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 Name 11, Feb, 2014 Signature I Date 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 familymy 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 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 II 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 Introduction 1.1 Noble metal nanoparticles 1.1.1 Localized Surface Plasmon Resonance 1.1.2 Plasmon Coupling and its Optical Properties 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 III 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 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 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 IV 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 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 Conclusion 128 V 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 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 VI 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 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, which induce 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 orders of magnitude Most importantly, this two-photon sensing assay can be applied to detection of VII 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 VIII 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 IX 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) 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 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 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 Figure 1.5 Jablonski energy diagram for one-photon and two- photon absorption 23 Figure 1.6 Optical setup of a TPPL experiment 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 X 26 Figure 5.4 Power dependence of the TPPL of Ag NPs under excitation at 810 nm in presence of 60 nM thrombin (Power density: 23, 30, 36, 43, 47 mW) The two-photon excitation nature of the observed emission was confirmed by measuring the integrated emission intensity as a function of the laser excitation power (Figure 5.4) The best-fitting straight line of log-log plot gave a slope of 2.1, confirming the two-photon excitation nature of the observed emission 116 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 For an excellent sensing system, high selectivity is a matter of necessity To make sure that the TPPL enhancement is mainly due to the binding event between aptamer and silver nanoparticles, not due to other reasons, BSA was chosen as the control sample The TPPL obtained with 70 nM of BSA exhibited only twice enhancement (Figure 5.5) This result clearly demonstrated that our TPPL assay is highly selective to thrombin and it can distinguish from BSA This high selectivity was attributed to the high specificity of the thrombin aptamer In order to check if the enhancement of TPPL is due to specific binding between TBA15 and thrombin, we have tested random DNA sequences that not bind to thrombin The random DNA leads to only fold enhancement of TPPL under the same experiment condition These results demonstrate that the enhancement of TPPL results from binding events between thrombin and aptamer Random DNA and BSA can not influence the performance of the sensing platform Thus, the two-photon sensing platform displays very good specificity for thrombin detection 117 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 118 [thrombin] in undiluted serum The inset shows that the enhancement factor of TPPL is direct proportion to [thrombin] in the range of low concentration It is very important to detect thrombin in the blood serum as thrombin generally exists in blood, which is a more complicated media than the buffer solution The above TPPL scheme was tested in the presence of fetal bovine serum (20% volume ratio of the total sample) The TPPL signals in the complex media were found to steadily increase with increasing concentration of thrombin (Figure 5.6) The enhancement factor of TPPL is direct proportion to the concentration of thrombin in the low concentration range (Figure 5.6c) The LOD of this TPPL method was calculated to be 0.10 nM Fetal bovine serum is a complicated biological fluid containing a large number of proteins and other materials that may contribute extra background signal and result in a relatively poor sensitivity compared to that in the buffer solution.51 Because of the highly selective and high-affinity interactions between thrombin and the thrombin-binding aptamer, this sensing scheme can discriminate against nonspecific binding and thus readily detects physiological thrombin levels, even in complex, contaminant–ridden samples such as blood serum The extinction method was conducted under the same experimental condition for direct comparison The LSPR moved from 395 to 364 nm in serum due to change of dielectric media The extinction ratio A483nm/A364nm was plotted versus [thrombin] as Figure 5.7 The extinction methods gave an LOD of 12.2 nM (Figure 5.7) The LOD of our TPPL method is orders of magnitude better than the extinction method In addition, this TPPL based detection of thrombin in the complex media is also highly selective against BSA and by using random DNA sequence as shown in Figure 5.8 119 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 shows that A483nm/A364nm is direct proportion to [thrombin] in the low concentration range 120 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 Furthermore, we performed the spiking experiment The experiment was conducted as follow: spike certain human α–thrombin into diluted fetal bovine serum, the sample was kept for several minutes Then, the TPPL spectra of the sample were measured The used relationship curve was displayed in Figure 5.9 Results in Table 5.1 show that the unpretreated 10-fold diluted serum samples, resulted in a recovery to 88% The serum used is 10-fold diluted It is known that there are lots of proteins in serum Even in the presence of these 121 proteins, the recoveries obtained (75%-88%) were acceptable We can speculate that diluted serum samples almost can not influence the detection of thrombin In determination of proteins by using antibodies or aptamers, 10-fold dilution of serum is often used.13, 51-53 The excellent sensing specificity could be attributed to advantages of two-photon photoluminescence, which includes less background and deep penetration Figure 5.9 The relationship curve for spiking thrombin into serum experiment Table 5.1 Recovery of Human α–Thrombin Spiked into Fetal bovine serum samples Actual thrombin Detected thrombin concentration (nM) concentration (nM) 46.8 39.6 85 62.5 47.1 75 78.1 68.7 88 Recovery (%) 122 5.4 Conclusions We have demonstrated a label free, fast and highly sensitive 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, TBA15, which induce aggregation of Ag NPs and result in significantly enhanced TPPL The LOD of our TPPL assay is as low as 3.1 pM in the buffer solution, more than 36 times lower than that of the extinction method (1.3 nM) The dynamic range of this method covers more than orders of magnitude Most importantly, this TPPL assay can be applied to detection of thrombin in fetal bovine serum with LOD of 0.1 nM Furthermore, 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 123 References Wang, Y Y.; Liu, B., Langmuir 2009, 25, 12787-12793 Nishino, A.; Suzuki, M.; Ohtani, H.; Motohashi, O.; Umezawa, K.; Nagura, H.; Yoshimoto, T., J Neurotraum 1993, 10, 167-179 Serruys, P W.; Vranckx, P.; Allikmets, K., Int J Clin Pract 2006, 60, 344- 350 Wei, H.; Li, B L.; Li, J.; Wang, E.; Dong, 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J., Nature 1992, 355, 564-566 46 Li, H.; Rothberg, L J., J Am Chem Soc 2004, 126, 10958-10961 126 47 Chen, C.; Song, G T.; Ren, J S.; Qu, X G., Chem Commun 2008, 46, 6149-6151 48 Li, H.; Rothberg, L J., Anal Chem 2004, 76, 5374-5377 49 Jiang, C F.; Zhao, T.; Yuan, P.; Gao, N.; Pan, Y.; Guan, Z.; Zhou, N.; Xu, Q.-H., ACS Appl Mater Interfaces 2013, 5, 4972-4977 50 Li, F.; Du, Z.; Yang, L.; Tang, B., Biosens Bioelectron 2013, 37, 907-910 51 Li, L L.; Ge, P H.; Selvin, P R.; Lu, Y., Anal Chem 2012, 84, 7852-7856 52 Fischer, N O.; Tarasow, T M.; Tok, J B H., Anal Biochem 2008, 373, 121-128 53 Centi, S.; Tombelli, S.; Minunni, M.; Mascini, M., Anal Chem 2007, 79, 1466-1473 127 Chapter Conclusion Noble metal nanoparticles, such as Au and Ag, display many unique optical properties, including localized surface plasmon resonance (LSPR), which results from collective oscillation of the conduction band electrons The LSPR band is dependent on the morphology of particle (size and shape) and dielectric environment surrounding the particles Plasmon coupling of aggregated metal nanoparticles can induce the LSPR peak red or blue shifted The generation of “hot spots” in gap region can enhance the local electrical field significantly 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 induce significant enhancement of TPPL 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, aggregation effects on two-photon induced photoluminescence and singlet oxygen generation of Au nanospheres and Au nanorods of two different aspect ratios were invested in Chapter Aggregation effects of noble metal nanoparticles are generally believed to be adverse to biomedical applications, however, aggregated Au nanospheres and short Au nanorods were found to display enhanced two-photon induced photoluminescence and singlet oxygen generation capabilities compared to the un-aggregated ones 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 128 photoluminescence and singlet oxygen generation of the aggregated long Au nanorods were found to be lower than the un-aggregated 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 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, a label free, fast, highly sensitive and selective two-photon sensing scheme for selective detection of thrombin on the picomolar level was demonstrated The assay is based on selective interactions between thrombin 129 and a DNA aptamer, which induce 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 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 this thesis, aggregation effects on two-photon induced photoluminescence and singlet oxygen generation of Au nanospheres and Au nanorods of two different aspect ratios were systematically studied Then, a series of cross linking and non-cross linking aggregation based TPPL sensing assays were designed and successfully applied to dopamine, mercury ions and thrombin In addition to the unique advantages of two-photon sensing such as deep penetration and localized detection, TPPL based sensing methods could be potentially combined with two-photon microscopy to offer additional advantages of 3D detection and mapping for potential in-vivo sensing applications 130 ... optical properties of noble metal nanoparticles and two- photon photoluminescence, the aggregation of noble 32 metal nanoparticles induced enhanced two- photon photoluminescence and their applications. .. 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. .. microfabrication,129 two- photon excited imaging,130 two- photon excited photoluminescence, 131, 132 and two- photon related chemical and biological applications. 133, 134 These interesting and potentially useful applications