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 FLUORESCE

WATER QUALITY CONTROL USING DIVERSITY

ORIENTED FLUORESCENCE LIBRARY

APPROACH

XU WANG

NATIONAL UNIVERSITY OF SINGAPORE

2015

WATER QUALITY CONTROL USING DIVERSITY

ORIENTED FLUORESCENCE LIBRARY

2015

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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

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

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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 1 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

IV

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 eco-efficiency 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

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

1.1 Water contamination and its quality control 2

1.2 Fundamentals of fluorescence and fluorescent sensors 7

1.3 Diversity oriented fluorescence library approach 14

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

VI

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

Chapter 4: Construction of a fluorescent “tongue” for rapid heavy metal ion detection and its application in biological

systems 107

4.1 Introduction to heavy metal ion pollution 108

4.2 Rational design of a styryl-based fluorescent sensor array for heavy metal ions 111

4.3 Photo-physical properties measurements of sensors and their responses to heavy metal ions 114

4.4 Principle component analysis to afford quantitative data measurement 119

4.5 Establishing a “safe zone” prototype 126

4.6 Application of fluorescent metal sensor in biological systems 130

4.7 Summary 133

4.8 Experimental Details 134

References 173

Chapter 5: High efficiency multiplexed detection of Zn2+ by dye assembled upconversion nanoparticles 182

5.1 Brief introduction to biological zinc and upconversion nanoparticles 183

5.2 Rational design of a dye assembled upconversion nanoparticle system 188

5.3 Photo-physical properties measurements of the zinc detection system and zinc responses 192

VIII

5.4 Biological applications 194

5.5 Summary 199

5.6 Experimental Details 200

References 206

Chapter 6: Conclusion and prospects 212

6.1 Conclusion: an optimization path of fluorescent sensor generation 213

6.2 Prospects 216

Environment and nature are always amongst the top list of people’s concerns whereas environmental pollutants are more and more attracting intensive research throughout the world Small molecule fluorescent sensors have stood out due to their high sensitivity and selectivity, cheap and easy measurement instruments, and facilely tunable emission range and structures However, a large amount of both environmental and biological species lack their specific sensors and the designing is hampered by their 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 us a comprehensive understanding of dye properties and dye responses to the analytes Caffeine orange (CO) was selected as a representative sensor developed through this method After then, to quickly adapt the loads of fluorescent dyes to other species without the need of conducting the whole unbiased screening process again, we developed the screening format to an image-based hyper throughput screening approach The setup of the whole screening system is simple and adaptable to any other dye and through this system, we have developed sensors for a variety of species, including food safety and social security species One of the most outstanding sensor is for milk fat, which is no doubt its first fluorescent sensor

X

The success of these stories has inspired to incorporate the image-based system with spectrometer system, and through designing of molecules, we have constructed a Singapore Tongue (SGT) sensor array targeting heavy metal ions in water systems Not only are the SGT able to visualize and qualify multiple heavy metal ions, it is also capable of semi-quantifying these species Most importantly, we have come up with a “safe zone” prototype that

is able to exclusively and comprehensively detect any harmful heavy metal ion species from the drinking water samples This could serve as a tool to replace the current complicated instrumental analysis As a conclusion, the progress of fluorescent sensor development from DOFLA could trigger the enhancement

of environmental monitoring

List of Tables

Table 1.1.1 Summarization of waste species and their

concentrations in drinking water

2

Table 1.2.1 Summary of fluorescent sensors targeting

various heavy metal ions

Table 3.5.1 Comparison of fat concentrations acquired

from fluorescence measurement and ingredient tables listed on milk package

86

Table 4.5.2 Estimated Sample Details based on PCA Plots 129

Table 5.1.1 Summary of dye assembled UCNP detection

systems

185

Figure 1.2.2 The location of Gemini 4 spacecraft using

fluorescent dye fluorescein

9

Figure 1.3.1 Illustration of target oriented approach and

diversity oriented fluorescence library approach and their comparison

Figure 2.1.1 Health effects of caffeine on human beings 31

Figure 2.2.1 Format of the unbiased high-throughput

screening platform

34

Figure 2.2.2 A representative color bar graph of one single

fluorescent sensor to all the analytes tested

35

Figure 2.3.1 The color bar graph of one single fluorescent

sensor (BD-185) to all the analytes tested

36

Figure 2.3.2 The structures of BD-185 and its analogs existing

in DOFLA library used in caffeine test

37

Figure 2.3.3 Screening of indole containing BODIPY dyes

from DOFLA fluorescent dye sets towards six concentrations of caffeine

38

Figure 2.3.4 The structures of potent BD-185 derivatives used

in caffeine test

39

Figure 2.3.5 Screening of the potent BD-185 derivatives

towards seven concentrations of caffeine

40

Figure 2.4.1 Fluorescence responses of CO against caffeine 41

Figure 2.5.1 Mass spectrometry tests of CO and caffeine

interactions

42

Figure 2.5.2 FT-IR spectra of CO-caffeine interactions 43

Figure 2.5.3 Proton NMR titration experiments of CO against

Figure 2.5.6 TEM measurements of CO-caffeine interactions 47

Figure 2.6.1 In-house screening heatmaps of CO towards

bio-related molecules

51

Figure 2.6.2 Selectivity tests of CO against caffeine analogs 52

Figure 2.7.1 Constitution of reverse phase syringe used for

caffeine extraction and detection

53

Figure 2.7.2 Applicability tests of caffeine detection kit 54

Figure 2.8.1 The design and constitution of microfluidics

device for caffeine extraction and measurement

58

Figure 2.8.2 Test of microfluidics caffeine extraction 59

Figure 3.1.1 The chemical structures of (A) triglyceride and 72

XIV

(B) lactose

Figure 3.2.1 Diagram of each components in the imaging

black box

76

Figure 3.2.2 The image of the whole high-throughput

screening and confirmation set

77

Figure 3.2.3 Scheme of the image based hyper-throughput

screening approach

78

Figure 3.3.2 MO identification and photo-physical properties 80

Figure 3.4.1 19F NMR of MO with various concentrations of

extracted triglyceride

82

disaggregation from self-aggregates in water

83

Figure 3.5.1 Selectivity and versatility test of MO towards

milk from various origins

84

Figure 3.5.2 Image of MO towards various brands of milk

under irradiation of UV lamp

85

Figure 3.6.1 The schematic diagram of the rapid quantitative

milk fat detector

87

Figure 3.6.2 The fluorescence response of milk fat detector

against milk fat

88

Figure 3.8.2 19F NMR of MO in D2O (1% DMSO-d6) with

no fat

94

Figure 3.8.3 19F NMR of MO in D2O (1% DMSO-d6) with

Figure 4.3.1 Integrated fluorescence responses of SGT

towards heavy metal ions

116

Figure 4.3.2 Absorbance spectra of sensors to heavy metal

ions

118

Figure 4.4.1 PCA processing of heavy metal ions responses

from all five sensors

120

Figure 4.4.2 PCA processing of heavy metal ions responses

from SGT1-SGT3 sensors

122

Figure 4.4.3 PCA processing of heavy metal ions responses

from SGT1, SGT2 and SGT3 respectively

124

Figure 4.5.1 Dose-dependence PCA graph of seven selected 127

XVI

heavy metal ions

Figure 4.5.2 Individual PCA plots that describe the responses

of the SGT sensor array towards seven different heavy metal ions

128

Figure 4.6.1 Metal detection in Alzheimer’s disease brain with

SGT-3

132

Figure 4.8.20 Fluorescence response of SGT5 to Hg2+ 170

Figure 5.1.1 A generalized scheme of neuronal zinc functions 183

Figure 5.1.2 A list of various morphologies and emission

colors UCNP can achieve

184

Figure 5.2.3 The design principle and performance of dye

assembled UCNP detection system

190

Figure 5.2.5 UCL signals of UCNPs in the presence of SGT3 192

Figure 5.3.1 Zn2+ responses of the nanoprobe and its

Figure 5.4.1 Cell cytotoxicity assay of the nanoprobe 195

Figure 5.4.2 Cellular Zn2+ imaging of the nanoprobe 196

Figure 5.4.3 Amyloid Zn2+ imaging using the nanoprobe 197

Figure 5.4.4 Imaging Zn2+ distribution in zebrafish using

UCNP

199

fluorescent sensor generation

214

XVIII

XX

List of Acronyms

Ultraviolet UV

Dynamic light scattering DLS

2-dimensional 2D 3-dimensional 3D

XXII

Dopamine- and cAMP-regulated neuronal

Boron-dipyrromethene BODIPY

Guanosine-5'-triphosphate GTP

Guanosine-5'-monophosphate GMP

Glutathione GSH

Nicotinamide adenine dinucleotide 2'-phosphate,

reduced

NADPH

Monosodium glutamate MSG

Ethanol EtOH

Hertz Hz Dichloromethane DCM

N, N, N’, N’-tetrakis(2-pyridylmethyl)ethylenediamine TPEN

6-Methoxy-(8-p-toluenesulfonamido)quinoline TSQ

Paraformaldehyde PFA

XXIV

Methanol MeOH

Cysteine Cys Homocysteine Hcy

2

1.1 Water contamination and its quality control

Rapid development throughout the world has seen the thriving economies

of domestic society However, alongside the boosting social modernization

level and industrial advancement, their adverse impacts over natural

environment, especially water resources have to be seriously coped with

Thousands of tons of waste water are poured into natural water systems every

day, which contain various pollutants, including heavy metal ions, organic

accumulation of these species in the natural resources and ultimately, in our

bodies, has led to numerous health hazards and serious attention throughout

the world Developed countries such as the United States and Singapore have

conducted comprehensive research on the water quality control process to

ensure safe drinking water Typical water pollutants published by major

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 (WHO), US Environmental Protection Agency (EPA) and Singapore Public Utility Board

(PUB) The table is adapted and summarized from references 3-5

0.5

dosed at 20 mg/L

in a month

<1

than 5% of samples positive

in a month, MCLG 2 =

stands for maximum contaminant level goal

The listed major environmental pollutants, if inhaled, would cause a series

of diseases or body disorders For example, heavy metal ions are raised from both anthropological and natural sources while after absorption, they mostly accumulate in developing brains and function to disrupt the protein/peptide

secondary structures, thus causing the formation of diseased conformations,

Exposure to BPA contaminated waste water leads to cardiac disorder, cancer,

such as cryptosporidium oocysts have haunted the United States of America and United Kingdom decades ago and their traces can still be found in the

categories of contaminants-Inorganic wastes, organic wastes and microbiological pathogens-has attracted intensive research interests

Currently, instrumental analysis and biotechnology are the most prevalent techniques applied to monitor water contaminants To determine inorganic contaminants such as heavy metal ions, researchers have developed various approaches For instance, spectroscopic detection methods such as atomic absorption spectrophotometry utilizes the characteristic absorption bands of

Luminescent recombinant bacteria sensors combine the sensitive luminescence measurement with bacteria-based metal chelators to achieve

techniques, such as absorptive stripping voltammetry measurements and DNA-based biosensors, have all added up to the choices of inorganic

chromatography (HPLC/GC) is one of the most widely used monitoring

6

sample injection and facile data output and analysis have rendered HPLC/GC widespread usage Especially in professional utilities and governmental agencies, HPLC/GC is regarded as routine monitoring instruments for majority of the contaminants In terms of microbiological pathogens, traditional detection methods require a tedious period of sample preparation that costs days to weeks The researchers need to collect contaminated water samples from the field, purify them and then culture them in the prepared media or agar plates After several days of incubation, they then can determine the bacteria identity based on the morphologies or by certain staining methods,

generations of instruments such as surface enhanced Raman spectroscopy (SERS) has been developed, which utilizes the characteristic Raman spectra of certain bacteria species to achieve qualification.22

Although to some extent, all of these water contaminants have been addressed using various instruments or techniques; we should admit that there

is still a long path to pave Intensive and careful sample preparation processes, complicated handling expertise of instruments and the cost required to purchase such instruments have significantly hampered their application, especially in resource limited regions We, normal human beings, are currently dwelling in such a world where rapid or even instant analysis of any substance

is required when facing more and more extreme cases For instance, the outburst of several notorious infectious diseases, including severe acute respiratory syndrome (SARS), malaria, influenza A virus subtype H1N1 or even Ebola has clearly shown the lagging response of our current environmental monitoring system In other situations, severe environmental

pollution such as oil leakage or even underlying terrorist assaults that deal with chemical or biological weaponries could severely harass our mother planet The lack of efficient monitoring processes and techniques have rung the alarm to human beings that we simply cannot afford days or even hours using current methods and just wait for the testing results when our people are exposed to the risks A more robust environmental detection method is at utmost demand and we are turning our attention to other techniques, such as fluorescent sensors

1.2 Fundamentals of fluorescence and fluorescent sensors

The phenomenon of fluorescence was discovered almost five hundred years

the famous experiment of light refrangibility (wavelength change) conducted

by Professor George G Stokes, he observed bathochromic shift of ultraviolet (UV) light created by uranium glass to the visible blue light after passing through fluorspar This phenomenon, of which an invisible light changed to visible light, was named fluorescence by Professor Stokes Nowadays researchers have understood the underlying mechanism of this phenomenon (Figure 1.2.1) Among each fluorophore, multiple electronic states exist and accompanying each state are a series of vibrational sub-states derived from vibration of chemical bonds Not surprisingly, when excitation energy is given

to the fluorophore, ground state electrons tend to migrate to the higher state when provided with sufficient energy Very rapidly, the excited state fluorophore tend to release energy through the constant vibration and rotation

of its own chemical bonds, thus relaxing to the lowest excited state While

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Figure 1.2.2 The location of Gemini 4 spacecraft using fluorescent dye

fluorescein This green dye is highly fluorescent under sunshine and clearly visible from the air Figure adapted from reference 27

The superior characteristics of fluorescence technique have triggered extensive research on its applications Among the various usage approaches, small molecule fluorescence sensors show unique potential for high sensitivity and selectivity, cheap and easy measurement instruments, and facilely tunable emission range and structures Versatile fluorescent sensors have been

have also evolved from early-period spectroscopy based sensing to stage direct real time naked-eye observation, which significantly facilitates the monitoring process of any target

current-There are two factors to be considered regarding the design and construction of small molecule fluorescent sensors: the receptor and the reporter (fluorophores in this case) The receptor is the key to sensor selectivity while the reporter is responsible for signal output (emission wavelength, signal range, etc.) This point can be illustrated more clearly with one series of fluorescent sensors: heavy metal ion sensors The majority of heavy metal ion sensors follow closely to the rule: a receptor linking to a reporter Designed chemical recognition of heavy-metal ions serves as the basis for a proper sensing event The specific interactions between heavy-metal ions and the selected receptors usually involves non-covalent binding forces, such as metal coordination, hydrogen bonding, hydrophobic forces, π-π

The reporters, on the other hand, are usually fluorophores with substantial

quantum yields Through careful design of their structures, these fluorophores can be quenched by introducing photo-induced electron/energy transfer (PET) moieties or intramolecular charge transfer (ICT) moieties These moieties, upon binding with heavy metal ions, will release the strain of the fluorophores and thus their emission will be turned on or their emission maximum will shift towards bathochromic or hypochromic directions Through such design, numerous small molecule fluorescent heavy metal ion sensors have been

Table 1.2.1 Summary of fluorescent sensors targeting various heavy metal

ions The sensors are categorized according to their targets The colorful moieties indicate the reporters of these sensors and the black moieties indicate their receptors Figure adapted and summarized from reference 33 under copyright permission

Metal

Zn 2+

Cu +

12