WATER QUALITY CONTROL USING DIVERSITY ORIENTED FLUORESCENCE LIBRARY APPROACH

WATER QUALITY CONTROL USING DIVERSITY ORIENTED FLUORESCENCE LIBRARY APPROACH XU WANG NATIONAL UNIVERSITY OF SINGAPORE 2015         WATER QUALITY CONTROL USING DIVERSITY ORIENTED FLUORESCENCE LIBRARY APPROACH XU WANG (M Sc., National University of Singapore, Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2015     Blank Page   Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of Professor Young-Tae Chang (in the Chemical Bioimaging Lab, S9-03-03), Chemistry Department, National University of Singapore, between 08/01/2011 and 05/01/2015 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) Peng, J.†, Xu, W.†, Teoh, C L., Han, S., Kim, B., Samanta, A., Er, J C., Wang, L., Yuan, L., Liu, X., Chang, Y T.*, High-efficiency In Vitro and In Vivo Detection of Zn2+ by Dye-assembled Upconversion Nanoparticles, J Am Chem Soc., 2015, 137, 2336−2342 2) Xu, W.†, Ren, C.†, Teoh, C L.†, Peng, J., Gadre, S H., Rhee, H W., Lee, C L., Chang, Y T.*, An artificial tongue fluorescent sensor array for identification and quantitation of various heavy metal ions, Anal Chem., 2014, 86, 8763–8769 3) Xu, W., Bai, J., Peng, J., Samanta, A., Divyanshu, Chang, Y T.*, Milk quality control: instant and quantitative milk fat determination with a I   BODIP PY sensor-b based fluoreescence detector, Chem m Commuun 2014, 50 0, 10398-10410 H Zhai, D.,, Er, J C., Zhang, Z L., Kale, K A A.,, Agrawallaa, 4) Xu, W , Kim, T H., B K., Cho, Y K., Chang, Y Y T.*, Mak ke Caffeinee visible: a fluorescen nt caffeinee "traffic lig ght" detectoor, Sci Rep 2013, 3, 22 255   2015-5-4 Xu Wang Name Signatuure Daate II   Acknowledgement Not many Ph D candidates are privileged to have a supervisor parallel to Prof Young-Tae Chang, my Ph D mentor, who has pioneered this fluorescence field with his profound knowledge, enthusiastic and perpetual spirit of exploration and most importantly, his strict yet supportive help to the students I am deeply grateful to his support, both academically and personally, during my stay in the chemical bio-imaging laboratory throughout the past five years Prof Chang’s careful mentoring and patient work has grown me up from a freshman of science to a competent researcher, capable of solving complicated scientific problems In this regard, I am fortunate because I have a supervisor who is a good scientist, good teacher and a man of integrity and honor Hail to you, Prof Chang! I met a lot of good scientists and researchers during my PhD period and we’ve been working very well together Dr Animesh Samanta, the intelligent and meticulous No student of Chang lab; Dr Duanting Zhai, the beautiful Chinese lady who taught me how to “survive” here; Dr Krishna Kanta Ghosh, our funny senior with a whole abdomen of knowledge (fat); Dr Raj Kumar Das, the silent warrior with super synthesis skill; Dr Cheryl Kit Mun Leong, tiny little girl, but a huge ego! Dr Dongdong Su, my best drinking companion; Dr Chai Lean Teoh, super biologist who did all the biological experiments for me; Dr Changliang Ren, a great friend and a great mentor to me! Dr Lin Yuan, super chemist, I admire you! Mr Jun Cheng Er, talented young scientist who kept our bench so clean and ordered In fact he is like a laborious hamster who knows how to store instruments that could last years! Mdm Meiling III   Zhang, our secretary and babysitter! And finally, Dr Juanjuan Peng, whom I regard as one of my best friends here, is a very caring and beautiful lady always smiling at others She thinks that she is a little fat but in fact she is not We have worked in a lot of projects and I sincerely hope our friendship lasts the lapse of time I would not list all the other members because there are too many here But thank you all for the kind support and it is truly fun working with you guys! I have acknowledged my financial supporter several times in the publications and I would like to thank them again because they have all the way supported my research Hail to you, the Singapore Peking Oxford Research and Enterprise (SPORE)! You have truly helped the water ecoefficiency of Singapore and the world! Last but not least, I would love to express my deepest emotion to my parents, Dr Qingyu Xu and Mrs Su Li, and my wife, Dr Xian Qin I cannot be an integral person without you, my dearest family, let alone continuing my research and completing my thesis I love you and I would endeavor to become better for you IV   Table of Contents Summary IX List of Tables XI List of Figures XII List of Schemes XVIII List of Acronyms XIX Chapter 1: Introduction 1.1 Water contamination and its quality control 1.2 Fundamentals of fluorescence and fluorescent sensors 1.3 Diversity oriented fluorescence library approach 14 1.4 Scope and outline 21 References 23 Chapter 2: Make caffeine visible: Development of a fluorescent traffic light caffeine detector 28 2.1 Introduction to caffeine and available detection methods 29 2.2 Development of an unbiased high-throughput screening platform 33 2.3 Identification of potential fluorescence caffeine sensors and structure activity relationship studies 35 2.4 Photo-physical properties of the optimized caffeine sensor 40 2.5 Interaction mechanism studies 41 V   2.6 Selectivity and applicability studies 48 2.7 Development of a hand-held caffeine detection kit 52 2.8 Development of an automated caffeine detection kit 57 2.9 Summary 60 2.10 Experimental Details 61 References 65 Chapter 3: Instant and Quantitative Fat Amount Determination in Milk Quality Control Using a BODIPY Fluorescent Sensor-based Detector 70 3.1 Introduction to milk fat and milk quality control 71 3.2 Development of an image based hyper throughput screening platform 74 3.3 Identification of a fluorescent milk fat sensor and its photo-physical properties 78 3.4 Interaction mechanism studies 81 3.5 Selectivity and applicability studies 83 3.6 Development of a simplified milk fat detector 86 3.7 Summary 89 3.8 Experimental Details 90 References 104 VI   Cell Culture: The HeLa cell lines and OSCC cell lines were grown in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 U mL-1 antibiotic /100 mg mL-1 antimycotic) in a humidified atmosphere at 37oC with 5% (v/v) CO2 Cytotoxicity of 1-PAA-UCNPs: To study the cytotoxicity, we dispensed 100 L of cell suspension (~5000 cells/ well) in a 96-well plate The cells were pre-incubated for 24 hr in high glucose media (DMEM) with 10 % fetal bovine serum (FBS) and 1% Anti-Anti with in a humidified incubator (37 °C, %CO2) Next, different concentrations of UCNPs, 1-PAA-UCNPs (0, 100, 200, 300, 400, 500, and 600 g/mL, diluted in Roswell Park Memorial Institute (RPMI) 1640 medium) were then added to the wells The cells were subsequently incubated for 12 or 24 hr at 37 °C under 5% CO2 Then, MTT (20 L; mg/mL) was added to each well, and the plate was incubated for an additional hr at 37 °C under 5% CO2 After the addition of 100 L DMSO, the assay plate was allowed to stand at room temperature for hr The optical density OD570 value (Abs) of each well, with background subtraction at 690 nm, was measured by means of a fluorimeter and UV/Vis instrument, SpectraMax M2, Molecular Devices The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean Abs value of treatment group/mean Abs value of control) × 100% Cell Imaging: HeLa and OSCC cell lines were maintained at 37 oC in 5% CO2 in DMEM media respectively, both supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin The cells were plated at around 60-70% confluence 24 hr before imaging experiments in 35 206   mm culture dishes Prior to imaging experiments, the cancer cells were incubated with UCNPs (500 µg/mL), 1-PAA-UCNPs (500 µg/mL) for hr and the 1-PAA-UCNPs treated cells incubated with Zn2+ (0.25 mM) solution for 30 mins Both cell lines were washed for three times with cell culture media The cell lines were further washed using cell culture media and subsequently imaged at ambient temperature Biological sample preparation: 15-20 months-old triple transgenic mice (APPsw/P301L tau/PSENM146) were sacrificed for tissue harvesting The animals were either perfused with % (w/v) paraformaldehyde (PFA) or the brain tissue was rapidly frozen and stored at -80 °C immediately after extraction Free-floating sections were prepared from the PFA-perfused brain using vibratome (Leica) and 40 m slices were stored in anti-freeze solution at -20 °C until required Separately, 10 m sections of rapidly frozen brain were prepared using a cryostat and picked up on coated slides and stored in a -20 °C freezer Brain tissue UCL in vivo imaging: Brain tissue sections were treated with 500 g/mL 1-PAA-UCNPs for hr at room temperature, rinsed and mounted on slides Separately, sections were treated with 1-PAA-UCNPs for hr at room temperature, or pre-treated with mM EDTA or 100 M Zn2+ solution for hr before staining with 1-PAA-UCNPs Imaging was performed with an inverted fluorescence microscope Ti (Nikon) mounted with a 980 nm diode laser Tracing Distribution of Zn2+ in Zebrafish: Zebrafish were kept at 28 °C and maintained at optimal breeding conditions For mating, male and female zebrafish were maintained in one tank at 28°C on a 12 hr light/12 hr dark 207   cycle, and then the spawning of eggs was triggered by giving light stimulation Zebrafish were maintained in E3 embryo media Zebrafish larva at day were incubated with 500 g/mL 1-PAA-UCNPs for hr at 28°C Alternatively, day zebrafish were exposed to mM EDTA for hr at 28 °C to remove intact Zn2+ in zebrafish firstly, then zebrafish were further incubated with 1-PAAUCNPs for hr at 28 °C The nanoprobe treated zebrafish were imaged by an Olympus BX51 microscope with a xenon lamp adapted to a 980 nm diode laser References: Baldridge, A et al Inhibition of twisting of a green fluorescent protein-like chromophore by metal complexation Chem Comm 46, 5686-5688 (2010) Burdette, S C & Lippard, S J Meeting of the minds: Metalloneurochemistry P Natl Acad Sci USA 100, 3605-3610 (2003) Walkup, G K., Burdette, S C., Lippard, S J & Tsien, R Y A New Cell-Permeable Fluorescent Probe for Zn2+ J Am Chem Soc 122, 5644-5645 (2000) Kimura, E., Aoki, S., Kikuta, E & Koike, T A macrocyclic zinc(II) fluorophore as a detector of apoptosis P Natl Acad Sci USA 100, 3731-3736 (2003) Que, E L.; Domaille, D W.; Chang, C J "Metals in Neurobiology: Probing Their Chemistry and Biology with Molecular Imaging", Chem Rev 108, 1517-1549 (2008) Atwood, C S et al Dramatic Aggregation of Alzheimer Aβ by Cu(II) Is Induced by Conditions Representing Physiological Acidosis J Biol Chem 273, 12817-12826 (1998) Xu, Z et al Zn2+-Triggered Amide Tautomerization Produces a Highly Zn2+-Selective, Cell-Permeable, and Ratiometric Fluorescent Sensor J Am Chem Soc 132, 601-610 (2009) 208   Qian, F et al Visible Light Excitable Zn2+ Fluorescent Sensor Derived from an Intramolecular Charge Transfer Fluorophore and Its in Vitro and in Vivo Application J Am Chem Soc 131, 1460-1468 (2009) Datta, B K., Mukherjee, S., Kar, C., Ramesh, A & Das, G Zn2+ and Pyrophosphate Sensing: Selective Detection in Physiological Conditions and Application in DNA-Based Estimation of Bacterial Cell Numbers Anal Chem 85, 8369-8375 (2013) 10 Wang, H et al New molecular probe for the selective detection of zinc ion Analyst 138, 5576-5579 (2013) 11 Wong, B A., Friedle, S & Lippard, S J Solution and Fluorescence Properties of Symmetric Dipicolylamine-Containing Dichlorofluorescein-Based Zn2+ Sensors J Am Chem Soc 131, 71427152 (2009) 12 Xu, Z., Yoon, J & Spring, D R Fluorescent chemosensors for Zn2+ Chem Soc Rev 39, 1996-2006 (2010) 13 Burdette, S C., Walkup, G K., Spingler, B., Tsien, R Y & Lippard, S J Fluorescent Sensors for Zn2+ Based on a Fluorescein Platform:  Synthesis, Properties and Intracellular Distribution J Am Chem Soc 123, 7831-7841 (2001) 14 Guo, Z., Kim, G.-H., Shin, I & Yoon, J A cyanine-based fluorescent sensor for detecting endogenous zinc ions in live cells and organisms Biomaterials 33, 7818-7827 (2012) 15 Xu, Y et al A Highly Selective Fluorescence-Based Polymer Sensor Incorporating an (R,R)-Salen Moiety for Zn2+ Detection Chem Eur J 16, 12898-12903 (2010) 16 Meng, X., Wang, S., Li, Y., Zhu, M & Guo, Q 6-Substituted quinoline-based ratiometric two-photon fluorescent probes for biological Zn2+ detection Chem Comm 48, 4196-4198 (2012) 17 Satapathy, R., Wu, Y.-H & Lin, H.-C Novel Thieno-imidazole Based Probe for Colorimetric Detection of Hg2+ and Fluorescence Turn-on Response of Zn2+ Org Lett 14, 2564-2567 (2012) 18 Wang, F & Liu, X G Recent advances in the chemistry of lanthanidedoped upconversion nanocrystals Chem Soc Rev 38, 976-989 (2009) 19 Zhou, J., Liu, Z & Li, F Upconversion nanophosphors for small209   animal imaging Chem Soc Rev 41, 1323-1349 (2012) 20 Boyer, J C., Vetrone, F., Cuccia, L A & Capobianco, J A Synthesis of colloidal upconverting NaYF4 nanocrystals doped with Er3+, Yb3+ and Tm3+, Yb3+ via thermal decomposition of lanthanide trifluoroacetate precursors J Am Chem Soc 128, 7444-7445 (2006) 21 Zhang, F et al Rare-Earth Upconverting Nanobarcodes for Multiplexed Biological Detection Small 7, 1972-1976 (2011) 22 Feng, W., Sun, L D., Zhang, Y W & Yan, C H Synthesis and assembly of rare earth nanostructures directed by the principle of coordination chemistry in solution-based process Coord Chem Rev 254, 1038-1053 (2010) 23 Auzel, F Upconversion and anti-stokes processes with f and d ions in solids Chem Rev 104, 139-173 (2004) 24 Ju, Q et al Amine-Functionalized Lanthanide-Doped KGdF4 Nanocrystals as Potential Optical/Magnetic Multimodal Bioprobes J Am Chem Soc 134, 1323-1330 (2011) 25 Lehmann, O., Kompe, K & Haase, M Synthesis of Eu3+-doped core and core/shell nanoparticles and direct spectroscopic identification of dopant sites at the surface and in the interior of the particles J Am Chem Soc 126, 14935-14942 (2004) 26 Park, Y I et al Theranostic Probe Based on Lanthanide-Doped Nanoparticles for Simultaneous In Vivo Dual-Modal Imaging and Photodynamic Therapy Adv Mater 24, 5755-5761 (2012) 27 Zhang, F et al Fabrication of Ag@SiO2@Y2O3:Er Nanostructures for Bioimaging: Tuning of the Upconversion Fluorescence with Silver Nanoparticles J Am Chem Soc 132, 2850-2851 (2010) 28 Sivakumar, S., Boyer, J C., Bovero, E & van Veggel, F C J M Upconversion of 980 nm light into white light from sol-gel derived thin film made with new combinations of LaF3:Ln(3+) nanoparticles J Mater Chem 19, 2392-2399 (2009) 29 He, G S., Tan, L S., Zheng, Q & Prasad, P N Multiphoton absorbing materials: Molecular designs, characterizations, and applications Chem Rev 108, 1245-1330 (2008) 30 Cheng, L et al Facile Preparation of Multifunctional Upconversion 210   Nanoprobes for Multimodal Imaging and Dual-Targeted Photothermal Therapy Angew Chem., Int Ed 123, 7523-7528 (2011) 31 Wang, F & Liu, X Recent advances in the chemistry of lanthanidedoped upconversion nanocrystals Chem Soc Rev 38, 976-989 (2009) 32 Zhang, P., Rogelj, S., Nguyen, K & Wheeler, D Design of a highly sensitive and specific nucleotide sensor based on photon upconverting particles J Am Chem Soc 128, 12410-12411 (2006) 33 Kumar, M & Zhang, P Highly Sensitive and Selective Label-Free Optical Detection of DNA Hybridization Based on Photon Upconverting Nanoparticles Langmuir 25, 6024-6027 (2009) 34 Achatz, D E., Meier, R J., Fischer, L H & Wolfbeis, O S Luminescent Sensing of Oxygen Using a Quenchable Probe and Upconverting Nanoparticles Angew Chem Int Ed 50, 260-263, (2011) 35 Yao, L., Zhou, J., Liu, J., Feng, W & Li, F Iridium-Complex-Modified Upconversion Nanophosphors for Effective LRET Detection of Cyanide Anions in Pure Water Adv Funct Mater 22, 2667-2672 (2012) 36 Liu, J et al Iridium(III) Complex-Coated Nanosystem for Ratiometric Upconversion Luminescence Bioimaging of Cyanide Anions J Am Chem Soc 133, 15276-15279 (2011) 37 Mader, H S & Wolfbeis, O S Optical Ammonia Sensor Based on Upconverting Luminescent Nanoparticles Anal Chem 82, 5002-5004 (2010) 38 Liu, Q., Peng, J., Sun, L & Li, F High-Efficiency Upconversion Luminescent Sensing and Bioimaging of Hg(II) by Chromophoric Ruthenium Complex-Assembled Nanophosphors ACS Nano 5, 80408048 (2011) 39 Liu, Y et al A Cyanine-Modified Nanosystem for in Vivo Upconversion Luminescence Bioimaging of Methylmercury J Am Chem Soc 135, 9869-9876 (2013) 40 Li, Z., Lv, S., Wang, Y., Chen, S & Liu, Z Construction of LRETBased Nanoprobe Using Upconversion Nanoparticles with Confined Emitters and Bared Surface as Luminophore J Am Chem Soc 137, 211   3421-3427 (2015) 41 Cen, Y et al Phospholipid-modified upconversion nanoprobe for ratiometric fluorescence detection and imaging of phospholipase D in cell lysate and in living cells Anal Chem 86, 7119-7127 (2014) 42 Wang, F et al Tuning upconversion through energy migration in coreshell nanoparticles Nat Mater 10, 968-973 (2011) 43 Boyer, J C., Manseau, M P., Murray, J I & van Veggel, F C J M Surface Modification of Upconverting NaYF4 Nanoparticles with PEG-Phosphate Ligands for NIR (800 nm) Biolabeling within the Biological Window Langmuir 26, 1157-1164 (2010) 44 Zhao, L Z et al Near- Infrared Photoregulated Drug Release in Living Tumor Tissue via Yolk-Shell Upconversion Nanocages Adv Funct Mater 24, 363-371 (2014) 45 Bogdan, N., Vetrone, F., Ozin, G A & Capobianco, J A Synthesis of Ligand-Free Colloidally Stable Water Dispersible Brightly Luminescent Lanthanide-Doped Upconverting Nanoparticles Nano lett 11, 835-840 (2011) 46 Cheng, L., Yang, K., Chen, Q & Liu, Z Organic Stealth Nanoparticles for Highly Effective in Vivo Near-Infrared Photothermal Therapy of Cancer ACS Nano 6, 5605-5613 (2012) 47 Cirrito, J R Extracellular Amyloid-β Protein Dynamics in Alzheimer's Disease Microdialysis in Drug Development., Vol 163-178 (2012) 48 Adlard, P A & Bush, A I Metals and Alzheimer's disease J Alzheimers Dis 10, 145-163 (2006) 49 Crichton, R R., Dexter, D T & Ward, R J Metal based neurodegenerative diseases-From molecular mechanisms to therapeutic strategies Coordin Chem Rev 252, 1189-1199 (2008) 50 Barnham, K J & Bush, A I Metals in Alzheimer's and Parkinson's diseases Curr Opin Chem Biol 12, 222-228 (2008) 51 Bush, A I., Pettingell, W H., Deparadis, M., Tanzi, R E & Wasco, W The Amyloid Beta-Protein Precursor and Its Mammalian Homologs Evidence for a Zinc-Modulated Heparin-Binding Superfamily J Biol Chem 269, 26618-26621 (1994) 52 Hutchinson, R W et al Imaging and spatial distribution of β-amyloid 212   peptide and metal ions in Alzheimer’s plaques by laser ablation– inductively coupled plasma–mass spectrometry Anal Biochem 346, 225-233 (2005) 53 Brustein, E., Marandi, N., Kovalchuk, Y., Drapeau, P & Konnerth, A "In vivo" monitoring of neuronal network activity in zebrafish by twophoton Ca2+ imaging Pflug Arch Eur J Phy 446, 766-773 (2003) 54 Grant, K A., Raible, D W & Piotrowski, T Regulation of latent sensory hair cell precursors by glia in the zebrafish lateral line Neuron 45, 69-80 (2005) 55 Xu, W et al An Artificial Tongue Fluorescent Sensor Array for Identification and Quantitation of Various Heavy Metal Ions Anal Chem 86, 8763-8769 (2014) 56 Wang, F., Wang, J A & Liu, X G Direct Evidence of a Surface Quenching Effect on Size-Dependent Luminescence of Upconversion Nanoparticles Angew Chem Int Ed 49, 7456-7460 (2010)     213   Chapter 6: Conclusion and prospects 6.1 Conclusion: an optimization path of fluorescent sensor generation 214 6.2 Prospects 217 214   6.1 Conclusion: an optimization path of fluorescent sensor generation Ever since the earliest day man has walked the planet, our expedition to the heart of nature-to understand nature, to utilize nature and to live with nature in harmony-began Giants of the ancient would shout: “Give me a place to stand on, and I will move the earth!” Others, humble, obedient to the ferocious temper of nature Thousands of years have passed, what were once dreams have come true owing to the explosive development of science and technology For instance, when encountering something new, we once asked questions like “What is this stuff”, “What is it made of” and “Is it safe to eat or drink”, etc Nowadays, the level of inquiry has become more sophisticated and we now become curious about “What are the constituents?”, “How much of each component?”, and “Is there a disease vector present?” Such concerns have raised in both environmental and biomedical studies In most cases, we can give the answers now; however, state-of-the-art analytical instrumentation is required for such analysis Often the analyses involve considerable time with sample preparations that require a high level of expertise Nevertheless, we are still living in such a world that demand rapid or instant analysis when facing an increasingly more extreme cases Biomedically, the emergence of multiple infectious diseases such as H1N1, malaria or even Ebola has imposed severe pressure on our analytical system Other more humane situations, environmental pollution, food safety or even possible terrorist attack using chemical or biological weapons that harass the planet have also rung the alarm to current monitoring processes With such demand, we cannot afford days or even hours to wait for the testing results and allow the people exposed to the risks The modern situation has been linked to 215   an age-old dream that, being ablee to pick a sample of any a ilk, be iit biologicaal (bacteria, parasites, p etc.) or chem mical (heavy y metals, orrganic moleecules, etc.)), and know its composittion in real ttime, remains in the miind of chem mists Small molecule m flu uorescent seensors offer us a perfecct manner tto tackle thee complicatedd systems owing o to thheir non-inv vasive naturre, superiorr sensitivity y, fine spatiootemporal resolution r aand emission/target tu unability, eetc Simply y speaking, with w fine tun ning of senssor structurres and prop perties, we w will be ablee to selectiveely and senssitively obsserve any su ubstance Prof Youngg-Tae Chang g pioneered “diversity “ oriented o fluuorescence library approach (DOFFLA)” as a new paraddigm for sensor s devvelopment and demonstrated itts universaal applicationn to a broad d range of analytes, both biological and envvironmentaal (Figure 6.1.1) Figure 6.11.1 The op ptimization path of DOFLA D bassed fluoresscent sensor generation To rapiddly generatte fluoresceent sensors from DOFLA, our enndeavor hass undergone a series off evolution One of th he earliest struggles rrelies on an n 216   unbiased screening process Generally speaking, we split each fluorescent dye as a single unit corresponding to a variety of biologically important species Thus by measuring the fluorescence spectra of each fluorescent dye against the biological species and comparing their photo-physical properties to that of the control sample, we are able to understand clearly that which fluorescent dye can be lighted on or quenched by which analyte This method offers a stable and integrated screening format to conduct research on all of our thousands of fluorescent dyes towards a huge number of analytes From this approach, the first fluorescent “turn on” sensor for caffeine has been developed Though highly applicable as it is, the unbiased screening platform requires large level of analyte preparation and the spectrometer measurement is relatively time consuming to cover thousands of fluorescent dyes Therefore, in order to promote the fluorescent sensor generation, we developed a new platform-the image based hyper throughput screening platform We rationalize and simplify the fluorescence procedure as follows: to trigger fluorescence, we need an excitation lamp that delivers energy to the fluorophore and as a result we will observe emission light at a longer wavelength This excitationrelaxation-emission process has inspired us to evolve and optimize the DOFLA application: instead of embedding the analytes into each plate and adding individual fluorescent dye, the sensors have already been embedded into the 96-well plates with regards to their structures and properties By irradiating the sensor plate with an excitation lamp we can readily obtain the background image of the library using an off-the-shelf camera The excitation lamp is an ultraviolet (UV) lamp because UV is able to trigger fluorescence 217   signals from majority of the fluorophores After addition of any analyte-be it environmental or biological-to the sensor plate, we can shoot one more image using the same setup Both images are lined up and compared side by side to reveal any tiny change of intensity or emission color from the fluorescent dye Any dye bearing fluorescence change will be picked out as a hit and subjected to further analysis This platform has greatly accelerated our fluorescent sensor discovery process, and a series of sensor have been developed therein: food safety-bisphenol A and milk fat; social security-date rage drugs such as GHB and GBL The combination of image based hyper throughput screening platform with the spectrometer analysis has inspired us to tackle even more difficult species With combinatorial dye designing involved, we have built a fluorescent sensor array that differentiates and semi-quantitatively designates seven heavy metal ions at the same time Furthermore, by incorporating a variety of drinkable water into the test, we could establish a safezone test chart while any species lying inside the safezone is deemed as safe to drink, any species outside of the safezone is deemed potentially hazardous and requires special attention This safezone model is accumulative because the more samples we test, the more precise the zone can be This model can be employed to any water quality control process and could save the cost of expensive machines in resource limited regions This concludes our efforts to optimize the fluorescent sensor development up to now 6.2 Prospects 218   It should be noted that although a series of environmental and biomedical fluorescent sensors have been discovered, in view of science, a more important task should be to summarize them and then raise the common feature that causes these fluorescence changes Thus we would be able to replicate the successful results, improve it and even apply it to other similar studies The progress of both DOFLA and fluorescence technique should lead us further in the field of science and hopefully one day we would be able to claim: “Give me a fluorescent sensor, and I will illuminate the world” 219   220   [...]... assembled UCNP detection 185 systems XI   List of Figures Figure 1.2.1 The Jablonski diagram of fluorescence 8 The location of Gemini 4 spacecraft using 9 phenomenon Figure 1.2.2 fluorescent dye fluorescein Figure 1.3.1 Illustration of target oriented approach and 16 diversity oriented fluorescence library approach and their comparison Figure 1.3.2 Illustration of DOFLA derived fluorescent 18 sensors... Figure 4.8.1 Fluorescence response of SGT1 to Cr3+ 151 Figure 4.8.2 Fluorescence response of SGT1 to Cu2+ 152 Figure 4.8.3 Fluorescence response of SGT1 to Hg2+ 153 Figure 4.8.4 Fluorescence response of SGT2 to Cd2+ 154 Figure 4.8.5 Fluorescence response of SGT2 to Cr3+ 155 Figure 4.8.6 Fluorescence response of SGT2 to Cu2+ 156 Figure 4.8.7 Fluorescence response of SGT2 to Hg2+ 157 Figure 4.8.8 Fluorescence. .. 4.8.9 Fluorescence response of SGT2 to Zn2+ 159 Figure 4.8.10 Fluorescence response of SGT3 to Cd2+ 160 Figure 4.8.11 Fluorescence response of SGT3 to Hg2+ 161 Figure 4.8.12 Fluorescence response of SGT3 to Pb2+ 162 Figure 4.8.13 Fluorescence response of SGT3 to Zn2+ 163 Figure 4.8.14 Fluorescence response of SGT4 to Cd2+ 164 Figure 4.8.15 Fluorescence response of SGT4 to Fe3+ 165 Figure 4.8.16 Fluorescence. .. contamination and its quality control    2  1.2 Fundamentals of fluorescence and fluorescent sensors   7  1.3 Diversity oriented fluorescence library approach  14  1.4 Scope and outline    21  References    23  1   1.1 Water contamination and its quality control Rapid development throughout the world has seen the thriving economies of domestic society However, alongside... have conducted comprehensive research on the water quality control process to ensure safe drinking water Typical water pollutants published by major entities are summarized and listed in Table 1 shown below.3-5 Table 1.1.1 Summarization of common water waste species and their allowed concentrations in drinking water Data is summarized from the drinking water standards published by World Health Organization... eccentric properties Diversity oriented fluorescence library approach (DOFLA) has come to tackle this issue from a different angle and this thesis summarizes the evolution of our in vitro screening approaches First of all, we designed an unbiased high-throughput screening method to conduct integrated screening of thousands of fluorescent dyes towards more than fifty biological analytes This approach has allowed... 4.8.16 Fluorescence response of SGT4 to Hg2+ 166 Figure 4.8.17 Fluorescence response of SGT4 to Pb2+ 167 Figure 4.8.18 Fluorescence response of SGT4 to Zn2+ 168 Figure 4.8.19 Fluorescence response of SGT5 to Cd2+ 169 XVI   Figure 4.8.20 Fluorescence response of SGT5 to Hg2+ 170 Figure 4.8.21 Fluorescence response of SGT5 to Pb2+ 171 Figure 4.8.22 Fluorescence response of SGT5 to Zn2+ 172 Figure 5.1.1 A generalized... chromatography HPGC Surface enhanced Raman spectroscopy SERS Severe acute respiratory syndrome SARS Ultraviolet UV Photo-induced electron/energy transfer PET Intramolecular charge transfer ICT Diversity Oriented Fluorescence Library Approach DOFLA -hydroxybutyric acid GHB -Butyrolactone GBL Structure activity relationship SAR Caffeine orange CO Nuclear magnetic resonance NMR Transmission electron microscopy TEM... Methanol MeOH Fluorescence/ Förster resonance energy transfer FRET Upconversion luminescence UCL Polyacrylic acid PAA Oleic acid OA X-ray diffraction XRD Cysteine Cys Homocysteine Hcy Methyl thiazolyl tetrazolium MTT Oral squamous cell carcinoma OSCC Anterior lateral-line ALL XXIV   Chapter 1: Introduction   1.1 Water contamination and its quality control    2  1.2 Fundamentals of fluorescence and... hyper-throughput 78 screening approach Figure 3.3.1 Milk auto -fluorescence background 79 Figure 3.3.2 MO identification and photo-physical properties 80 Figure 3.4.1 19F NMR of MO with various concentrations of 82 extracted triglyceride Figure 3.4.2 Fluorescence light-on induced by MO 83 Selectivity and versatility test of MO towards 84 disaggregation from self-aggregates in water Figure 3.5.1 milk from ... fluorescent sensors, other tricks should come into play 1.3 Diversity oriented fluorescence library approach Diversity Oriented Fluorescence Library Approach (DOFLA) was first proposed and reported by... Introduction 1.1 Water contamination and its quality control 1.2 Fundamentals of fluorescence and fluorescent sensors 1.3 Diversity oriented fluorescence library approach 14 1.4... Introduction   1.1 Water contamination and its quality control    2  1.2 Fundamentals of fluorescence and fluorescent sensors   7  1.3 Diversity oriented fluorescence library approach  14