Faculty of Computer Science, Electronics and Telecommunications
Department of Electronics
Doctoral Thesis
Silicon Photomultipliers Applied for Fluorescence Detection of
Biomarkers in the System with Self Calibrated Gain
M.Sc. Eng. Piotr Dorosz
Supervisor: Prof. Wojciech Kucewicz, PhD, Eng. Kraków 2017
I would like to thank my supervisor Prof. Wojciech Kucewicz for constant support in my scientific and academic challenges. Moreover, for creating many opportunities for my self-development including this dissertation.
I would also like to thank my colleagues PhD Eng. Łukasz Mik and M.Sc. Eng. Mateusz Baszczyk for invaluable help in designing electronic and optic parts of the measurement system.
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INTRODUCTION ... 7
1. SCOPE OF WORK AND THESES ... 8
1.1. FLUORESCENCE ... 10 2. JABŁOŃSKI DIAGRAM ... 13 2.1. STOKES SHIFT ... 16 2.2. QUANTUM YIELD,FLUORESCENCE LIFETIME,ABSORPTION AND EMISSION COEFFICIENTS ... 17
2.3. FLUOROPHORES ... 20
2.4. MAIN PHOTODETECTORS USED IN FLUORESCENCE DETECTION ... 23
2.5. SILICON PHOTOMULTIPLIER ... 24
3. STRUCTURE AND OPERATION MODES ... 24
3.1. KEY PARAMETERS OF THE SIPM ... 28
3.2. ELECTRICAL MODEL OF THE SIPM... 35
3.3. FRONT-END ELECTRONICS ... 38
4. PREAMPLIFIER ... 39
4.1. FOUR CHANNELS ASIC WITH POLE-ZERO CANCELLATION ... 46
4.2. MULTI-CHANNEL ASICBASED ON SUPER COMMON GATE AMPLIFIER ... 52
4.3. DATA ACQUISITION SYSTEM FOR THE SIPM AND DETECTION METHODS ... 65
5. THERMAL GENERATION IN MEASUREMENT DATA ... 70
5.1. GAIN STABILIZATION IN SIPMAPPLICATIONS ... 73
5.2. SELECTION OF A SUITABLE ASIC FOR FLUORESCENCE DETECTION. ... 83
5.3. OPTICAL SYSTEM ... 84
5.4. 5.4.1. STATIONARY SYSTEM WITH PHOTOMETRIC CUVETTES ... 84
5.4.2. MICROFLUIDIC SYSTEM WITH GLASS MICRO-CAPILLARY ... 86
5.4.3. MICROFLUIDIC SYSTEM WITH µ–SLIDE ... 88
CALCULATING THE VALUE OF SIGNAL FROM THE SIPMBASED ON HISTOGRAM ... 89
5.5. INFLUENCE OF THE EXCITATION LIGHT DURATION ON THE MEASURED SIGNAL ... 91
5.6. INFLUENCE OF THE FLOW SPEED ON THE FLUORESCENCE INTENSITY ... 92 5.7.
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FLUORESCENCE MEASUREMENT RESULTS ... 94
6.
SAMPLE PREPARATION AND INTERPRETATION OF RESULTS ... 95 6.1.
MEASUREMENTS IN STATIONARY SYSTEM ... 98 6.2.
MEASUREMENTS IN MICROFLUIDIC SYSTEM ... 101 6.3.
MEASUREMENT OF ANTIBODY-FLUOROPHOR COMPOUND ... 112
6.4.
SUMMARY AND CONCLUSIONS ... 116
7. ACKNOWLEDGMENT ... 118 BIBLIOGRAPHY ... 118 8. LIST OF FIGURES ... 129 8.1. LIST OF TABLES ... 137 8.2.
APPENDIX A. INVERSION REGIONS OF MOS TRANSISTOR – EKV MODEL... 139
APPENDIX B. DETERMINATION OF THE INPUT TRANSISTOR SIZE FOR THE MINIMIZATION OF THE WHITE SERIES NOISE ... 141
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NTRODUCTION1.
The first observation of fluorescence took place in the 19th century [1]. The phenomenon have been vastly used and developed especially in biomedicine. The demand for applications based on fluorescence to a large extent is a result of continuous improvement and miniaturization of measurement equipment, hence availability and facilitation of carrying out the measurement itself. Each year new method of fluorescence detection is introduced which slightly improves achieved results. Although, this permanent development is relevant, a significant progress can be made when the limit of detection is decreased. This refers to the ability to measure extremely low fluorescence light intensity – single photon. Until recently, such high measurement precision could be reached mainly by the Photomultiplier Tube (PMT). Large gain of this device is occupied at the expense of complex design, substantial size, susceptibility to the effects of external magnetic field, biasing voltage ~kV and high production costs. An alternative for the PMT that was introduced at the end of 20th century is the Silicon Photomultiplier (SiPM) [2-3]. Besides the ability to measure single photons, it is much smaller in size, requires biasing lower than 100V, is resistant to magnetic field and mechanically strong. Nevertheless, the SiPM is not solely the substitute for the PMT. The SIPM has advantages that allow it to detect low light intensity in medical, chemical, biological applications but also to build integrated, portable microfluidic systems [4-7]. Microfluidic setup consists of a microchannels, reaction and emission chambers which can be integrated in form of a multi-purpose Lab-on-chip or Micro Total Analysis System (μTAS). The advantages of microfluidic system is miniaturization. It consumes less reagents, has faster response, increases the efficiency of light detection. The SiPM can be integrated into the flow setup, improving the measurement performance. The miniaturization of the optic and electronic parts of the readout system allows to design a mobile application for fluorescence detection. The immediate result of the measurement enables to obtain a preliminary point-of-care diagnosis. The simplicity of the design results in lower price of the application. Thus, the availability of the measurement can be increased by the institutions and end-users which cannot purchase the expensive diagnostic laboratory equipment.
8 SCOPE OF WORK AND THESES
1.1.
The purpose of this dissertation was to design a measurement device based on the SiPM that can detect fluorescence of biomarkers. The use of a microfluidic system to measure this phenomenon is greatly advantageous due to the small volume of tested sample. Because of the extremely low intensity of measured light the device had to be properly calibrated. The gain of the SiPM is temperature dependent, hence a stabilization method has been developed to keep the gain constant, independently of the temperature. The data acquisition system for the SiPM has been designed. It consists of a dedicated application specific integrated circuit (ASIC) required to amplify and shape signals from the photodetector. The device has been designed with the aim of transforming it into a portable application.
Preliminary measurements proved that the SiPM could be applied in fluorescence detection. Thus, the following theses have been defined:
1. The data acquisition system equipped with a designed application specific integrated circuit can be successfully used in measurements with the Silicon Photomultiplier. Silicon Photomultiplier’s gain stabilization method can be implemented in the system, hence improving its parameters.
2. The Silicon Photomultiplier can be applied in the measurements of fluorescence of biomarkers.
The dissertation presents in detail all steps that have been taken in order to confirm the theses. Firstly, the main principles of fluorescence phenomenon had to be studied (Chapter 2). It resulted in the choice of biomarker detection method which is based on ELISA protocol (Enzyme-Linked Immunosorbent Assay). The method uses a compound of an antibody and a fluorophor. The fluorophor is responsible for the emission of fluorescent light while the antibody binds with sought antigen. The implementation of this method in a mobile measurement system becomes the main purpose of the dissertation. The system is based on the Silicon Photomultiplier because of its many advantages (Chapter 3). A significant amount of work has been put into developing an electronic system which is able to read signals from the photodetector. An Application Specific Integrated Circuit (ASIC) for the SiPM has been designed (Chapter 4) together with two Data Acquisitions Systems (DAQ). The DAQs are placed on four-layer PCB (Printed Circuit Board). They consist of the ASIC, FPGA (Field Programmable Gate Array) and USB or PCI drivers (Chapter 5). The chapter 5.2 describes the
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SiPM’s gain stabilization algorithm which is crucial from the point of the mobile application because it enables the system to adapt to changing temperature conditions. The design of the measurement system concludes with three variants of its optical part (one stationary and two microfluidic). Chapter 6 describes the fluorescence detection with the system presented in Chapters 4 and 5. The aim of the measurements is to reduce the volume of measured sample and choose the best optical system in terms of detection limit. Afterwards, the most suitable version is used in an antibody detection. Chapter 7 summarizes the dissertation and describes the future development and possible applications of the measurement system.
