Aggregation enhanced two photon excited photoluminescence of noble metal nanoparticles and their chemical and biological applications

This thesis presents a study on the application of aggregation induced enhancement of two-photon photoluminescence of noble metal nanoparticles.. Aggregation effects of noble metal nanop

Declaration Page

I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of Associate Professor Xu Qing-Hua, (in the laboratory S7-04-07), Chemistry Department, National University of Singapore, between 3/08/2009 and 14/08/2013

I have duly acknowledged all the sources of information which have been used in the thesis

This thesis has also not been submitted for any degree in any university previously The content of the thesis has been partly published in:

1) Cuifeng Jiang, Zhenping Guan, Siew Yin Rachel Lim, Lakshminarayana Polavarapu and Qing-Hua Xu Two-photon ratiometric sensing of Hg2+ by using cysteine functionalized Ag nanoparticles Nanoscale, 2011, 3, 3316-3320

2) Cuifeng Jiang, Tingting Zhao, Peiyan Yuan, Nengyue Gao, Yanlin Pan, Zhenping Guan, Na Zhou, and Qing-Hua Xu Two-Photon Induced Photoluminescence and Singlet Oxygen Generation from Aggregated Gold Nanoparticles ACS Appl Mater Interfaces 2013, 5, 4972 − 4977

3) Cuifeng Jiang, Tingting Zhao, Shuang Li, Nengyue Gao, and Qing-Hua Xu Highly Sensitive Two-Photon Sensing of Thrombin in Serum Using Aptamers and Silver Nanoparticles ACS Appl Mater Interfaces, 2013, 5, 10853-10857

Jiang Cuifeng

11, Feb, 2014

ACKNOWLEDGEMENTS

This section is dedicated to these important persons who play very important roles over the past four years I am grateful to them

The first person I want to express deep and sincere gratitude is my supervisor-Prof

Xu Qing-Hua for his support, encouragement and guidance both in research area and life I have learnt a lot from his detailed and constructive comments His enthusiasm, hard work and rigorous methodology in scientific research always encouraged me More importantly, Prof Xu has provided much value advice on facing problems in research and life These advices helped me to get through a hard time I really appreciate Prof Xu

I would like to acknowledge the financial, academic and technical support of the National University of Singapore, and its staff I also thank the Department of Chemistry and its academic and administrative staff for the kind support and assistance since the start of my studies at NUS

I wish to thank all my past and present lab mates, Dr Lakshminaraya Polavarapu,

Dr Ren Xinsheng, Dr Li Lin, Dr Lee Yih Hong, Dr Yu Kuai, Dr Shen Xiaoqin, Dr Zhao TingTing, Ms Ye Chen, Mr Guan Zhenping, Ms Yuan Peiyan, Mr Chen Jianqiang, Mr Gao Nengyue, Ms Jiang Xiaofang, Ms Zhou Na, Mr Pan Yanlin,

Ms Li Shuang, Mr Ma Rizhao, Ms Han Fei, Dr Tang Fu, Dr Wang Qisui and all

my friends in NUS for their help, continuous encouragement and support

Finally but most importantly, I would like to express my appreciation to my family-

my parents, my husband, my daughter, my parents-in law and my sister My parents always support me in the past 30 years It is my sister who always look after my parents when I am absent for most time My husband encouraged me almost everyday

in the past 4 years His continuous efforts on baby, parents and family are biggest support for me My daughter is a lovely girl and she is my motivation in the past year

My parents in-law support me wholeheartedly to finish the four years’ study, their help on taking care of my daughter make me focus on my research I am lucky to have these family and no words can express the appreciation to them

TABLE OF CONTENTS

THESIS DECLARATION ···I

ACKNOWLEDGEMENT ···II

TABLE OF CONTENTS ···IV

SUMMARY ···VI

LIST OF TABLES ···IX

LIST OF FIGURES ···X

LIST OF SCHEMES ···XIV

LIST OF PUBLICATIONS ···XV

Chapter 1 Introduction 1

1.1 Noble metal nanoparticles 1

1.1.1 Localized Surface Plasmon Resonance 1

1.1.2 Plasmon Coupling and its Optical Properties 5

1.2 Singlet Oxygen and its Application 11

1.3 Noble Metal Nanoparticles Based Sensing Method 13

1.3.1 Overall introduction of nanoparticles in sensing 14

1.3.2 Cross linking aggregation based sensing 16

1.3.3 Non-cross linking aggregation based sensing 20

1.4 Two-Photon Excited Photoluminescence 23

1.4.1 Two-Photon Absorption 23

1.4.2 Plasmon Coupling Induced Enhancement of Two-Photon Excited Photoluminenscence 28

1.4.3 Measurement of Two-Photon Excited Photoluminescence 30

1.5 Applications of Two-Photon Photoluminescence 30

1.6 Thesis Outline 32

References 35

Chapter 2: Two-Photon Induced Photoluminescence and Singlet Oxygen Generation from Aggregated Gold Nanoparticles 45

2.1 Introduction 46

2.2 Experiment Section 47

2.2.1 Materials 47

2.2.2 Preparation and assembly of gold nanoparticles 48

2.2.3 Detection of singlet oxygen generation 48

2.2.4 Instrumentations and characterizations 49

2.3 Results and discussion 49

2.4 Conclusions 61

References 63

Chapter 3 Simple Two-Photon Sensing of Dopamine using Au nanoparticles 67

3.1 Introduction 68

3.2 Experiment Section 69

3.2.1Materials 69

3.2.2 Preparation of Au nanoparticles: 70

3.2.3 Detection of dopamine: 70

3.2.4 Instrumentations and characterizations 70

3.3 Results and Discussion 71

3.4 Conclusions 78

References 79

Chapter 4 Two-Photon Ratiometric Sensing of Hg2+ by Using Cysteine Functionalized Ag Nanoparticles 82

4.1 Introduction 83

4.2 Experiment Section 84

4.2.1 Materials 84

4.2.2 Preparation of cysteine functionalized Ag nanoparticles 85

4.2.3 Cell Culture and preparation of samples for two-photon excitation microscopy: 86

4.2.4 Instrumentation and characterizations 86

4.3 Results and Discussion 88

4.4 Conclusions 100

References 102

Chapter 5 Highly Sensitive Two-photon Sensing of Thrombin in Serum using Aptamers and Silver nanoparticles 105

5.1 Introduction 106

5.2 Experiment Section 108

5.2.1 Materials 108

5.2.2 Preparation of silver nanoparticles 108

5.2.3 Detection of thrombin 108

5.2.4 Instrumentations and characterizations 109

5.3 Results and Discussion 110

5.4 Conclusions 123

References 124

Chapter 6 Conclusion 128

Summary

As noble metal nanoparticles, Au and Ag nanoparticles exhibit some unique optical properties, such as localized surface plasmon resonance (LSPR), which results from the conduction band electrons’s collective oscillation The LSPR band is dependent on the morphology (size and shape) of particles, and dielectric environment

of the particles Plasmon coupling of adjacent noble metal nanoparticles can also result in a red or blue shifted localized surface Plasmon resonance peak and significantly enhanced local electrical field within the gap region The enhanced local electrical field can enhance the two-photon photoluminescence (TPPL) It is of great importance to investigate Plasmon coupling enhanced TPPL and their application in chemical and biological application

Plasmon coupling of noble metal nanoparticles can enhance two-photon photoluminescence (TPPL) significantly Two photon emission advantages one photon emission for its narrow beam of high intensity light and deeper penetration This thesis presents a study on the application of aggregation induced enhancement of two-photon photoluminescence of noble metal nanoparticles

Firstly, two-photon excited photoluminescence and two-photon induced singlet oxygen generation of Au nanospheres and two ratios of Au nanorods before and after aggregation were invested in Chapter 2 Aggregation effects of noble metal nanoparticles are generally believed to be adverse to biomedical applications, however, Au nanospheres and short Au nanorods displayed enhanced two-photon excited photoluminescence and singlet oxygen generation efficiency after aggregation The two-photon photoluminescence of Au nanospheres and short Au nanorods were enhanced by up to 15.0- and 2.0-fold upon aggregation, and the corresponding two-photon induced singlet oxygen generation capabilities were enhanced by 8.3- and 1.8-fold, respectively The two-photon induced photoluminescence and singlet oxygen generation of the aggregated long Au nanorods were found to be lower than the unaggregated ones These results support that the change in their two-photon induced photoluminescence and singlet oxygen generation originate from aggregation

modulated two-photon excitation efficiency Based on these results, we designed a series of TPPL assay for detection of dopamine, mercury ions and thrombin

In chapter 3, a non-cross linking aggregation based TPPL assay for dopamine was demonstrated Protonated dopamine molecules can bind bidentately to surface of gold atoms through the catechol group The adsorption of dopamine displaces citrate groups, which stabilize the Au NPs, and neutralize the charge of solution, leading to non-cross linking aggregation of Au NPs When Au NPs solution was mixed with dopamine, TPPL intensity increases by about 47 times The TPPL assay was highly selective to dopamine and it can distinguish from uric acid, ascorbic acid and metal ions

A novel cross-linking based two-photon sensing strategy to detect mercury ions with high selectivity and sensitivity was developed in Chapter 4 This sensing approach is based on the observation that addition of Hg2+ into a cysteine functionalized Ag nanoparticle solution could significantly enhance their two-photon emission An enhancement factor up to 100 folds was obtained when mercury was added The sensitivity and sensing range can be easily tuned Compared to the conventional colorimetric or extinction spectra based methods, this scheme offers improved sensitivity, quantitative detection of Hg2+ with a larger dynamic range, and allows detection deep into biological environments such as cells and tissues where deep penetration is required The sensitivity could be further improved by using two-photon microscopy with the additional advantages of 3D detection and mapping

In chapter 5, we demonstrated a label free, fast, highly sensitive and selective two-photon sensing scheme for selective detection of thrombin on the picomolar level The assay is based on selective interactions between thrombin and a DNA aptamer, whichinduce aggregation of Ag NPs and result in significantly enhanced two-photon photoluminescence.The LOD of our two-photon sensing assay is as low as 3.1 pM in the buffer solution, more than 360 times lower than that of the extinction method (1.3 nM) The dynamic range of this method covers more than 4 orders of magnitude Most importantly, this two-photon sensing assay can be applied to detection of

thrombin in fetal bovine serum with LOD of 0.1 nM In addition to the unique advantages of two-photon sensing such as deep penetration and localized detection, this method could be potentially combined with two-photon microscopy to offer additional advantages of 3D detection and mapping for potential in-vivo sensing applications

List of Tables

Table 1.1 Penetration depth (mm) at various wavelength 25

Table 2.1 Optical properties of Au nanoparticles 54

Table 5.1 Recovery of Human α–Thrombin Spiked into Fetal bovine serum samples

116

List of Figures

Figure 1.1 Au nanocrystals with different shapes and sizes: (A) nanospheres; (B)

nanocubes; (C) nanobranches; (D—F) nanorods with increasing aspect

ratios; (G—J) nanobipyramids with increasing aspect ratios (K)

Normalized extinction spectra of the nanospheres (black), nanocubes

(red), and three nanorod samples (green, blue and purple) (L)

Normalized extinction spectra of the four nanobipyramid samples (red,

green, blue and purple) and nanobranches (black) 3

Figure 1.2 (A, B) Normalized extinction spectra and (C, D) TEM images of isolated

Au NSs (panels A and C) and Ag NSs (panels B and D) of different

sizes 4

Figure 1.3 Electrodynamic modeling calculations for Au nanoparticles (A) Change

of extinction spectra for 20 nm diameter particles with inter-particle

distance Inset is the peak shift vs inter-particle distance (B) Influence

of Au nanoparticle diameter on the extinction spectra at fixed (0.5 nm)

interparticle diameter (C) Extinction spectra of “line aggregates” of

varying number Inset is the peak shift against the number of Au

particles in the line aggregate 7

Figure 1.4 (a) Hot spots in a Raman image of Au nanoparticles arising from (b)

condensed nanoparticle pairs (c) FDTD calculations of adjacent

nanoparticle pairs showing a hot spot in the junction for incident

polarization along the inter-particle axis; (d) for incident polarization

orthogonal to the inter-particle axis, no hot spot occurs 9

Figure 1.5 Jablonski energy diagram for one-photon and two- photon absorption

23 Figure 1.6 Optical setup of a TPPL experiment 26

Figure 2.1 TEM images of isolated (a, c, e) and aggregated (b, d, f) Au NSs (a, b),

short Au NRs (c, d) and long Au NRs (e, f) 46

Figure 2.2 Extinction spectra of Au NSs (a) short Au NRs (b) and long Au NRs (c)

before and after addition of different amounts of cysteine 48

Figure 2.3 TPPL spectra of (a) Au NSs (b) short Au NRs and (c) long Au NRs

under excitation at 800 nm using femtosecond laser pulses (power

density: 50 mW); (d) excitation power dependence of TPPL for

aggregated Au NSs, short and long Au NRs Error bars represent

standard deviations for measurements taken from three independent

experiments 48

Figure 2.4 Extinction spectra of ABDA in the presence of (a) isolated Au NSs (b)

aggregated Au NSs (c) isolated short Au NRs (d) aggregated short Au

NRs (e) isolated long Au NRs (f) aggregated long Au NRs under

illumination of 800 nm femotosecond laser 51

Figure 2.5 Photo-oxidation of ABDA as a function of irradiation time in the

presence of isolated (un-aggregated) and aggregated Au NSs (a) short

Au NRs (b) and long NRs (c) A0 and A is the absorbance of ABDA at

380 nm before and after laser irradiation, respectively Error bars represent standard deviations for measurements taken from three independent experiments 53

Figure 2.6 Enhancement factor of two-photon photoluminescence (TPPL) and

two-photon induced 1O2 generation (TP-1O2) for Au NSs, short Au NRs and long Au NRs upon aggregation Error bars represent standard deviations for measurements taken from three independent experiments

55 Figure 3.1 (a) Photographs of Au NPs in the presence of 0, 8, 25, 36 mM of

dopamine; (b) Extinction spectra of Au NPs with different

concentration of dopamine; (c) Plot of the extinction ratio A736nm/A518nm

versus [dopamine] The inset shows that A736nm/A518nm is direct proportion to [dopamine] in the low concentration range

66 Figure 3.2 TEM images of Au NPs in the (a) absence and (b) presence of 36 mM

dopamine 68

Figure 3.3 TPPL spectra of Au NPs solution (a, b) upon addition of different

concentration of dopamine; (c) Plot of TPPL enhancement factor versus [dopamine].The inset exhibits that enhancement factor of TPPL

is direct proportion to [dopamine] in a low concentration range

70 Figure 3.4 The power dependence of the TPPL of Au NPs under excitation at

810nm for a sample with 36 mM of dopamine 70

Figure 3.5 (a) TPPL spectra and (b) TPPL enhancement factors for Au NPs in the

presence of 25 mM dopamine, or 250 mM other analytes 71

Figure 4.1 (a) Photographs of Ag NPs with different concentration of Hg2+; (b)

Extinction spectra of Cys-Ag NPs in the presence of different

concentration of Hg2+ (c) Plot of extinction ratio A550nm/A360nm versus

[Hg2+] 84

Figure 4.2 TEM images of cys-Ag in the (a) absence and presence of (b) 3 mM (c) 5

mM (d) 35 mM of Hg2+ 85

Figure 4.3 (a) TPPL spectra of cys-Ag NPs in the absence and presence of Hg2+; (b)

Plot of TPPL enhancement factor versus [Hg2+] The inset is the linear

dependence in the low Hg2+ concentration regime [cysteine] = 35μM

86

Figure 4.4 Power dependence of the TPPL of cys- Ag NPs under excitation at 800

nm for the sample with 20 mM of Hg2+ [cysteine] = 35 μM 87

Figure 4.5 (a) TPPL spectra of cys-Ag in the absence and presence of Hg2+ and (b)

Plot of TPPL enhancement factor versus [Hg2+] ([cysteine] = 7 μM) (c)

TPPL spectra of cys-Ag in the absence and presence of Hg2+ and (d) Plot

of TPPL enhancement factor versus [Hg2+] ([cysteine] = 175 μM) The

inset is the linear dependence in the low Hg2+ concentration regime

89

Figure 4.6 TPPL enhancement factor for cys-Ag in presence of various metal ions

Inset is the corresponding TPPL spectra [Metal ions] = 68 μM

90

Figure 4.7 Two-photon excitation microscopy of cys- Ag NPs in the absence (a) and

presence of 0.5 mM (b) and 10 mM(c) of Hg2+ 91

Figure 4.8 UV-Vis extinction spectra of citrate capped Ag NPs in presence of Hg2+

with different concentrations 92

Figure 4.9 (a) TPPL spectra of citrate-Ag NPs in the presence of different

concentration of mercury ions (b) Plot of TPPL enhancement factor versus [Hg2+] 93

Figure 4.10 (a) Extinction spectra of cys- Au NPs in absence and presence of Hg2+

(b) Plot of extinction ratio A600nm/A526nm versus [Hg2+] using cys-Au NPs 93

Figure 4.11 (a) TPPL spectra of cys-Au NPs in the presence of different

concentration of mercury ions (b) Plot of TPPL enhancement factor versus [Hg2+] 94

Figure 5.1 (a) Photographs and (b) Extinction spectra of Ag NPs in the presence of

different concentrations of thrombin; The extinction ratio A483nm/A395nm

versus [thrombin] was plotted in (c) The inset shows that A483nm/A395nm

is direct proportion to [thrombin] in the low concentration range

106

Figure 5.2 TEM images of Ag NPs (a) in the absence and (b) presence of 70 nM

thrombin 107

Figure 5.3 (a, b) TPPL spectra of Ag NPs solution upon addition of different

concentration of thrombin; (c) Plot of TPPL enhancement factor versus

[thrombin] The inset exhibits that the TPPL enhancement factor is

direct proportion to [thrombin] in the low concentration range 108

Figure 5.4 Power dependence of the TPPL of Ag NPs under excitation at 810 nm in

resence of 60 nM thrombin (Power density: 23, 30, 36, 43, 47 mW)

110

Figure 5.5 (a) TPPL spectra and (b) TPPL enhancement factor of Ag NPs solution

upon addition of 70nM thrombin, 70nM BSA or using random DNA

sequence replacing TBA15 111

Figure 5.6 (a, b) TPPL spectra of Ag NPs solution upon addition of different

concentration of thrombin; (c) Plot of TPPL enhancement factor versus [thrombin] in undiluted serum The inset exhibits that enhancement factor of TPPL is direct proportion to [thrombin] in the low

concentration range 113

Figure 5.7 (a) Extinction spectra of Ag NPs with different concentration of

thrombin in serum media; (b) Plot of the extinction ratio A483nm/A364nm

versus [thrombin] in serum The inset exhibits that A483nm/A364nm is direct proportion to [thrombin] in the low concentration range

114

Figure 5.8 (a) TPPL spectra and (b) TPPL enhancement factor of Ag NPs solution

upon addition of 70nM thrombin, 70nM BSA or using random DNA

sequence replacing TBA15 in serum 115

Figure 5.9 The relationship curve for spiking thrombin into serum experiment

116

Scheme 1.3 Diagram of type II photochemistry in one-photon and two-photon

induced 1O2 generation (ISC: Inter-Syetem Crossing) 12

Scheme 1.4 Schematic representation of the inter-particle cross-linking aggregation

and color change of Au NPs induced by target DNA 15

Scheme 1.5 Schematic depiction of metal NPs functionalized with chelating ligands

induced aggregation 16

Scheme 1.6 Schematic depiction of non-cross linking aggregation of Au NPs in the

presence of target DNA and salt 19

Scheme 1.7 Scheme for the engineered cocaine aptamer and the visual detection of

cocaine based on the red-to-blue color change of gold nanoparticles

20

Scheme 2.1 Scheme of two-photon induced photoluminescence and generation of

singlet oxygen from aggregated Au NPs 45

Scheme 3.1 Schematic description of TPPL assay for detection of dopamine based

on non-cross linking aggregation of Au NPs 65

Scheme 4.1 Schematic description of cross-linking aggregation of Ag NPs based

TPPL sensing assay for detection of mercury ions 82 Scheme 5.1 Schematic description of TPPL method sensing thrombin by using Ag

NPs and TBA15. 104

List of Publications:

1 Cuifeng Jiang, Zhenping Guan, Siew Yin Rachel Lim, Lakshminarayana Polavarapu and Qing-Hua Xu Two-photon ratiometric sensing of Hg2+ by using cysteine functionalized Ag nanoparticles Nanoscale, 2011, 3, 3316-3320

2 Cuifeng Jiang, Tingting Zhao, Peiyan Yuan, Nengyue Gao, Yanlin Pan, Zhenping Guan, Na Zhou, and Qing-Hua Xu Two-Photon Induced Photoluminescence and Singlet Oxygen Generation from Aggregated Gold Nanoparticles ACS Appl Mater Interfaces 2013, 5, 4972 − 4977

3 Cuifeng Jiang, Tingting Zhao, Shuang Li, Nengyue Gao and Qing-Hua Xu Highly sensitive two-photon sensing of thrombin in serum using aptamers and silver nanoparticles ACS Appl Mater Interfaces, 2013, 5, 10853-10857

4 Xiao-Fang Jiang, Yanlin Pan, Cuifeng Jiang, Tingting Zhao, Peiyan Yuan, T Venkatesan, and Qing-Hua Xu Excitation Nature of Two-Photon Photoluminescence of Gold Nanorods and Coupled Gold Nanoparticles Studied

by Two-Pulse Emission Modulation Spectroscopy J Phys Chem Lett 2013, 4,

1634 − 1638

5 Yee hong Lee, Lakshminarayana Polavarapu, Cuifeng Jiang, Peiyan Yuan, Qing-Hua Xu Recent Advances in Metal Enhanced Optical Properties COSMOS 2010, 6, 167

6 Cuifeng Jiang and Qing-Hua Xu Simple Two-Photon Sensing of Dopamine using Au NPs In preparation

Chapter 1 Introduction

A general overview on the optical properties and applications of noble metal nanoparticles is given at the beginning of this thesis to provide a framework for my research conducted A brief discussion is given on the localized surface plasmon resonance, noble metal based sensor, followed by an introduction on two-photon photoluminescence At the end of this chapter, an outline of the research that I have conducted is presented

1.1 Noble metal nanoparticles

In this section, the optical properties of gold nanoparticles will be discussed Metal nanoparticles have gained much attention in recent years owing to their unique physical and chemical properties as well as their applications in catalysis, optoelectonics, along with biological and chemical sensing

1.1.1 Localized Surface Plasmon Resonance

Novel properties and potential applications emerge when bulk gold is finely divided into the nanoscale particles One of the most noteworthing and useful changes is in gold nanoparticles interaction with light Upon irradiation with light, gold nanoparticles exhibit strong absorption at specific resonant wavelength, which is generally dependent on the morphology and dielectric environment of the gold nanoparticle.1 This phenomenon now known as localized surface plasmon resonance (LSPR), is the origin of many new applications of gold nanoparticles This phenomenon has been systematically studied in 1857 when Michael Faraday demonstrated the synthesis of gold colloids in an aqueous medium.2 This study has paved the way in preparing gold nanoparticles in the scope of investigating its various potentially useful applications

Scheme 1.1 shows the creation of a surface plasmon oscillation.3 For a spherical nanoparticle, the ionic core is much heavier compared to the free electrons When the incoming light wave is irradiating, the free conduction electrons will be polarized Assume the positive charges in the particle to be immobile, then, the negative charges comprising of the conduction band electrons, can move under the influence of external fields generated by incoming light Therefore, when the metallic nanoparticle is placed in an electric field, a displacement of the negative charges from the positive charges can occur, resulting in a net charge difference around the nanoparticle, which brings about a linear restoring force to the nanoparticle Consequently, surface plasmon oscillation of the electrons is created and this dipolar oscillation has a specific time period In a word, the electromagnetic radiation with the metal nanoparticles induces a dipole The collective oscillation of the electrons is also sometimes called “dipole particle plasmon resonance” to differentiate of plasmon excitations that occur in bulk metal surfaces Thus, the term surface plasmon resonance refers to the cosistent excitation of all the “free” electrons within the conduction band, resulting in an in-phase oscillation

terms of preparing novel structures as well as in the control of shapes and sizes

of these NPs.8-10 In general, the relationship between LSPR and metal NPs are: (1) the LSPR intensity increases and the wavelength redshifts with increasing the size of spherical metal nanoparticles; (2) the LSPR wavelength for non-spherical particles redshifts with increasing corner sharpness and particle anisotropy; (3) the intensity of the resonance peak increases if charges separate with mirror symmetry; (4) the number of resonance peaks increases with the number of ways that the particle can be polarized; and (5) the LSPR wavelength red shifts with increasing the refractive index of the surrounding medium Figure 1.1 exhibits LSPR of different shapes of gold nanoparticles.11Figure 1.2 displays the LSPR of different sizes of gold and silver nanoparticles.12

Figure 1.1 Different shapes or sizes of Au nanocrystals: (A) nanospheres; (B) nanocubes; (C) nanobranches; (D—F) nanorods with increasing aspect ratios; (G—J) nanobipyramids with increasing aspect ratios (K) Normalized extinction spectra of the nanospheres (black), nanocubes (red), and three

nanorod samples with different ratios (green, blue and purple) (L) Normalized extinction spectra of the four nanobipyramid samples (red, green, blue and purple) and nanobranches (black) Reprinted with permission from ref 11 Copyright 2010 Elsevier

Figure 1.2 (A, B) Normalized extinction spectra and (C, D) TEM images of individual Au NSs (panels A and C) and Ag NSs (panels B and D) of different sizes Reprinted with permission from ref 12 Copyright 2012 American Chemical Society

Au nanostructures with LSPR located at near infrared (NIR) range have essential applications, because NIR falls within the biological transparency window (700-1000) The light in this range can penetrate deeper into biological tissues.13, 14 Thus, among the development of gold nanoparticles, gold nanorods are mostly used because their LSPR could be tuned to NIR by adjusting the parameter ratio.15 Gold nanorods typically have two LSPR peaks, one peak for the transverse mode around 520 nm and the other one for the longitudinal mode, and the position depends strongly on the aspect ratio of the rod These structures are typically synthesized in solution-phase, by a

seed-mediated method, in which small gold seeds are added to a series of growth solutions By adjusting the concentrations of key reagents and the size

of the initial gold seeds, 16 it is possible to prepare nanorods with strong LSPR-based absorption across the visible spectrum and into the NIR On the other hand, the LSPR of gold nanoshells can also be tuned into the NIR by adjusting the thickness of gold walls surrounding a dielectric or hollow core.17,

18

One typical nanoshell structure was created by depositing small gold nanoparticles onto the surface of a silica sphere or big gold nanoparticles, followed by deposition small gold nanoparticles The LSPR depends on the ratio between the diameter of the particle and the thickness of the deposited gold layer Based on the above review, complex synthetic methods have to be explored both for gold nanorods and nanoshells in order to achieve the required NIR absorption, and the preparation process was time consuming However, there is no such problem for gold nanospheres and its ease in preparation makes us particularly interested in them

1.1.2 Plasmon Coupling and its Optical Properties

Besides the shape, size and dielectric environment of the nanoparticles, the LSPR of metal nanoparticles can also be significantly interfered by adjacent particles with inter-particle distance shorter than half of the particle diameter When the individual nanoparticles come into close proximity, the electron oscillation generated by one nanocrystal will induce another nanoparticle to generate the electron oscillation The plasmon resonances of the two nanocrystals are then coupled together This interaction energy is so strong that the assembled metallic nanoparticles can shift their wavelengths to longer

or shorter range compared to each individual nanoparticles.13 Take gold colloids as example, the color of solution changed from red to blue upon aggregation For silver nanoparticles, the aggregation-induced colour change

is observed to be from yellow to brown

The Mie theory cannot be directly applied for a coupled system because of the complexity of the aggregates and its interference effects.19 Thus, in the calculation of optical properties for an aggregate, influence of all neighboring nanoparticles have to be considered.3 Theoretical calculations reveal that all factors including particle size, distance between nanoparticles and aggregate size have significant influence on the properties of a coupled system.3

The distance between nanoparticles is found to have a significant influence

on the absorption spectra of the coupling system.20 Figure 1.3A shows the calculated absorption spectra for Au NPs (d = 20 nm) coupled system with different distances Individual gold nanospheres exhibit only one peak known

as LSPR, while coupled Au NPs have two peaks The first peak is attributed to the surface plasmon excitation of individual Au nanospheres, and the second peak at longer wavelength can be ascribed to the dipole plasmon resonance of the metal nanoparticles The oscillating electrons in one particle experience the electric field generated by the oscillations of a second particle, resulting in

a combined plasmon oscillation of the coupled system With decrease in separation distance, the intensity of the first peak decreases while the second peak is intensified and shifts to a longer wavelength It is referred that classical single-particle Mie theory can be applicable when the separation distance is beyond about 5 times the particle radius

On the other hand, the absorption spectra of the coupled system are evaluated to be size-dependent.20 Figure 1.3B displayed the calculated size-dependent effect for Au nanoparticles The interparticle spacing in this Figure is set as 0.5 nm The peaks varied from a single broad peak to two distinct peaks, for particles with diameter of 5 nm to 36 nm

In addition, the optical property of aggregates can also be influenced by the aggregates size.20 Figure 1.3C shows the calculated absorption spectra of aggregated Au nanoparticles with linear morphology It reveals that, different numbers of particles show different peaks With an increase in the number of aggregates, the peak at longer wavelength shows a red shift In summary,

these mathematically evaluated results demonstrate that factors such as large metal nanoparticles or short separation distances should result in higher optical response A number of experiments have been performed to investigate the influence parameters and obtained the similar trends.12, 21, 22

Figure 1.3 Electrodynamic modeling calculations for Au nanoparticles (A)

Extinction spectra for gold nanoparticles (diameter: 20 nm) with different

inter-particle distance Inset is the ratio of peak shift vs inter-particle distance;

(B) Extinction spectra for different sizes of Au nanoparticles with fixed inter-particle distance (0.5 nm); (C) Extinction spectra for linear Au nanoparticles aggregates with different numbers of particles Inset is the ratio

of peak shift against the number of Au particles Reprinted with permission from ref20 Copyright 2004 American Chemical Society

Novel optical properties such as plasmon coupling lead to wider applications of metal nanoparticles For example, gold nanospheres are usually not suitable for applications in the field of medicine because its LSPR is located in the UV-Visible range, which has an adverse effect on tissues However, the LSPR of aggregated gold nanospheres can extend to NIR range

by adjusting the aggregation morphology In addition, plasmon coupling can

be used to monitor distances between single pairs of Au or Ag nanoparticles.23Moreover, assembly of metal nanoparticles has attracted various interests in application such as sensors, catalysis, medical diagnostics, and more The extinction coefficients of gold nanoparticles are much higher compared to traditional organic compounds For example, the extinction coefficient of 13

nm gold particles is 2.7108 (at 520 nm), which is 3-5 orders of magnitude higher than that of traditional organic dyes.24 This combination of high extinction coefficients and distance-dependent optical property makes metal nanoparticles an ideal color reporter When gold nanoparticles close to each other, colors of the nanoparticles change from red to blue Thus, a wide range

of assays, taking advantage of plasmon coupling have been developed for detection of nucleotides, proteins, and cells based on the color change A group of studies will be further discussed in detail on section 1.3.1

d c

Figure 1.4 (a) Hot spots in a Raman scattering image of coupled Au nanoparticles arising from (b) zoom-in view of selected hot spots, arising from condensed nanoparticle pairs; (c) FDTD calculations for adjacent nanoparticle pairs A hot spot appears in the junction for incident polarization along the inter-particle axis and (d) for incident polarization orthogonal to the inter-particle axis, no hot spot occurs Reprinted with permission from ref 27 Copyright 2011 American Chemical Society

Besides change of LSPR, another important property generated by plasmon coupling is generation of “hot spots” In 1977, raman scattering spectra of an adsorbed molecule were found to be extraordinarily large enhanced after

aggregation.25 After numerous studies on this effect, it was deduced that the huge enhancements were induced by the strongly interacting between metallic nanoparticles.26 The junction between adjacent nanoparticles, can generate highly strong and localized electromagnetic fields after excitation by incident light from the appropriate polarization Therefore, the inter-junction regions were named “hot spots” Halas et al have clearly identified bright SERS signals to result from adjacent nanoparticle pairs by using a combination of single-particle Raman and AFM experiments as shown in Figure 1.4.27Single-molecule detection using SERS was reported in 1997 and has attracted much attention.28, 29

Due to the existence of “hot spots”, plasmon coupling between noble metal nanoparticles has been therefore employed for enhancing second harmonic generation (SHG), two-photon excited fluorescence and two-photon photoluminescence (TPPL) Roch and co-workers showed that a dimer made

up of two gold nanospheres exhibits a remarkable efficiency for SHG Our research group found out that the two-photon photoluminescence of Ag NPs was significantly enhanced after aggregation, induced by conjugated polymers.30

The synthetic strategies to couple gold NPs can be generalized as two major methods: (a) direct formation of Au NPs aggregates; (b) induced aggregation of pre-preparation Au NPs In this thesis, we will focus on assembling already formed Au NPs to aggregate Metal nanoparticles prepared

by citrate method are generally stable because of a charged double layer (citrate) in solution that surrounds each colloidal nanoparticle, producing a coulomb barrier against aggregation As proposed in numerous prior studies, the driving forces for aggregation could be attributed to electrostatic forces, van der Waals interaction, chemical bonding, hydrogen bonding, magnetic fields, or a combination of these forces The detailed strategy to induce plasmon coupling will be discussed in section 1.3

1.2 Singlet Oxygen and its Application

Singlet oxygen (1O2), the first excited state of molecular oxygen, is an intermediat with high reactivity It plays important roles in chemical and biological processes, such as polymer degradation and cell death.31, 32 As shown in scheme 1.2, oxygen’s electronic ground state, is a spin triplet Thus,

it often exhibits radical-like behavior in chemical reactions Two of oxygen’s lowest-energy excited electronic states, 1O2 (a1 g ) and 3O2 (b1

 g + ), are spin singlets The lower energy a1 g state, commonly known as “singlet oxygen”, has rich chemical properties.33 The singlet oxygen has high reactivity It is because two electrons paired into one 2pπ* orbital, producing the high oxidative activity of the unoccupied π* orbital

of light and photosensitizers to give rise to a cytotoxic effect to diseased tissue Upon absorption of a photon, the photosensitizer is promoted to its singlet excited state, then, after inter-system crossing, transfer to its excited triplet state The triplet can participate in a one- electron oxidation- reduction

reaction (Type I photochemistry) with a neighbouring molecule, producing free radical intermediates that can react with oxygen to produce various reactive oxygen species (ROS).35 Alternatively, the triplet state photosensitizer can also transfer its energy to ground state oxygen (Type II photochemistry), generating singlet molecular oxygen, a highly oxidative specie that can react with many biological molecules, including proteins, lipids, and nucleic acids (Scheme 1.3) These reactions occur only in the localized area of the light-absorbing photosensitizers Therefore, one advantage of PDT is that biological responses to the photosensitizers are activated only in the selected area of issues The generation of singlet oxygen can be measured directly by detecting the photoluminescence of singlet oxygen at about 1270 nm.36Alternatively, the ability of singlet oxygen generation efficiency can be examined by monitoring the rate of oxidation by means of a chemical probe (eg: ABDA) This chemical probe can be oxidized by singlet oxygen, causing

an observable drop in absorbance

Energy Transfer

hindered because of its limited light penetration depth into tissues in the visible range and its lack of selectivity in the z - direction The newly found phenomenon of two-photon induced generation of singlet oxygen resolved these problems.37 Excitation of a given molecule can also occur upon the simultaneous absorption of two photons, when use pulsed laser sources This process is often described as proceeding through a so-called virtual state Two-photon induced generation of 1O2 has attracted much attention because of its numerous advantages in various applications.38-36 Two-photon photodynamic therapy (TPPDT) is its most widely used application, which will be discussed further on section 1.5

1.3 Noble Metal Nanoparticles Based Sensing Method

It is important and urgent to develop detection methods for chemical and biological agents in many fields, which include environmental, biomedical sciences and more Noble metal nanoparticles, such as Au and Ag, are excellent candidates for developing sensors due to their unique chemical and physical properties.24, 37-43 They can be prepared by simple chemical reduction

by NaBH4 and possess excellent biocompatibility when used with appropriate ligands.44 In addition, their optical properties are dependent on their morphology (eg: size and shape) and surrounding environment Thus, the required optical properties can be obtained by tuning these parameters For example, a colloidal solution containing 13nm diameter Au nanoparticles appears red in color because their LSPR locates at 520 nm However, plasmon coupling results in significant red-shifting and spectra broadening of the LSPR, which is accompanied by a color change from red to blue This color change

of Au NPs provides a good platform for colorimetric based sensing for metal ions, small molecules, proteins, nucleic acids, and cancer cells

1.3.1 Overall introduction of nanoparticles in sensing

Lots of efforts have been made on use of nanoparticles in sensing, by improving synthesis method or developing novel detection approach

The most popular method to prepare Au NPs is developed in 1951 using citrate reduction of HAuCl4 in H2O, where citrate group functions as both reducing and stabilizing agent.45 Diameter of nanoparticles prepared by this method is around 20 nm Then, 20 years later, Frens G found the size of nanoparticles can be tuned by adjusting the volume of citrate salt.46 Because of its easy preparation and biocompatibility of citrate prepared Au NPs, this method becomes the most popular route and have been used to prepare Au NPs for sensing assays

In comparision to the stability of preparation method, different detection methods have been studied, including colorimetric, 47-49 fluorescence, 50, 51 surface plasmon resonance 52-54 and SERS methods 55-57 Colorimetric is the most popular approach for its versatility and will be discussed in detail on section 1.3.2 &1.3.3

Fluorescence method is widely used in sensing systems When a dye closes to Au NPs, the fluorescence of the dye will be quenched due to Forster resonance energy transfer (FRET).58 By taking advantage of this principle, Au NPs can serve as sensing probe for FRET-based assay The traditional strategy

is Au NPs based molecular beacons.59-61 Typically, Au nanoparticles are conjugated with a single stranded DNA and the other end of the DNA is attached with a dye.62 The fluorescence of dye will be quenched due to FRET because single stranded DNA rolls around the nanoparticles However, upon addition of complementary DNA, DNA will undergo conformational change (rodlike or G-quarter), and a distance appears between dye and nanoparticles

In this case, the fluorescence is turned on and the signal is proportional to the concentration of target DNA By employing similar principle, different structures of DNA have been detected.63-66

However, the detection range of Au NPs based molecular beacons is limited to DNA In order to expand the application of fluorescence sensing, semiconductor quantum dots as stable fluorophores have been combined with

Au NPs.67 For example, Guo and co-workers have employed QDs and Au NPs

to sense Pb2+.68 The negatively charged QDs and positively charged Au NPs assmble together by electrostatic interaction Upon addition of Pb2+, Pb2+ induces aggregation of Au NPs, inhibiting the FRET, and releases the QDs, which results in the increase of fluorescence signal accordingly

It is known that bigger signal change favors better sensitivity In this case, SERS is a powerful tool for good sensitivity detection because of its high enhancement fold and analytes’ unique fingerprint The Raman scattering singal can be enhanced largely by the presence of rough surface.69 Based on this principle, various assays have been designed to sense small organic molecules,55 oligonucleotides70 and proteins71 For example, Ray and co-workers have detected TNT by using cysteine modified Au NPs as the probe.55 The interaction between cysteine and TNT caused aggregation of Au NPs and the “hot spots” in the gap region of coupling results in high SERS enhancement, giving a sensitivity of picomolar level Additionally, SERS methods have been successfully used for detection of target DNA,72 thrombin,

71

protease73 and so on

As discussed previously, localized surface plasmon resonance of noble metal nanoparticles are dependent on their size, shape and surrounding environment The influence of surrounding media on LSPR is used to detect chemical or biological agents.74-77 LSPR wavelength of nanoparticles exhibits red-shift or blue-shift upon adsorption of analytes on their surface Besides in solution phase, this kind of assay can be performed on substrates by immobilizing nanoparticles on it ITO glass, 78 optical fibers79 and quartzs 80 have been used as the substrates to detect streptavidin, 78 human IgG, 79 BSA 80 and so on

Although numerous detection platforms have been developed, challenge

still exists for sensing Nanoparticles are unstable and get aggregation easily in biological environment due to existence of salt and proteins, which limits the application of the above methods Thus, modification of nanoparticles is necessary For example, Au NPs can be functionalized with antibody, aptamer, small biomolecule and silica to change the property of their surfaces Alternatively, novel detection techniques taking advantage of their unstability

in biological environement can be developed, such as two-photon photoluminescence technique

In this thesis, we will mainly focus on cross-linking aggregation based sensing and non cross-linking aggregation based sensing Although there are many systems work based on aggregation of noble metal nanoparticles, they can be divided to two main groups: cross-linking aggregation based sensing and non cross-linking aggregation based sensing Aggregation of Au nanoparticles in sensors can be induced by an inter-particle cross-linking in which the enthalpic benefits of inter-particle bond formation can overcome inter-particle repulsive forces On the other hand, aggregation can be induced

by the controlled loss of colloidal stability in a non-cross linking aggregation mechanism Such mechanisms are also extended to silver nanoparticles

1.3.2 Cross linking aggregation based sensing

Cross linking aggregation is the most common approach in Au NPs based colorimetric sensing

The pioneering work for Au NPs based colorimetric method was developed by Mirkin and co-workers.81, 82 DNA can be covalently attached to

Au NPs through a thiol-group When the complementary DNA was added, Au NPs will assemble together due to the hybridization between two DNA strands, and the color changed from red to purple in the colloidal solution as shown in scheme 1.4 Fabrication of coordinate bonds between DNA bases and chemicals is another way to form crosslinkers Mirkin et al reported a

colorimetric sensor for Hg2+ by using Au NPs and DNA.83 This sensing system takes advantage of the selective coordination between Hg2+ and bases that of thymidine-thymidine (T-T) type Hg2+ can bind to the T-T sites selectively and raise the Tm of the mismatched structures When the temperature was raised beyond that of the melting temperature of Au NPs, the solution containing Hg2+ remains purple in colour, while the other solution changed to red.83

In addition to the Au NPs-DNA system, other biological recognition events and some chemical reactions can also be used as cross-linking aggregation based colorimetric sensors 24, 91

Noble metal nanoparticles based colorimetric sensing assay for heavy

metal ions generally requires the modification of Au NPs with chelating ligands Upon addition of heavy metal ions, the nanoparticles aggregate with the chelating agents act as crosslinkers shown in scheme 1.5

Scheme 1.5 Schematic depiction of metal NPs functionalized with chelating ligands induced aggregation

Fang Chai et al have reported a simple colorimetric strategy for the sensing of Hg2+ utilizing L-cysteine functionalized 16 nm Au NPs.92 The color change is induced by a mercury-chelation interaction in which the surface carboxylate molecules serve as receptors of mercury ions The observed color change becomes faster with the assistance of UV radiation No colorimetric response was observed in the presence of other divalent metal ions This assay gave a detection limit of 100 nM Colorimetric detection of Hg2+ and Cu2+have been achieved using Au NPs modified with peptide and cysteine.93 Moreover, Au nanoparticles have provided a successful application for colorimetric detection of proteins.94 The immunoassay based on noble metal nanoparticles is a straightforward one-step procedure Particles functionalized with a ligand will form aggregates in the presence of a specific target This

process could be monitored by colorimetric change or by obtaining an extinction spectra change Rosenzweig et al have demonstrated the high sensitive and specificity for detection of anti-protein A using gold nanoparticles based on aggregation-induced color change.95 Au nanoparticles are coated with protein antigen, upon addition of corresponding antibody, the

Au NPs will assemble together due to antibody-antigen interaction In addition, the authors investigated the influence of experiment condition (eg: pH, temperature and concentration of Au nanoparticles) on the sensitivity of the sensing system The limit of detection of this assay is 1mg/mL Colorimetric detection of IgG was achieved by Zeng and co-workers, using Au NPs and cysteine or histidines modified peptide linker connecting scFv.96 Suri and co-workers reported a one-step homogeneous colorimetric immunoassay format coupled with zeta potential measurements for the determination of specific diabetic biomarker glycated hemoglobin (HbA1c).97 It is of utmost importance to detect proteins sensitively and specifically because many diseases are often related to the appearance of certain biomarker proteins or abnormal protein concentrations

Besides the cross-linking assay system for colorimetric sensing, using targets to break the cross-linkers between Au NPs is another viable strategy

To break the DNA cross-linkers, the simplest method is to use endonuclease to hydrolyze the DNA-duplex interconnection The first study on enzyme-responsive system to disassemble aggregated Au NPs is reported by Mirkin et al Since then, many studies have been reported on the detection of endonuclease A blue-to-red color change sensor for Pb2+ was developed by Chen and co-workers.99 In this system, Au NPs were bifunctionalized by (crown-5) CH2O(CH2)4SH and thioctic acid (TA) Initial aggregation of Au NPs was formed due to hydrogen bonding interaction between carboxylic acid and organic solvent molecules The addition of Pb2+ metal ions disrupts the assembly by its association with the crown ether moiety, leading to a blue-to-red color change

DNA structure conformational change induced by target can also be employed to disassemble the complex, besides cleavage of nucleic acid linkages Lu et al reported the structure-switching aptamer into the sensing system to disassemble Au NPs and applied the new sensor for colorimetric detection of adenosine and cocaine.100 When adenosine or cocaine was added, the aptamer switches its structure spontaneously to bind preferentially target adenosine and cocaine molecules As a result, only a few base pairs were left free, making the complex unstable Consequently, the aggregated particles were dissociated and the color changed from purple to red The disassembly process can be finished within seconds, and is much faster than the assembly process

1.3.3 Non-cross linking aggregation based sensing

For the colorimetric sensors stated in the previous subsection, the mechanism was based on the assembly and disassembly of cross-linking aggregation of Au NPs However, non-cross linking aggregation proved to be another effective colorimetric sensing route.101, 102 In preparation of Au NPs, the surface charges, combined with the counter ions (eg: citrate groups), form

a repulsive electric double-layer, stabilizing colloids against van der Waal attraction-induced aggregation Electrostatic repulsion is mainly sensitive to the bulk ionic strength, and decreases significantly at high salt concentration Therefore, citrate capped Au NPs assembled together when the salt concentration is high enough Based on this principle, non-cross linking aggregation has been used for biodetection

Aptamer based non-cross linking sensing arrays have proved to be efficient.105 For example, Fan and co-workers have designed a novel strategy

to detect small molecules based on non-cross linking aggregation.106 In this method, aptamer was specially designed Two-pieces of random, coil-like single stranded DNA were designed as one aptamer Upon addition of target

molecules, the two species DNA reassemble to an intact aptamer tertiary structure After addition of salt, the solution with target molecules will form aggregates and a color change to purple is observed (scheme 1.7) Using this method, cocaine was detected on the low micromolar level The significant advantage is that this method can be extended to detection of other small molecules, such as adenosine and potassium

Scheme 1.7 Schematic description of non-cross linking based assay for detection of cocaine using engineered cocaine aptamer and Au NPs Reprinted with permission from ref 70 Copyright 2012 Wiley

In addition to salt, conjugated polyelectrolytes have also been observed to lead to non-cross linking aggregation of Au NPs Recently, Heeger and co-workers reported a novel detection platform which employs single stranded DNA, unmodified Au NPs, and a positively charged, water-soluble conjugated polyelectrolytes to detect a series of targets, which include DNA sequences, small molecules, proteins and inorganic ions.107 Principle of this sensor is that, single stranded DNA can prevent the aggregation of Au NPs, while the conjugated polyelectrolytes can specifically inhibit the ability However, double stranded or other “folded” DNA structures does not show this kind of inhibition ability This colorimetric method is convenient and sensitive to low picomolar concentrations of target DNA, additionally, it can be applied in blood serum

The process of aggregation for non-cross linking system is much faster

than that of cross-linking system Different aggregation mechanisms contribute to this difference For non-cross linking system, the aggregation was induced by van der Waals forces of attraction between the nanoparticles This attractive force works in close proximity, and results in rapid aggregation

On the other hand, random collisions of metal NPs will induce the aggregation

in non-cross linking system, which is much faster than the Brownian motion.108

The above cross-linking and non-cross linking mechanism have been extended to Ag NPs However, much less attention has been given to the application of Ag NPs in colorimetric sensing because of its instability Nonetheless, Ag NPs are more cost-effective in their preparation and exhibit much higher extinction coefficients than Au NPs of the same size and have been used for detection of metal ions,109, 110 small molecules,111, 112 DNA sequences,113 and protein.114

1.4 Two-Photon Excited Photoluminescence

1.4.1 Two-Photon Absorption

M Göppert-Mayer proposed the concept of two-photon absorption (2PA) process in her doctoral thesis, which is the first time to mention 2PA.115 She predicted that a molecular can complete its transition from a lower energy level to the higher one by simultaneous absorbing two photons.116 The key point of her theory was the concept of an intermediate state However, the experiment confirmation of two-photon excited process was developed until the appearance of laser device 117 In 1961, Kaiser and Garrett reported the first experimental confirmation of two-photon absorption excited frequency-upconversion fluorescence.118 Since then, lots of studies have been reported on two-photon excited fluorescence in many fields, which include chemistry, physics and optics With development of different kinds of laser

devices, higher-order multiphoton excitation associated processes have been realized, not only 2PA related processes

Since the development of two-photon excited process, basically, two theories can explain multi-photon absorption processes the semiclassical theory and the quantum electrodynamical theory.119-121

In the semiclassical theory, the media are depicted by the theory of quantum physics, while the light fields are explained by use of the classical Maxwell theory.122 The expression for the nonlinear electric polarization of an optical medium is the most essential feature of the semiclassical theory P is the electric polarization vector of a medium In a certain volume, all the light fields can induce electric dipole moment vectors Summation of all the vectors

of the molecules generated by the light field is defined as P In a linear process,

P is linearly proportional to the applied electric field E.121 For the nonlinear absorption (take two-photon as an example), P is proportional to the square of

E.123 However, in the semiclassical theory, the light waves are described as classical electromagnetic fields, and no concept of quantization of light, which limited the application of semiclassical theory in two-photon excited process

In contrast, different from semiclassical theory, the medium was described quantum-mechanically, as well as the optical field in quantum theory 124-126Thus, the quantum theory is a more rigorous approach to describe two-photon absorption process both qualitatively and quantitatively In this theory, intermediate state is very important to understand the two-photon absorption process, which takes the combined system of the photon field and a molecular system into consideration

Figure 1.5 shows the schematic diagram for the process of two-photon excited fluorescence The dashed line represents an intermediate state between two real energy states of the molecule Thus, different from one-photon excited fluorescence process, two-photon excited fluorescence can be understood as a “two-step” process: (i) Firstly, one photon is absorbed to excite the molecule to an intermediate state from its initial state Eg (ii) In the

second step, the same molecule will be excited from the intermediate state to the final real state Ef by absorbing another photon In this case, the intermediate state is the key connection between the two processes However, the molecular status in intermediate state is not certain, which means the molecule can stay in any possible energy states (except Eg and Ef) Thus, there

is a certain distribution probability in each energy state Because the probability distribution range is very large, the residence time on each energy level, including intermediate state, should be very short, even can be ignored

In this case, we can speculate that “two steps” of two-photon absorption actually occur at the same time

Figure 1.5 Jablonski energy diagram for one-photon and two- photon absorption

TPA has different selection rules with that of one photon absorption For a molecule, TPA probability at certain position is proportional to the square of the local light intensity, while in one photon absorption, the absorption is

linearly proportional to the light intensity If we define I as the local light intensity with the sample, z as the position along the beam propagation