審定書 i
ACKNOWLEDGEMENTS ii
中文摘要 iii
ABSTRACTS v
TABLE OF CONTENTS vii
LIST OF FIGURES xi
LIST OF TABLES xvii
Chapter 1 Introduction and Literature Review 1
1.1 General Background Information 1
1.1.1 Dopamine associated with psychiatric and neurological disorders 1
1.1.2 Detection of DA in clinical diagnosis of Parkinson’s disease 2
1.2 Introduction of Biosensor 5
1.2.1 The specific interaction of bioreceptor 7
1.2.2 Aptamers SELEX 8
1.3 Biosensor System Based on Different Transducer 11
1.3.1 Electrochemical biosensors 11
1.3.2 Optical biosensors 13
1.3.3 Fluorescent biosensors 14
1.4 Biosensors for the Detection of Small Molecule 17
1.5 Research Purpose 24
Chapter 2 Experimental Section 26
2.1 Materials and Chemicals 26
2.2 Apparatus 29
2.3 Mechanism in This Project 31
2.4 Fabrication of Biosensor 32
2.4.1 Surface cleaning 32
2.4.2 Surface modification 33
2.4.3 Surface modification analysis 35
2.4.4 Quantitative analysis of amine groups on modified chips 36
2.4.5 Fabrication of DBA functional-biosensor 36
2.4.6 Agarose gel electrophoresis analysis 38
2.5 Synthesis of Red Fluorescent BSA-Au NCs 38
2.6 Detection of DA by DBA-modified Chip bPEI 39
2.7 Specificity and Selectivity Experiments 41
2.7.1 Interference substances 41
2.7.2 Non-specific aptamer for DA 41
2.8 Stability and recovery experiments 42
2.8.1 Stability tests 42
2.8.2 Detection of DA in CSF and FBS for recovery experiments 43
Chapter 3 Discussion and Results 44
3.1 Preparation of Functionalized Biochips 44
3.1.1 Molecular Dynamics simulation 46
3.1.2 TNBS assay 49
3.1.3 Quantitative analysis of -NH2 on Chip bPEI and Chip APTES 52
3.2 Immobilization of DBA on Biochip 54
3.2.1 Agarose gel electrophoresis 55
3.2.2 Conjugation rate of DBA on amine-modified chips 58
3.3 Investigation of DBA-Functionalized chip 60
3.3.1 AFM analysis for Chips 60
3.3.2 XPS analysis for surface characterization of biochips 62
3.4 DA Detecting Based on Colorimetric System 64
3.4.1 Oxidation of DA standard solution induced by aqueous NaOH 64
3.4.2 Linear regression based on colorimetric system 65
3.4.3 Selectivity studies (colorimetric system) 66
3.5 DA Detecting Based on Fluorescence Quenching System 68
3.5.1 Preparation of BSA-Au NCs 69
3.5.2 Quenching by DA solution and mechanism 71
3.5.3 The quenching time of BSA-Au NCs 74
3.5.4 Control experiments 75
3.5.5 Aqueous NaOH influences 76
3.5.6 Linear regression based on quenching system 78
3.5.7 Selectivity studies (fluorescence quenching system) 80
3.5.8 Biosensor stability 82
3.6 Recovery Experiment 83
Chapter 4 Conclusion 84
References 85

Motivation:　
Dopamine (DA), one of the most prominent catecholamine neurotransmitter existing human’s circulation system and cerebrospinal fluid, plays a crucial role in several neurological disorders. According to research, the normal content of DA in healthy people was in the range of 1.3 - 2.6 μM (i.e. 0.26 μg/mL - 0.54 μg/mL), which indicated that when the DA concentration was abnormal in human plasma, it could lead to certain neurological disease such as schizophrenia, or Parkinson’s disease (PD).
Purpose:
In clinical diagnosis, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) are harnessed to detect changes in striatal DA level. On the other hand, ELISA Kit and electrochemical detection (ECD) for HPLC are extremely selective and sensitive detection techniques, however, they are actually time-consuming, costly and complicated in clinical detection. Thus, we aimed to design a fabrication for a biochip detecting for DA would be so facile and cheap that have an inclination to be employed for the clinical detection in the near future.
Methods:
We immobilized the DBA onto amine groups-modified chips by covalent binding with GA linker. The amine groups modified chips were fabricated with branched polyethylenimine (bPEI). After that, we employed two sensing system for detection as soon as DA was captured by DBA. In the first system, was adopted based on the oxidation reaction of DA induced by sodium hydroxide (NaOH), causing the color of supernatant altering from transparent to brownish accompanied by a specific absorbance spectrum.
The second system the BSA-Au nanoclusters (BSA-Au NCs) can be quenched with the increasing concentration of DA based on PET mechanism.
Results:
In this study, the biochip prepared was able to detect DA with linear range from 1 ng/mL, to 10 mg/mL and a limit of detection (LOD) as low as 0.1 ng/mL based on fluorescence quenching system, which was in the detecting range of DA in human serum concentration. Also the results of interference test (e.g. ascorbic acid, glucose, alanine, uric acid, etc.) showed that our designed system was selective and specific. With those advantages, the biochip for DA detection certainly has potentiality to be applied in the clinical detection in the near future.

審定書 i
授權書 ii
ACKNOWLEDGEMENTS iii
中文摘要 iv
ABSTRACTS vi
TABLE OF CONTENTS viii
LIST OF FIGURES xii
LIST OF TABLES xviii
Chapter 1 Introduction and Literature Review 1
1.1 General Background Information 1
1.1.1 Dopamine associated with psychiatric and neurological disorders 1
1.1.2 Detection of DA in clinical diagnosis of Parkinson’s disease 2
1.2 Introduction of Biosensor 5
1.2.1 The specific interaction of bioreceptor 7
1.2.2 Aptamers SELEX 8
1.3 Biosensor System Based on Different Transducer 11
1.3.1 Electrochemical biosensors 11
1.3.2 Optical biosensors 13
1.3.3 Fluorescent biosensors 14
1.4 Biosensors for the Detection of Small Molecule 17
1.5 Research Purpose 24
Chapter 2 Experimental Section 26
2.1 Materials and Chemicals 26
2.2 Apparatus 29
2.3 Mechanism in This Project 31
2.4 Fabrication of Biosensor 32
2.4.1 Surface cleaning 32
2.4.2 Surface modification 33
2.4.3 Surface modification analysis 35
2.4.4 Quantitative analysis of amine groups on modified chips 36
2.4.5 Fabrication of DBA functional-biosensor 36
2.4.6 Agarose gel electrophoresis analysis 38
2.5 Synthesis of Red Fluorescent BSA-Au NCs 38
2.6 Detection of DA by DBA-modified Chip bPEI 39
2.7 Specificity and Selectivity Experiments 41
2.7.1 Interference substances 41
2.7.2 Non-specific aptamer for DA 41
2.8 Stability and recovery experiments 42
2.8.1 Stability tests 42
2.8.2 Detection of DA in CSF and FBS for recovery experiments 43
Chapter 3 Discussion and Results 44
3.1 Preparation of Functionalized Biochips 44
3.1.1 Molecular Dynamics simulation 46
3.1.2 TNBS assay 49
3.1.3 Quantitative analysis of -NH2 on Chip bPEI and Chip APTES 52
3.2 Immobilization of DBA on Biochip 54
3.2.1 Agarose gel electrophoresis 55
3.2.2 Conjugation rate of DBA on amine-modified chips 58
3.3 Investigation of DBA-Functionalized chip 60
3.3.1 AFM analysis for Chips 60
3.3.2 XPS analysis for surface characterization of biochips 62
3.4 DA Detecting Based on Colorimetric System 64
3.4.1 Oxidation of DA standard solution induced by aqueous NaOH 64
3.4.2 Linear regression based on colorimetric system 65
3.4.3 Selectivity studies (colorimetric system) 66
3.5 DA Detecting Based on Fluorescence Quenching System 68
3.5.1 Preparation of BSA-Au NCs 69
3.5.2 Quenching by DA solution and mechanism 71
3.5.3 The quenching time of BSA-Au NCs 74
3.5.4 Control experiments 75
3.5.5 Aqueous NaOH influences 76
3.5.6 Linear regression based on quenching system 78
3.5.7 Selectivity studies (fluorescence quenching system) 80
3.5.8 Biosensor stability 82
3.6 Recovery Experiment 83
Chapter 4 Conclusion 84
References 85

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