Fluorescent multiple chemical sensing using time-domain fluorescence lifetime imaging [Elektronische Ressource] / vorgelegt von Stefan Nagl
153 Pages
English
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Fluorescent multiple chemical sensing using time-domain fluorescence lifetime imaging [Elektronische Ressource] / vorgelegt von Stefan Nagl

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153 Pages
English

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Published 01 January 2008
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Language English
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Fluorescent Multiple Chemical Sensing using Time-
Domain Fluorescence Lifetime Imaging



Dissertation zur Erlangung des Doktorgrads der Naturwissenschaften
(Dr. rer. nat.)

der Fakultät Chemie und Pharmazie
der Universität Regensburg












vorgelegt von
Stefan Nagl
Regensburg, im Mai 2008
Diese Doktorarbeit entstand in der Zeit vom Juni 2004 bis zum April 2008 am Institut für
Analytische Chemie, Chemo- und Biosensorik der Universität Regensburg.

Die Arbeit wurde angeleitet von Prof. Otto S. Wolfbeis















Promotionsgesuch eingereicht am: 27.05.2008
Kolloquiumstermin: 19.06.2008
Prüfungsausschuß: Vorsitzender: Prof. Hans-Helmut Kohler
Erstgutachter: Prof. Otto S. Wolfbeis
Zweitgutachter: Prof. Achim Göpferich
Drittprüfer: Prof. Bernhard Dick
Contents
CONTENTS



CHAPTER 1
Introduction
1.1. Time-resolved Fluorescence Imaging...........................................................................1
1.2. Chemical Sensors and Biosensors.................................................................................4
1.3. Optical Multiple Chemical Sensing ............................................................................10
1.4. Sensor Miniaturization and Microarray Technology ...............................................14
1.5. Aim of the Research......................................................................................................16
1.5. References ......................................................................................................................18

CHAPTER 2
Record Response Optical Trace Oxygen Sensing and Imaging
2.1. Introduction...................................................................................................................22
2.2. Results and Discussion..................................................................................................23
2.2.1. Fluorescence spectra...........................................................................................................23
2.2.2. Fluorescence and phosphorescence spectra at low temperatures.......................................25
2.2.3. Singlet oxygen luminescence spectra and lifetimes...........................................................26
2.2.4. Scanning electron micrographs..........................................................................................29
2.2.5. Fluorescence lifetime imaging...........................................................................................30
2.2.6. Comparison of oxygen quenching efficiencies..................................................................33
2.3. Conclusion ....................................................................................................................34
2.4. Experimental Section....................................................................................................35
2.5. References......................................................................................................................36

I Contents
CHAPTER 3
A Dual Fluorescence Sensor for Trace Oxygen and Temperature with a
Large Temperature Range and Unmatched Oxygen Sensitivity
3.1. Introduction...............................................................................................................39
3.2. Experimental Section ................................................................................................42
3.2.1. Materials.............................................................................................................................42
3.2.2. Methods..............................................................................................................................42
3.3. Results and Discussion ..............................................................................................45
3.4.1. Composition of the dual sensors ...................................................................................45
3.4.2. Luminescence spectra...................................................................................................47
3.4.3. Calibration of the dual sensors .....................................................................................49
3.4.4. Response times of the dual sensors to oxygen..............................................................56
3.4.5. Derivation and validation of a bivariate calibration function for all temperatures and
oxygen concentrations covered.....................................................................................58
3.4. Conclusion......................................................................................................................63
3.5. References.......................................................................................................................64

CHAPTER 4
A Method for Simultaneous Luminescence Sensing and Imaging of Two
Species Using Optical Probes of Different Luminescence Decay Time
4.1. Introdcution...............................................................................................................70
4.2. Results and Discussion ..............................................................................................72
4.2.1. Experimental design ..........................................................................................................72
4.2.2. Material selection...............................................................................................................75
4.2.3. Calibration of the temperature sensitivity ..........................................................................77
4.2.4. Calibration of the oxygen sensitivity .................................................................................79

II Contents
4.2.5. Measurement of oxygen consumption caused by enzymatic catalysis at varying
temperatures.......................................................................................................................82
4.3. Conclusion...................................................................................................................... 83
4.4. Experimental Section ................................................................................................84
4.4.1. Materials.............................................................................................................................84
4.4.2. Preparation of the dual sensing film...................................................................................85
4.4.3. Calibration of the dual sensor ............................................................................................86
4.4.4. Enzymatic oxygen consumption measurements ................................................................87

4.5. References......................................................................................................................89

CHAPTER 5
Luminescent Polymer Nanoparticles as Probes for Protein, Oxygen and
Temperature
5.1. Introduction...............................................................................................................92
5.2. Experimental part .....................................................................................................94
5.2.1. Materials.............................................................................................................................94
5.2.2. Polymer syntheses..............................................................................................................95
5.2.3. Nanoparticle syntheses.......................................................................................................96
5.2.4. Instruments.........................................................................................................................98

5.3. Metalloporphyrin-doped phosphorescent PD nanoparticles as optical probes.....100
5.3.1. Platinum porphyrin-doped nanospheres displaying FRET to red-emitting cyanine
dyes......................................................................................................................... .........100
5.3.2. Palladium benzoporphyrin-doped nanospheres for NIR applications..............................106
5.4. Temperature-sensitive doped PMAN nanospheres................................................108
5.4.1. Optical spectra and nanoparticle size...............................................................................109
5.4.2. Temperature sensitivity....................................................................................................109
5.5. Dye-doped polystyrene-based nanobeads for oxygen sensing................................110
5.4.1. Optical spectra and nanoparticle size...............................................................................111

III Contents
5.4.2. Oxygen sensitivity............................................................................................................112
5.6. Conclusion.....................................................................................................................114
5.7. References......................................................................................................................116

CHAPTER 6
Microarray Analysis of Protein-Protein Interactions Using Subnanosecond-
resolved Fluorescence Lifetime Imaging
6.1. Introduction.............................................................................................................118

6.2. Experimental part ...................................................................................................120
6.2.1. Protein labeling ................................................................................................................120
6.2.2. Microarray production and incubation.............................................................................121
6.2.3. Fluorescence excitation and detection .............................................................................121
6.2.4. Image acquisition and analysis ........................................................................................122

6.3. Results and Discussion ............................................................................................124
6.4. Conclusion.....................................................................................................................128
6.5. References.....................................................................................................................130
7. Summary......................................................................................................................133
8. Abbreviations, Acronyms and Symbols....................................................................136
9. Curriculum Vitae........................................................................................................140
10. Publications, Presentations and Abstracts................................................................141
11. Acknowledgements......................................................................................................145




IV CHAPTER 1 Introduction

CHAPTER 1
INTRODUCTION


1.1. Time-resolved Imaging
One of the first people who used time-resolved imaging was the photographer Eadweard
Muybridge in 1878, who had been hired by the university founder Leland Stanford to prove a
bet that there were moments during horse galloping, when horses have all four hooves off the
ground (Fig. 1.1).

Fig 1.1. Horse galopping, imaged with a timing resolution of approx. 50 ms, from Ref. 1.

Those images used fast mechanical shutters. In the early twentieth century a novel technique
for fast imaging emerged that was based on repeated exposition of a photographic film with
[2]short light pulses (“stroboscopic imaging”). The step towards timescales required for most
molecular light emission processes was made possible by, among many other developments,
[3]the invention of digital charge-coupled device (CCD) technology and the availability of
[4]
high-energy ultrashort laser pulses.


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1 CHAPTER 1 Introduction








Fig 1.2. The Jablonski diagram showing typical timescales of electronic transitions.

Characteristic timescales of electronic transitions are depicted in Fig. 1.2 (Jablonski diagram).
Fluorescence emission in organic molecules follows an exponential decay law and is mostly
found in the timescale of hundreds of picoseconds (ps) to a few nanoseconds (ns). Because
the transition to the triplet state violates a selection rule in quantum mechanics (Δs ≠ 0), the
timescale of phosphorescence is much longer and found to occur in microseconds (µs) to
milliseconds (ms).
The fluorescence decay is usually generally described by a multiexponential decay
[5,6]function,
−t /τ iI (t) = a ⋅e (Eq. 1.1) ∑ i
i
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2 CHAPTER 1 Introduction

[7]although other approaches such as using a stretched exponential decay exist. The
fluorescence lifetime can be measured in the frequency or in the time domain (Fig. 1.3). Both
approaches are suitable for imaging.


Fig 1.3. Fluorescence lifetime measurement techniques, from Ref. 8.

In a frequency-domain lifetime measurement a sinusoidally modulated light source is
employed, and the phase shift of the emission with respect to the excitation light is
determined. For time-domain fluorescence lifetime imaging, two main approaches exist. Time
correlated single photon counting (TCSPC) is a point scanning method that uses timing
information of many individual photons arriving at a photomultiplier to convolute a decay
curve. The image is then digitally calculated out of many point measurements (Fig. 1.4.a). It
is mainly used along with confocal microscopy. Gated CCD imaging on the other hand
records a number of images at different delays with respect to the excitation light pulse and
calculates lifetimes from the temporal light intensity behavior of each CCD pixel.

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3 CHAPTER 1 Introduction

a) b)






Fig 1.4. Time-domain imaging techniques. a) TCSPC deconstructing the decay curve at each point
using single photon arrival times, from Ref. 9, b) gated CCD imaging using fast intensifiers or other
modulators from Ref. 10.

1.2. Chemical Sensors and Biosensors
Sensors have become part of our daily life to an extent we are not aware of: temperature
sensors turn refrigerators on and off, pressure sensors display oil pressure in cars and
elsewhere, and photosensors turn on and off city lights, to mention only a few.
Most chemical sensors are around for only about 30 years only though, with some
notable exceptions such as the pH glass electrode reported in 1909 by Haber and
[11] [12] Klemensiewicz or Clark’s oxygen electrode in 1956. The first biosensor can also be
attributed to Clark when he described an experiment in 1962 using his oxygen electrode
[13] covered with a dialysis membrane filled with glucose oxidase. Nowadays, the most often
produced chemical sensor is the solid-state oxygen sensor (of the conductivity type, used by
the millions in catalytic converters, and capable of continuously and reversibly recording
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