Time-resolved quantitative assays and imaging of enzymes and enzyme substrates using a new europium fluorescent probe for hydrogen peroxide [Elektronische Ressource] / vorgelegt von Meng Wu
133 Pages
English

Time-resolved quantitative assays and imaging of enzymes and enzyme substrates using a new europium fluorescent probe for hydrogen peroxide [Elektronische Ressource] / vorgelegt von Meng Wu

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Time-resolved Quantitative Assays and Imaging of Enzymes and Enzyme Substrates Using a New Europium Fluorescent Probe for Hydrogen Peroxide Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (doktorum rerum naturalis, Dr. rer. nat.) der Fakultät Chemie und Pharmazie, der Universität Regensburg Bundesrepublik Deutschland vorgelegt von Meng WU aus Wuhan, China im December 2003 Time-resolved Quantitative Assays and Imaging of Enzymes and Enzyme Substrates Using a New Europium Fluorescent Probe for Hydrogen Peroxide By Meng WU A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy (Dr. rer. nat) Faculty of Chemistry and Pharmacy in University of Regensburg Federal Republic of Germany ©Copyright by Meng WU 2003 This study was performed at the Institute of Analytical Chemistry, Chemo- and Biosensors of the University of Regensburg between May 2001 and December 2003 under the supervision of Prof. Otto S. Wolfbeis. Date of defense: 18. 12. 2003 Committee of defense (Prüfungsausschuß): Chairperson (Vorsitzender) Prof. Dr. Manfred Liefländer First expert (Erstgutachter) Prof. Dr. Otto Wolfbeis Second expert (Zweitgutachter) Prof. Dr. Bernhard Dick Third expert (Drittprüfer) Prof. Dr.

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Published 01 January 2005
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Time-resolved Quantitative Assays and Imaging of
Enzymes and Enzyme Substrates Using a New
Europium Fluorescent Probe for Hydrogen Peroxide


Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften
(doktorum rerum naturalis, Dr. rer. nat.)

der Fakultät Chemie und Pharmazie,
der Universität Regensburg
Bundesrepublik Deutschland






vorgelegt von

Meng WU

aus Wuhan, China
im December 2003
Time-resolved Quantitative Assays and Imaging of
Enzymes and Enzyme Substrates Using a New
Europium Fluorescent Probe for Hydrogen Peroxide





By

Meng WU





A thesis submitted in conformity with the requirements
for the Degree of Doctor of Philosophy (Dr. rer. nat)


Faculty of Chemistry and Pharmacy
in University of Regensburg
Federal Republic of Germany


©Copyright by Meng WU 2003

This study was performed at the Institute of Analytical Chemistry, Chemo- and
Biosensors of the University of Regensburg between May 2001 and December 2003
under the supervision of Prof. Otto S. Wolfbeis.


















Date of defense: 18. 12. 2003

Committee of defense (Prüfungsausschuß):

Chairperson (Vorsitzender) Prof. Dr. Manfred Liefländer

First expert (Erstgutachter) Prof. Dr. Otto Wolfbeis

Second expert (Zweitgutachter) Prof. Dr. Bernhard Dick

Third expert (Drittprüfer) Prof. Dr. Claudia Steinem











子曰:「學而時習之,不亦悅乎?」
論語, 學而第一

Confucius said: "Isn't it a pleasure to study and practice what you have learned?"

Konfucius sagte: “Zu lernen und das Erlernte immer wieder auszuüben - ist das nicht eine Freude?”

From THE ANALECTS (Sayings)
Table of Contents I
Table of Contents
Graphical Abstract 1

Chapter 1. Introduction 5
1.1. Importance of Hydrogen Peroxide 5
1.2. Overview of Hydrogen Peroxide Based Enzymatic Assays 6
1.3. Overview of Methods for Determination of Hydrogen Peroxide 9
1.3.1. Analytical Methods Based on Physical Properties 10
1.3.2. Optical Analytical Methods Based on Chemical Reactions of H O 10 2 2
1.3.2.1. Determination based on simple oxidations 11
1.3.2.2. Determination based on enzyme coupling with peroxidases 11
1.3.2.3. Determination based on metal H O complexes 12 2 2
1.4. Time-resolved Fluorescentce Assays and Imaging 15
1.5. Aim of the Research 16
1.6. Reference 18

Chapter 2. Characterization of the Probe and Time-resolved Assay of H O 24 2 2
2.1. Introduction 24
2.2. Results and Discussion 25
2.2.1. Characterization of the Fluorescent Europium Probe 25
2.2.1.1. Absorbance, circular dichroism and fluorescence spectra 25
2.2.1.2. Lifetime characterization of EuTc-HP 27
2.2.1.3. pH, buffer, temperature and stability 29
2.2.1.4. Quenchers and interferents 32
2.2.2. Time-resolved Fluorescent Determination of Hydrogen Peroxide 33
2.2.2.1. Time-correlated single photon counting (TCSPC) method 33
2.2.2.2. Rapid lifetime determination (RLD) method 34
2.3. Experimental 38
2.3.1. Time-correlated Single Photon Counting Lifetime Determination 38
2.3.2. Rapid Lifetime Determination Assay of H O on Microplates 38 2 2
2.3.3. Time-resolved (gated) and Steady-state Fluorescence Assays 38
2.4. References 39
Table of Contents II
Chapter 3. Direct and Time-Resolved Enzymatic Detection of Glucose 41
3.1. Introduction 41
3.2. Results and Discussion 43
3.2.1. Assay Principle 43
3.2.2. Fluorescence Intensity-based Assays 45
3.2.3. Time-resolved (gated) Fluorescence Assay 48
3.2.4. Comparison 50
3.2.5. Analysis of Other Substrates of Oxidases 53
3.3. Experimental 54
Glucose Assay Protocol 54
3.4. References 55

Chapter 4. Fluorescence Imaging of the Activity of Glucose Oxidase 57
4.1. Introduction 57
4.2. Results and Discussion 58
4.2.1. Principle and Characterization of the Detection System 58
4.2.2. Imaging Setup and Analytical Schemes 59
4.2.3. Quantitative Aspects of GOx Imaging 63
4.2.4. Time-resolved Determination of GOx Using a Microplate Reader 64
4.2.5. Comparison 64
4.3. Experimental 66
4.4. References 68

Chapter 5. Determination of the Activity of Catalase 70
5.1. Introduction 70
5.2. Results and Discussion 72
5.2.1. Characterization and Optimization of the Assay 72
5.2.2. Inhibition and Denaturation of Catalase 76
5.2.3. Interferents 77
5.2.4. Discussion 78
5.3. Experimental 83
Recommended CAT Assay Protocol 83
5.4. References 84
Table of Contents III
Chapter 6. Further Applications and Structure of EuTc-HP 86
6.1. Application of GOx-based ELISA 86
6.2. The Catalase/Glucose Oxidase System 90
6.2.1. The Catalase/Glucose Oxidase System as a Platform for Screening 90
6.2.2. Detection of Catalase Independent of H O 93 2 2
6.3. Construction of Microplate Arrays and Sensors 94
6.4. Composition of the Fluorescent Probes 96
6.4.1. Stoichiometry and Structure 97
6.4.2. Combinatorial Approach for Discovery of New Lanthanide Probes 102
6.5. Experimental 105
6.5.1. Protocol of GOx-labeled Immunoassay 105
6.5.2. Coupled GOx-Catalase Enzymatic System 106
6.5.3. Construction of Microplate Sensors and Arrays 106
6.6. References 107

7. Materials and Instruments 109
7.1. Materials and Reagents 109
7.2. Instruments 110

8. Summary 112

9. Curriculum Vitae 114

10. Patent and List of Recent Publications 115

11. Appendix 117
11.1. Abbreviations and Symbols 117
11.2. Programs 118
11.3. Chinese Summary 125

12. Acknowledgements 127
Graphical Abstract
Graphical Abstract
Sensitive to H O at neutral pH2 2
A. Probe Study

OOxxiidasdaseses

+H O2 2
EuTc EuTcHP
-H O2 2

llooww fflluuoorrescenccee hhiigghh fflluuoorrescenescenccee
Catalase
1. The research is based on the finding of the reversible transformation of the weakly
fluorescent europium-tetracycline complex (EuTc) to highly fluorescent europium-
tetracycline-hydrogen peroxide complex(EuTc-HP) in neutral pH(see figure above).
3,01,0
EuTc-HP 2,5
0,8
2,0 EuTc
0,6
1,5
0,4 1,0

0,2 0,5

0,00,0
400 500 600 700 Wavelength (nm)

2. EuTc and EuTc-HP have shown not only merits that have made the lanthanide labeling so
versatile in bioanalysis, such as large Stokes shift, line-like emission (spectra, above), µs
range lifetime, but also compatibility with blue diode laser, as a H O fluorescent probe. 2 2
3. EuTc and EuTc-HP have been characterized through absorbance, circular dichroism,
fluorescence, pH response, temperature response, stability, and etc.
4. A combinatorial approach for discovery of new lanthanide probes has been tested.
1
Corrected Absorbance
Fluorescence (a.u.)Graphical Abstract
B. Assay Study
1. Substrate determinations
The substrates detected in this study are hydrogen peroxide, glucose as an example
of the substrates of oxidases (see figure below, a time trace of GOx + glucose with EuTc).

G30

25 F
20 E

15 D

C10
B

5 A
0 200 400 600 800 1000 1200 1400
T i m e ( s )

2. Enzyme analysis
Glucose oxidase (GOx) is studied in this thesis as a model enzyme of H O 2 2
producing oxidases, and catalase (CAT) as a model enzyme of H O consuming enzymes. 2 2
3. Different schemes of detections
Steady-state intensity-based detections, time-resolved “gated” detections (see figure
below), and lifetime-based detections of rapid lifetime determination method and time-
correlated single photon counting method have been studied in both microplates and cuvettes.
50000

40000
EuTc-HP
30000

2020000000


10000
EuTc

0 0 20 40 60 80 100 120 140 160 180 200
Integration µLag time Time ( s)
time
2
F l u o r e s c e n c e ( a. u.)Graphical Abstract
C. Imaging Study
“Seeing is believing”. Visualization through imaging is a better way for bioanalysis
(the image below showing “Glu” as glucose). The µs range lifetime of the fluorescent probes
has greatly facilitated the imaging here.







1. Four schemes of imaging (see figure below), that are the conventional fluorescence
intensity imaging (FII), the time-resolved ("gated") imaging (TRI), the phase delay ratioing
imaging (PDI), and the rapid lifetime determination imaging (RLI), have been used for
quantitative analysis.
A B50000 50000Fluorescence Intensity Imaging (FII) Time-resolved Gated Imaging (TRI)
40000 40000
EuTc-HP EuTc-HP
30000 30000
20000 20000W1
EuTc
10000 10000 EuTc
W1
0 0
0 40 80 120 160 200 240 0 40 80 120 160 200 240
Time ( µs) Time ( µs)

5000050000CD Rapid Lifetime Imaging (RLI)Phase Delay Imaging (PDI)
4000040000
EuTc-HP EuTc-HP
3000030000

2000020000 W1
1000010000 W2 W1 E uTc W2 EuTc
00 0 40 80 120 160 200 2400 40 80 120 160 200 240
Time ( s) Time ( s)µ µ
2. Hydrogen peroxide, glucose, and glucose oxidase have been determined by the fluore-
scence imaging system developed.
3