovine IgG molecules; and (B)-(D) the goat

anti-mouse IgG immobilized nanoprobes and MBs mixed with anti-mouse, human or bovine

IgG molecules.[141] 40

Figure 2.20 Schematic illustration of real-time multiplexed imaging using the FRES.[142] (A)

The mode of dual modal detection with fluorescence and Raman scattering (B)

Illustration of the in vivo multiplexed molecular imaging procedure 41

Figure 2.22 Polyol method for synthesizing Ag nanostructures In (A) the reduction of Ag+ ions

by ethylene glycol leads to the formation of nuclei that are highly volatile These

seeds are then grown into different nanostructures like (B) spheres, (C) cubes, (D)

truncated cubes, (E) right bipryamids, (F) bars, (G) spheroids, (H) triangular plates,

and (I) wires.[37] 47

Figure 2.23 Strategies for surface-coating SERS tags include coating with (A) denatured BSA,

(B) SH-PEG, (C) amphiphilic diblock copolymer, (D) liposome, and (E-H) silica

shells via different encapsulation routes.[170] 50

Figure 3.1 Schematic illustration of Ag NCs synthesis 52

Figure 3.2 Schematic illustration of Ag@SiO2 core-shell nanocube preparation 53

Figure 3.3 Schematic diagram for the preparation and chemical structures of the Ag@SiO2

/f-Al2O3/e-Al substrate 54

Figure 4.1 Characterizations of Ag NCs with different reaction times (A)-(D) SEM images;

(E) Uv-vis spectra; and (F) Linear relationship of maximum wavelength absorbance

and edge length of Ag NCs 62

Figure 4.2 Raman spectra with the excitation laser 532 nm of: (A) silicon wafer and 0.1 M R6G

on a silicon substrate (10 times); (B) SERS intensity and (C) SERS EF of Ag NCs

from various reaction times (excitation laser 532 nm) 64

Figure 4.3 (A) TEM image of Ag NCs (edge length ~50nm) (B) XRD pattern of Ag NCs – CB

72R mixture 65

Figure 4.4 Calibration curve of standard Ag+ solution 65

Figure 4.5 UV-Vis spectra of: (a) Ag NCs and Ag@SiO2 NCs prepared by adding different

chemicals ((b) 1mM MPTMS and 1mM TEOS, (c) 1mM TEOS, (d) 1mM MPTMS,

and (e) 1mM APTMS and 1mM TEOS, respectively) The inset is the UV-Vis

spectra, expanded in the range 454 nm to 466 nm 67

Figure 4.6 TEM images of Ag@SiO2 NCs prepared by adding different chemicals: (A) 1mM

MPTMS and 1mM TEOS, (B) 1mM TEOS, (C) 1mM MPTMS, and (D) 1mM

APTMS and 1mM TEOS, respectively 69

Figure 4.7 (A) SERS spectra (excitation laser 532 nm) of 10-5 M R6G on: (a) Ag NCs and

Ag@SiO2 NCs prepared by adding different chemicals ((b) 1mM MPTMS and

1mM TEOS, (c) 1mM TEOS, (d) 1mM MPTMS, and (e) 1mM APTMS and 1mM

TEOS, respectively) (B) SERS EF for 10-5 M R6G on Ag NCs and Ag@SiO2 NCs

with the excitation laser 532 nm 71

Figure 4.8 (A) SERS spectra with the excitation laser 632.8 nm of 10-5 M R6G on: (a) Ag NCs

and Ag@SiO2 NCs prepared by adding different chemicals ((b) 1mM MPTMS and

1mM TEOS, (c) 1mM TEOS, (d) 1mM MPTMS, and (e) 1mM APTMS and 1mM

TEOS, respectively) (B) SERS EF of 10-5 M R6G on Ag NCs and Ag@SiO2 NCs

with the excitation laser 632.8 nm 74

Figure 4.9 (A) MEPL spectra of 10-5 M R6G on: Si wafer, (a) Ag NCs, and Ag@SiO2 NCs

prepared by adding different chemicals ((b) 1 mM MPTMS and 1mM TEOS, (c) 1

mM TEOS, (d) 1 mM MPTMS, and (e) 1 mM APTMS and 1mM TEOS,

respectively) Inset: the amplified spectra of R6G on Si wafer (B) MEPL EF at 560

nm of R6G on Ag NCs and Ag@SiO2 NCs 75

Figure 4.10 Mechanism of coating silica shell by using APTMS coupling agent and TEOS 77

Figure 4.11 Mechanism of coating silica shell by using MPTMS coupling agent and TEOS.78

Figure 5.1 Characterization of Ag NCs (A) XRD pattern and (B) SEM image 84

Figure 5.2 UV-vis spectra of Ag@SiO2 NCs with various concentrations of TEOS 84

Figure 5.3 TEM images of Ag@SiO2 NCs with various concentrations of TEOS (A)-(A’) 0.5

mM (Ag@SiO2-0.5TE); (B)-(B’) 1 mM (Ag@SiO2-1TE); (C)-(C’) 12 mM

(Ag@SiO2-12TE); (D)-(D’) 20 mM (Ag@SiO2-20TE); (E)-(E’) 32 mM

(Ag@SiO2-32TE); and (F)-(F’) 50 mM (Ag@SiO2-50TE) 85

Figure 5.4 (A) Shell thickness distribution and (B) STEM profile of the Ag@SiO2-0.5TE

sample 86

Figure 5.5 Hydrodynamic particle size distributions of the Ag NCs and Ag@SiO2 NCs which

were dispersed in water 87

Figure 5.6 The signal of R6G on Ag@SiO2 NCs, which were prepared with various

concentration of TEOS: (A) SERS, (B) SERS EF (excitation laser 532 nm), (C) PL

spectra and (D) MEPL EF 88

Figure 5.7 The signal of R6G on the mixtures with various ratios of Ag@SiO2-1TE and

Ag@SiO2-20TE: (A) SERS, (B) SERS EF (excitation laser 532 nm), (C) PL spectra

and (D) MEPL EF 92

Figure 5.8 Normalized EF profiles of mixtures with various ratios of Ag@SiO2-1TE (X) and

Ag@SiO2-20TE (Y) 93

Figure 5.9 Stability of SERS intensity with respect to storage time for Ag NCs and Ag@SiO2

NCs 95

Figure 5.10 Stability of MEPL intensity with respect to storage time for Ag NCs and Ag@SiO2

NCs 96

Figure 5.11 SERS-MEPL stability of mixture X1Y5 (A) SERS; (B) MEPL; (C) Normalized

SERS EF (excitation laser 532 nm) and (D) Normalized MEPL EF 97

Figure 5.12 (A) SERS and (B) MEPL detection of various solutions on X1Y5 substrate (using

0.1 mM CR and 0.1 µM FAD; excitation laser 532 nm) 98

Figure 5.13 (A) SERS and (B) MEPL detection of Mix-CR&FAD (0.1 mM CR and 0.1 µM

FAD) on Ag@SiO2-1TE and Ag@SiO2-20TE (excitation laser 532 nm) 99

Figure 6.1 SEM and EDS of (A) Al foil, (B) Al foil after etching in HCl solution (named as

e-Al) and (C) e-Al after immersing in boiling water (named as f-Al2O3/e-Al) 103

Figure 6.2 Contact angle shapes of the water droplets on the substrates The error limit of the

contact angle value here is ~1oC 104

Figure 6.3 Ellipsometry spectra of (A) Si wafer, (B) Al foil, (C) Al2O3/Al substrate, and (D)

f-Al2O3/e-Al substrate (E) Refractive index (n) and extinction coefficient (k) of the

various substrates 105

Figure 6.4 (A) TEM image and (B) HR-TEM of Ag@SiO2 NCs; (C) SEM images of f-Al2O3

/e-Al (left) and Ag@SiO2/f-Al2O3/e-Al (right); (D) UV-vis spectra; and (E) XRD

spectra 106

Figure 6.5 UV-vis spectra of Ag@SiO2, Si wafer and Ag@SiO2/Si wafer 107

Figure 6.6 Characterization of Ag@SiO2 NCs on Si wafer and on f-Al2O3/e-Al substrate (A)

and (D): Schematic illustration of the orientation of Ag@SiO2 NCs (B) and (E):

SEM images (C) and (F): XRD spectra 109

Figure 6.7 (A) SERS and (B) MEPL of Al foil and f-Al2O3/e-Al (using [R6G] = 10-5 M)

Comparison (C) SERS EF and (D) MEPL of Al foil and f-Al2O3/e-Al substrate

before and after drop Ag@SiO2 (excitation laser 532 nm) 110

Figure 6.8 (A-B) SERS (excitation laser 532 nm) and (C-D) MEPL reproducibility of

Ag@SiO2/f-Al2O3/e-Al substrate at 10 random spots 111

Figure 6.9 Comparison SERS and MEPL activities of Ag@SiO2 NCs on various substrates

(A) SERS spectra, (B) SERS EF (excitation laser 532 nm), (C) MEPL spectra, and

(D) MEPL EF calculated by pristine sample and after 3-month storage 112

Figure 6.10 Plane-wave, refracting, and reflecting light at interfaces of various substrates: (A)

Si wafer; (B) Al foil; and (C) f-Al2O3/e-Al n1, n2, n3 and n4 are the refractive indices

of air, Si wafer, Al foil and Al2O3 (of f-Al2O3/e-Al) at 532 nm wavelength,

respectively The indices i, r, and e are for incident, refracted and reflected rays

113

Figure 6.11 Comparison SERS and MEPL activities of Ag@SiO2/Al2O3/Al and Ag@SiO2

/f-Al2O3/e-Al (probe molecules are 10-5 M R6G) (A) SERS, (B) MEPL, and (C) EFs

of SERS and MEPL 114

Figure 6.12 SEM and EDS of Al foil after 3-month storage under ambient condition 114

Figure 6.13 Comparison the SERS signals of biomarkers in unrine ([CR] = 10-4 M; [FAD] =

10-7 M) vs creatinine crystal (excitation laser 532 nm) 115

Figure 6.14 Concentration-dependent (A) SERS spectra of CR and (B) MEPL spectra of FAD

in urine recorded after dropping on Ag@SiO2/f-Al2O3/e-Al (C) Linear correlation

of normalized Raman intensities (at 682 cm-1) with the logarithm of CR

concentrations (D) Correlation of PL peak intensities with the logarithm of FAD

concentrations Fitted curves with error bar from 5 measurements 116

Figure 6.15 (A) SERS and (B) MEPL spectra of CR, FAD and mix-CR&FAD in urine after

dropping on Ag@SiO2/f-Al2O3/e-Al (using 10-4 M CR and 10-7 M FAD) 117

Figure 6.16 Detect mix-CR&FAD (10-4 M CR and 10-7 M FAD) by various substrates 118

List of Tables

Table 2.1 Comparison of the suitability of different metals for plasmonic applications.[37] 43

Table 2.2 Summary of the shapes, LSPR absorption peaks, demonstrated applications, and methods for synthesis of Ag nanostructures.[37] 45

Table 4.1 Summary of Ag NCs and Ag@SiO2 core-shell NCs prepared by various combinations of MPTMS, APTMS and TEOS 68

Table 5.1 Calculate the SiO2 thick and error of measurements for the Ag@SiO2-0.5TE sample 86

Table 5.2 Hydrodynamic particle size of the Ag NCs and Ag@SiO2 NCs 88

Table 5.3 The optimum thickness of shell for MEPL effect of various substrates 90

Table 5.4 Assignment of the SERS bands of CR and FAD.[270-272] 99

Table 7.1 Stability of various materials: comparative performance in SERS and MEPL 121

Denatured bovine serum albumin

Dynamic light scattering

Enzyme-linked immunosorbent assay

FRET Fluorescence resonance energy transfer

HCMUT Ho Chi Minh University of Technology

IPU Integrated practice unit

LSPR(s) Localized surface plasmon resonance(s)

National Taiwan University of Science and Technology

SAM Self-assembled monolayers

SERS

SPP(s)

Surface-enhanced Raman scattering

Surface plasmon polariton(s)

SPRi

SPR(s)

Surface plasmon resonance imaging

Surface plasmon resonance(s)

STEM

TEM

TEOS

Scanning transmission electron spectroscopy

Transmission electron spectroscopy

Chapter 1 Introduction

1.1 Overview about plasmonic technologies

Infectious and chronic diseases have considerable economic and societal impact across

the globe in both developed and developing countries Especially, in low-resource settings, it

is estimated that about 43% of the burden is from non-communicable diseases, e.g cancer,

cardiovascular diseases, and neuropsychiatric conditions, while 32% of the disease burden is

from communicable diseases, e.g acquired immune deficiency syndrome (AIDS), malaria, and

respiratory infections.[1] Therefore, demand for accessible and affordable healthcare present

significant challenges for providing effective and high-quality healthcare Traditional

healthcare models are expanding to include point-of-care (POC) diagnostics and

community-based approaches to respond to these challenges (Figure 1.1)

Figure 1.1 Models of healthcare delivery in chronic disease management [2] Where, primary care physician (PCP); integrated practice unit (IPU)

Diagnostic procedures are fundamental in the effective treatment of all diseases (Figure

1.2), and therefore a focus on diagnostic tools is one of the top priorities for improving

healthcare delivery.[3] The early diagnosis and monitoring will significantly reduce the disease

burden and potential for prevention or treatment Highly sensitive and specific bioassays, e.g

polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), are

available for diagnosis of many health symptoms However, the techniques have inherent

limitations, e.g many washing steps required with ELISA, while PCR-based diagnostics are

complicated, requiring for example amplification steps using primers, and are not generally

suited to rapid diagnostics Furthermore, progress in fundamental biological studies have

revealed a variety of new biomarkers that are either too low in abundance or are not suitable

for detection by traditional diagnostic tools.[4] Therefore, ultrasensitive biosensors for

multiplexed identification would help improve patient care.[5]

Figure 1.2 The outcomes from the diagnostic process [6]

The World Health Organization (WHO) identified the diagnostic devices’ characteristics that should be specific enough to biomarkers, sensitive, user-friendly,

affordable, and providing rapid tests to small amount of biological sample.[7] Optical biosensor

devices, which include refractive index change monitoring, absorption and spectroscopy, are

emerging as powerful biologic agent detection platforms satisfying these considerations.[8, 9]

Interestingly, plasmonics is an enabling optical technology with many applications in medical

diagnostics, disease monitoring, and biological imaging sensors As shown in Figure 1.3, the

plasmonic based biosensor platforms along with the underlying technologies are illustrated for

multiple targets

Figure 1.3 Plasmonic-based technologies for versatile biosensor applications [10]

In plasmonic technologies, optical nanostructures based on metals play important roles

as waveguides of routing light to expected locations with nanometer precision or as

nanoantennas to enhance electric fields (E-fields) These applications are made possible

through a strong interaction between incident light and free electrons in the nanostructures

Moreover, light can be effectively manipulated by good control over the nanostructures in

terms of shape and size.[11, 12] Although many new technologies stand to be realized from

plasmonics, with notable examples including invisibility cloaks,[13] superlenses,[14] and

quantum computing,[15] conventional technologies like surface-enhanced Raman spectroscopy

(SERS) and metal-enhanced photoluminescence (MEPL) could also be made significantly

faster and more efficient with the integration of plasmonic nanostructures.[16]

Generally, Raman spectroscopy is highly attractive in analytical science as it can

provide fingerprinting signals at the molecular level into a variety of physical, chemical and

biological surfaces It can be applied to in situ investigation of various interfaces and processes,

e.g solid-gas, solid-liquid and solid-solid, to which many other surface techniques are

inappropriate However, for diluted solutions (or solids) and low cross sections, the Raman

scattering is usually extremely weak Therefore, many applications based on Raman effect

were delayed for several decades, until the discovery of the laser together with the development

of more efficient detection systems substantially improved the sensitivity and scope of Raman

spectroscopy.[17] These issues have led researchers to concentrate to improve Raman

spectroscopy in surface science and related fields, which later was developed into SERS.[18, 19]

SERS has great influence on surface science and spectroscopy because the intrinsically low

inelastic scattering cross-section is overcome by plasmon enhancement A resonance condition

is reached when frequency of the incident light approximates the dipole plasmon from metal

nanostructures, leading to the strongest signal for the plasmon Such a condition is referred to

as surface plasmon resonance (SPR), or localized SPR (LSPR) for the case of metal

nanoparticle(s) [NP(s)].[20] SPR is considered for a flat surface or bulk material, the waves can

propagate along the surface LSPR is considered when the NPs are in the EM field, the wave

only can vibration around the NP’s surface The enhanced Raman scattering comes mainly

from the interaction of the plasmon band of metals, i.e roughened surfaces or NPs, with the

adsorbed molecules Therefore, maximum SERS enhancement can be achieved by optimizing

the plasmon and excitation wavelengths There is growing interest in using SERS to amplify

Raman signals for trace detections of specific molecules in many biomedical applications, e.g

cancer biomarkers, and live cells or animals.[21, 22] The substrate of SERS has been substantially

improved due to the rapid development of nanoscience However, only three noble metals (Au,

Ag, and Cu) could provide large enhancement in the visible region of the electromagnetic (EM)

spectrum (Figure 1.4), which has limited widespread applications involving other metallic

materials of both fundamental and practical importance.[23, 24] Fortunately, core-shell structure

NPs extended SERS to transition metals.[24] Currently, template and NPs based fabrication

strategies various SERS-active plasmonic nanostructures with controlled size, shape and

composition to improve the enhancement factor(s) [EF(s)]

Figure 1.4 A schematic illustration of the averaged EFs obtained in the visible region from

different types of SERS substrates: Au, Ag and Cu, transition metals, and core-shell (transition metals @ noble metals) NPs.[24]

On the other hand, photoluminescence (PL) spectroscopy is another well-known

spectroscopic method for selective detection of bioanalytes and proteins and as well as tracking

endogenous fluorophores or fluorescence labels in cell imaging and cancer diagnosis.[25, 26]

Similarly to the effect of SERS, the presence of noble metal nanostructures can enhance the PL

signal from local fluorophores The phenomenon, known as metal enhanced

photoluminescence (MEPL), has gained considerable research interest in recent years.[27, 28]

The origin of MEPL arises from the increase of excitation and/or radiative emission rate of

fluorophore as a result of the interaction between the fluorophore dipole moment and the

surface plasmon field of the metallic NP.[29]

It is interesting that by the local E-field enhancements in nanostructures sustaining

LSPR can enhance not only the SERS activity but also MEPL intensity.[30, 31] A dual-modal

plasmonic platform with a unique design that allows both the sensitive detection of PL via

MEPL and the specific Raman fingerprint via SERS is highly desirable to improve accuracy

and sensitivity in detection applications, and attracted increasing attention in recent years to

the study of the two effects on the same nanostructures.[32, 33]

1.2 Statement of the problem

The extent of plasmonic enhancement in LSPR is often related to NP composition, size

and morphology;[34] consequently, a wide array of NP shapes has been investigated, from

spheres to more complex shapes like cubes and octahedrons, as well as nanorods and nanowires

of varying lengths.[34, 35] Moreover, due to a strong dependence of E-field on the metal

substrate, a plethora of metals providing different plasmon resonant frequencies and EFs have

been investigated Au and Ag have been the most popular plasmonic metals, due to their SPR

bands located in the visible region of the EM spectrum, with Ag providing the strongest

enhancement due to its higher permittivity and quality factor (Q).[17, 36]

Furthermore, when compared with Au, Ag nanostructures produce a much stronger and

sharper SPR covering a broader spectrum from the ultraviolet (UV) to infrared (IR) region.[37]

It has long been recognized that the devices from Ag lead to more sensitive than from Au.[38]

However, poor chemical and structural stability has been a serious issue, limiting the further

practical applications, because the Ag nanostructures tend to evolve into spherical particles,

which results in the SPR band blue shift, under acids,[39] halides,[40] oxidants,[41] and heat.[42, 43]

Hence, there are not many reports about the plasmonic application of anisotropic Ag NPs with

relatively sharp corners Developing strategies for obtaining stable anisotropic Ag NPs, e.g

nanocubes (NCs), triangles or nanorods, while preserving their excellent plasmonic properties

for SERS and MEPL is highly desirable Another concern is that Ag nanostructures (or more

appropriately, Ag+ ions coming off the nanostructures) are considered to be toxic for in vivo

applications.[44]

The typical approach modifies the surface of the Ag nanostructure with a protecting

layer of inorganic or organic materials, e.g silica or titania,[45-47] Au,[48, 49] and self-assembled

monolayers (SAMs) of organic thiols,[50, 51] to keep Ag nanostructures away from the external

media Although the stability of protected Ag nanostructure has been improved to some extent,

these existing methods are still problematic For example, it is hard to obtain an anisotropic

Ag@silica core-shell nanostructure with a shell thickness of < 4 nm,[45, 52] which limits the use

of SERS Au deposition is initiated at specific sites of Ag NPs, so that the system is only stable

under mild conditions because of partial Au coverage.[48, 53] Thiol groups oxidize the surface

and corner of Ag, thus causing a large decrease in plasmonic activity.[54] On the other hand, the

optical properties of a PL material positioned in close proximity to metal NPs are affected by

the near-field electro-dynamical environment.[55] The modified fields cause an enhancement or

quenching of the PL relative to the native state, which depends sensitively on the distance

between analytes and metal surface An ideal shell layer should provide sufficient protection

against disturbance from the environment but should not significantly change the plasmonic

properties

Moreover, it is known that incident light interacts very strongly with metallic

colloids.[56] This property is shown in Figure 1.5 where a colloid is illuminated with a

wavelength light (λ) that matches the plasmon resonance λP (left) or with a wavelength longer than λP (right) The lines show the energy flow near the particle For a resonant wavelength,

the colloid has an optical cross section much greater than its physical cross section This effect

is seen by the energy flow into the particle, which results in the enhanced fields near illuminated

colloids For a nonresonant wavelength, the optical cross section can be similar to or smaller

than the physical cross section (Figure 1.5, right), which is typical of organic fluorophores and

semiconductor nanoparticles also known as quantum dots (QDs) A useful descriptive name

for metallic colloids is plasmon resonance particles (PRPs),[57] which accurately describes their

optical properties The PRPs of anisotropic metal NPs could be controlled not only by

geometrical characters, compositions and local dielectric environment but also by the NPs’ orientations.[58-60] However, a substrate for orientation-controlled NPs has still limited in

experimental research

Figure 1.5 Poynting vector or energy flow (lines) around a subwavelength metallic colloid

illuminated at the plasmon wavelength (left) and at a wavelength longer than the plasmon wavelength (right) The vertical lines on the left indicate the diameter of the cross sections for absorption.[56]

The LSPR property of plasmonic NPs exhibits a remarkable sensitivity to the proximal

environments of the nanomaterial such as dielectric function of their surrounding medium and

the substrate, as it can dramatically influence the plasmonic properties in a simple and

discernible manner.[61] Although the LSPR properties in general have been demonstrated very

well, relatively little attention has been paid to study the fundamental physics of the LSPR as

well as higher order multipolar plasmon resonance bands in very closely assembled plasmonic

NPs Moreover, based on the calculation results,[62] the substrate refractive index has a vital

role in redistributing the dipoles, which can significantly affect the LSPR and EM field

distribution around the NPs However, effects of the real substrates with varying refractive

indices on coupling plasmonic properties of metal NPs have not been investigated extensively

1.3 Objective of the study

Considering the sensitivity and specificity of assays, it would be desirable to develop

bifunctional sensors operating as both SERS and MEPL detection systems for multiple assays

The MEPL signals might be used as an immediate indicator of molecular recognition, while

the SERS signals could be used subsequently as the signature of specific molecular interactions

and quantifications Although the traditional SERS substrates, which are based on oxidation–

reduction cycle (ORC) of Au or Ag, can be easily fabricated, they still have many limitations

for SERS application For example, we cannot control the morphology and the gap of NPs The

nanoparticles have various sizes Therefore, their sensitivity and reproducibility are not high

enough for biomarker analysis at low concentrations.[63-65] Herein, my research work reports a

simple route for producing highly stable Ag NCs by varying the thickness of the silica shell

(from an ultrathin to a very thick layer) and demonstrate their potential application for SERS

and MEPL sensing The main purpose of over-coating with silica is to eliminate the

contribution of the former process and tune the EM field experienced by a probe, when bound

on the surface Moreover, silica is chemically inert and does not affect red-ox reactions at the

core surface, except through physical blocking of the surface Also, the silica shell is optically

transparent, therefore chemical reactions can be monitored by spectroscopies Moreover, in an

effort to address the above issues, the specific objectives of this research are as followed:

- Synthesize and apply the characterization techniques to observe the plasmonic

properties of Ag NCs

- Control the silica thickness of Ag@SiO2 core-shell NCs

- Design a new substrate for orientation-controlled Ag@SiO2 NCs

- Investigate the sensitivity and stability of Ag@SiO2 NCs and the new substrate

- Investigate the relationship between refractive index of substrate with plasmonic

coupling enhancement of Ag@SiO2 NCs

- Apply the dual SERS-MEPL functions of the new substrate for urinary biomarkers

detection

1.4 Significance of the study

This study will control the morphology and size of Ag NCs by using the polyol method

The Ag NCs’ optimum size for SERS intensity will be pointed out Moreover, uniform silica coating over Ag NCs from ultrathin to thick layers will develop by the Stöber condensation

reaction From the results, the study suggests the available mechanisms and the best condition

for ultrathin silica coating process Especially, the study will point out the optimum silica

thicknesses of Ag@SiO2 NCs for bifunctional SERS-MEPLS activities Furthermore, a new

substrate with high roughness and low refractive index for well-control orientated Ag@SiO2

NCs to edge-edge will be created based on flower-like alumina-coated etched Al foil

(f-Al2O3/e-Al) It is hoped that the new Ag@SiO2/f-Al2O3/e-Al substrate is able to provide a

coupling surface plasmon to generate new hotspots for both SERS and MEPL As a result, a

well distinguish and quality concentration of biomarkers in urine, e.g CR and FAD, can be

then detected by using the label-free SERS and MEPL techniques

1.5 Structure of the dissertation

This dissertation has been divided into 7 chapters Chapter 1 provides a general

description of the plasmonic technologies based on SERS and MEPL The challenges of

plasmonic researches and the main contributions of this study are also presented These two

ingredients – Raman scattering and plasmon resonances – are then connected in Chapter 2 to

describe in detail the fundamental principles and the EM mechanisms giving rise to SERS and

related plasmonic effects; such as MEPL Moreover, this chapter summarizes the applications

of SERS and MEPL techniques, illustrating the advantage for biodetection and bioimaging

The nanofabrication of metal nanostructures for plasmonic applications is shown clearly at the

end of Chapter 2 For experiments, Chapter 3 presents the precursors and procedures for

synthesis, characterization, and measurement of various substrates

The rest of the dissertation is then dedicated to a more practical results In Chapter 4,

the study focus on approaches to synthesize Ag NCs and coat uniform silica shell for SERS

and MEPL, and then discuss the role of coupling agents to silica thicknesses and SERS-MEPL

activities Chapter 5 illuminates how silica shell thickness influences the sensitivity and

stability of SERS intensity and MEPL signal, and the application towards detection of

creatinine (CR) and flavin adenine dinucleotide (FAD) by the mixture of two different silica

thicknesses Another aspect particularly emphasized throughout this study is the link between

SERS-MEPL and the wider research field of plasmonics, i.e the study and applications of the

plasmonic coupling of Ag NCs and various platforms (Chapter 6) In fact, SERS and MEPL

are based on the same raw ingredients of plasmonics (metals and light) A plasmonic coupling

substrate is designed based on the edge-edge orientation of Ag@SiO2 NCs on flower-like

alumina coated etched aluminum for detection of urinary biomarkers Finally, Chapter 7

summaries all of experimental results and focuses outlook of the field, which is directly related

to SERS-MEPL and their applications

Chapter 2 Literature Review

2.1 A brief overview of SERS and MEPL

A new spectroscopy technique, which uses the integrated SERS and MEPL signals, has

emerged as an optical tool with increased analytical capacity and sensitivity.[66-68] Considering

the different advantages of SERS and MEPL, this kind of dual mode spectroscopy holds great

potential in a variety of biomedical applications

2.1.1 Light scattering and optical properties of metals

2.1.1a Light scattering

Scattering of light in the form of propagating energy is one of the fundamental types of

interaction between light and material medium It is also thought of as the deflection of a ray

from a straight path, and involves absorption of an incident photon and instantaneous emission

of another photon This phenomena can be described in terms of EM radiation produced by

oscillating dipoles induced in the molecules by the incident EM field Generally, the scattering

process can be classified into two groups: elastic and inelastic scattering Energy of the incident

and scattered photons in elastic scattering are the same For such scattering involving objects

(particles, atoms and molecules) much smaller than the wavelength of incident light, it was

shown by Lord Rayleigh that the scattered intensity scales as the inverse fourth power of the

wavelength and the sixth power of its size.[69] Thus elastic scattering by atoms or molecules is

often referred to as Rayleigh scattering In detail, the intensity I (a.u.) of light scattered by any

one of the small spheres of diameter d (nm) and refractive index n from a beam of un-polarized

light of wavelength λ (nm) and intensity I0 (a.u.) is given by;[69]

In inelastic scattering, on the other hand, the incident and the scattered photons have

different energies Raman scattering is a well-known inelastic light scattering process, and can

provide specific structure and dynamic behavior of matter However, the light scattered

photons include mostly the dominant Rayleigh and the very small amount of Raman scattered

light.[70]

2.1.1b Optical properties of metals

A plasmon is a quantum quasi-particle representing the elementary excitations of

charge density oscillations in a plasma.[71] Unlike a photon, which is a real quantum particle, a

plasmon is always ‘lossy’ and highly interactive.[17] Thus, a plasmon will not be sustained in the absence of an external energy source Besides that, plasmonics is the study of the interaction

between EM field and free electrons The key component of plasmonics is a metal, because

plasmons are propagating on its surface, dispersive, EM waves coupled to the free electrons’

collective oscillations in the metal at a dielectric interface If the surface plasmons coupled with

photons then the resulting hybridized excitation is called surface plasmon polariton (SPP) The

schematic of a SPP wave is presented in Figure 2.1 These waves can propagate through the

surface of a metal until their energy is lost by absorption into the metal or radiation into other

directions However, if the wave vectors of the incident light and the SPP modes are different,

the photon energy cannot be coupled directly into the SPP mode.[17, 72] Surface plasmon

resonance (SPR) implies that the condition coupling takes place The coupling of the light to

the plasmon modes of the metal generates changes in transmission or reflection of the incident

light at this interface The SPR shows a strong effect by the dielectric environment and hence

has been utilized extensively in multiple biosensors.[73, 74]

Figure 2.1 Schematic diagram representing the SPP at a metal dielectric interface [20]

Normally, plasmon resonance is an optical phenomenon forming from the collective

oscillation of free electrons in a noble metal when the electrons are disturbed from their

equilibrium positions.[34] If the collective oscillation of conduction electrons is confined to a

finite volume, as with a metal NP, the corresponding plasma is called a localized surface

plasmon(s) [LSP(s)].[20] As opposed to surface plasmons, which are propagating in nature,

LSPs are non-propagating excitations of the conduction electrons of metallic nanostructures

coupled to EM fields.[72] As shown in Figure 2.2, the interaction of E-field with the incident

light induces a dipole moment in the particle The driven electrons are affected by the curved

surface of NPs, which along with the Coulomb forces, and add an effective restoring force;

therefore, the forces associated with the surface polarization In addition, the NPs exhibit strong

absorption and scattering of light at the resonance frequency, commonly known as a LSPR

mode.[75] Additionally, the position of the extinction maximum is sensitive to the intrinsic

dependence on the dielectric function of metal nanostructure and the surrounding environment

Thus, similar to the SPR, the LSPR can be used to sense the changes in the dielectric

environment surrounding the NPs, and hence this resulted in the development of many LSPR

based sensors.[20, 76, 77] Furthermore, LSPR can create intense local electric fields within a few

nanometers of a particle surface This kind of enhancement leads to the development of SERS

and MEPL.[78, 79]