Nanostructured Electrochemical Biosensors: Advanced Detection of Biomarkers and Pathogens

다국어 초록 (Multilingual Abstract)

The rapid development of electrochemical biosensors has transformed various fields, including disease diagnostics, environmental monitoring, and food safety. These sensors rely on the specific interactions between biomolecules and their target analytes to produce measurable electrical signals, enabling highly sensitive and label-free detection was introduced in Chapter 1. This thesis focuses on the creation and optimization of electrochemical biosensors designed to detect disease biomarkers and bacterial pathogens with enhanced sensitivity, selectivity, and reusability. In Chapter 2, an electrochemical impedance-based biosensor is developed to detect p-tau181, a key biomarker for Alzheimer’s disease, by functionalizing indium tin oxide (ITO) micro-electrodes with reduced graphene oxide/β-cyclodextrin and an anti-p-tau181 antibody, offering a non-invasive and highly sensitive detection method for early diagnosis. Building on this, the work progresses to the design of a biosensor for detecting Staphylococcus aureus in Chapter 3, utilizing a gold-interdigitated electrode with an innovative wave-shaped geometry to improve sensitivity and detection speed, which is essential for clinical diagnostics and food safety applications. Further advancements are made in Chapter 4 with the development of an antibody-free detection system, where vancomycin-coated silica nanoparticles are used in conjunction with a gold nanocluster-modified 8-channel electrode PCB, resulting in a highly sensitive and cost-effective approach for detecting Gram-positive bacteria. The sensor platform is further expanded in Chapter 5 to a 16-channel system with AuNPs@Ti3C2Tz nanocomposites and peptide-based architectures, enabling simultaneous detection of multiple bacterial pathogens, including Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus. This multi-analyte system demonstrates robust antifouling properties and a strong linear relationship between bacterial concentration and detection signals. Chapter 6 addresses the issue of sensor sustainability by introducing a vancomycin-functionalized interdigitated electrode (VF-IDEs) that allows for capacitive detection of Gram-positive bacteria in complex biological samples. The regeneration capability of this sensor ensures cost-efficiency and long-term usability, which is crucial for large-scale diagnostic applications. Finally, Chapter 7 broadens the scope of this research to investigate the potential of bacteria-based therapies in cancer treatment, focusing on their emerging role in tumor-targeted therapies and the challenges of translating these approaches into clinical practice. Overall, this thesis contributes to the advancement of electrochemical biosensor technology by introducing novel electrode designs, enhancing detection capabilities, and ensuring the sustainability and reusability of biosensors for both clinical diagnostics and environmental monitoring.

국문 초록 (Abstract)

전기화학 바이오센서 기술의 빠른 발전은 질병 진단, 환경 모니터링 및 식품 안전 분야를 포함한 다양한 분야에서 혁신을 가져왔다. 이러한 센서는 생체분자와 표적 분석물 간의 특이적인 상호작용을 이용하여 측정 가능한 전기적 신호를 생성함으로써 고감도이면서도 표지 없이도 검출이 가능한 기술로 제1장에서 소개되었다. 본 논문은 질병 바이오마커 및 박테리아 병원체를 감지하기 위해 고감도, 선택성 및 재사용성이 향상된 전기화학 바이오센서를 개발하고 최적화하는 데 중점을 둔다. 제2장에서는 인듐 주석 산화물(ITO) 마이크로 전극을 환원된 그래핀 산화물/β-사이클로덱스트린 (reduced graphene oxide/β-cyclodextrin)과 anti-p-tau181 항체로 기능화하여 알츠하이머병의 주요 바이오마커인 p-tau181을 감지하는 전기화학 임피던스 기반 바이오센서를 개발하였다. 이는 비침습적이고 고감도의 초기 진단 방법을 제공한다. 이를 바탕으로, 제3장에서는 Staphylococcus aureus를 감지하기 위해 혁신적인 파형(wave-shaped) 구조의 금 빗살형 (gold-interdigitated electrode) 전극을 활용하여 감도와 검출 속도를 향상시킨 바이오센서를 개발하였으며, 이는 임상 진단 및 식품 안전 분야에서의 실용적 응용 가능성을 보여주었다. 이어서 제4장에서는 항체를 사용하지 않는 검출 시스템을 개발하여, 반코마이신 코팅 실리카 나노입자(silica nanoparticles)를 금 나노클러스터(Au nanocluster)가 도포된 8채널 PCB 전극과 결합하여 그람 양성 박테리아를 감지하는 고감도이고 비용 효율적인 방법을 제시한다. 제5장에서는 AuNPs@Ti3C2Tz 나노복합체(nanocomposite)와 펩타이드 기반 아키텍처 (peptide-based architecture)를 사용하여 시스템을 16채널로 확장하였으며, Staphylococcus aureus, Bacillus cereus, Micrococcus luteus 등 여러 박테리아 병원체를 동시에 감지할 수 있는 능력을 보여주었으며, 이 다중 분석 시스템은 강력한 항오염 특성과 박테리아 농도와 검출 신호 간의 강한 선형 관계를 입증하였다. 제6장에서는 센서의 재사용성을 확보하기 위해 반코마이신 기능화 인터디지테이티드 전극(VF-IDEs)을 제시하여, 복잡한 생물학적 샘플에서 그람 양성 박테리아를 용량성 검출할 수 있게 하였다. 이 센서는 혈장 등 복잡한 생체 시료에서도 안정적으로 작동하며, 재생(regeneration) 기능을 통해 반복 사용이 가능하여 비용 효율적이고 실용적인 감염 진단용 플랫폼으로 활용될 수 있음을 입증하였다. 마지막으로, 제7장에서는 연구의 응용 범위를 확장하여 암 치료 분야에서의 박테리아 기반 치료(bacteria-based therapy) 가능성을 탐구하였다. 종양 표적 치료(tumor-targeted therapy)에서 박테리아의 새로운 역할을 조명하고, 임상 적용을 위한 기술적·윤리적 도전 과제들을 논의하였다. 본 논문은 새로운 전극 설계, 검출 능력 향상, 그리고 임상 진단 및 환경 모니터링을 위한 바이오센서의 지속 가능성과 재사용성을 보장함으로써 전기화학 바이오센서 기술의 발전에 기여하였다.

목차 (Table of Contents)

• CHAPTER 1. Introduction 1
• 1.1 The needs of electrochemical biosensor: Advanced strategies for biomarkers and pathogen applications 1
• 1.1.1 The history of electrochemical biosensor 2
• 1.1.2 Principle of electrochemical chemical methods 5
• 1.1.3 Label-based electrochemical detection 8
• 1.1.4 Label-free electrochemical detection 9
• 1.1.5 Specific clinical and pathogen detection needs 10
• 1.2 Inhibition electrochemical biosensor scenario 12
• 1.3 Explore the next generation: Micro/Nano electrode array sensors - Pushing the boundaries of fabrication techniques and bioanalysis applications 13
• 1.4 Recent advances and trends on electrochemical detection 16
• 1.4.1 Fabrication of electrodes 16
• 1.4.2 Nanomaterials 20
• 1.4.3 Computational biology and chemistry 21
• 1.5 Thesis outlines and research aim 24
• 1.5.1 Research aims 24
• 1.5.2 Thesis outlines 25
• References 28
• CHAPTER 2. Electrochemical impedance biosensor for the label- free assessment of plasma P-tau181 concentrations for precise clinical diagnosis of mild cognitive impairment and Alzheimer's disease 35
• 2.1 Introduction 35
• 2.2 Experiment 37
• 2.2.1 Examination of participants for the acquisition of human clinical plasma specimens 37
• 2.2.2 Chemicals and Materials 39
• 2.2.3 Functionalization of the label-free electrochemical impedance biosensor for the selective detection of the p-tau181 biomarker 40
• 2.2.4 Preparation of p-tau181 biomarker detection and quantification of plasma clinical samples 40
• 2.2.5 Statistical Evaluation 41
• 2.3 Results and discussion 42
• 2.3.1 Characterization of the fabrication of an electrochemical impedance- based biosensor utilizing EIS and CV 42
• 2.3.2 P-tau 181 biomarker detection: calibration curve and dissociation constant 44
• 2.3.3. Impedance-based biosensor's selectivity and interference test 47
• 2.3.4. Identification of individuals with healthy controls, patients with MCI, and AD using the impedance detection of p-tau181 in human clinical plasma samples 49
• 2.3.5. Assessment of p-tau 181 in human plasma clinical specimens with an impedance-based biosensor to differentiate between patients with AD, MCI, and healthy control subjects 51
• 2.3.6. Comparative analysis of the developed electrochemical impedance- based biosensor with participants' demographic characteristics for clinical disease stage identification 52
• 2.4. Conclusion 54
• 2.5 Supporting Information 55
• References 61
• CHAPTER 3. Ultrasensitive and Fast Staphylococcus aureus Detection Utilizing a Gold-Interdigitated Single-Wave-Shaped Electrode (Au-ISWE) Electrochemical Biosensor 67
• 3.1 Introduction 67
• 3.2 Experiment 70
• 3.2.1 Materials and Reagents 70
• 3.2.2 Instrumentation 70
• 3.2.3 Construction of the Au-ISWEs 71
• 3.2.4 Preparation of bacteria 71
• 3.2.5 Construction of the Biosensor Platform 72
• 3.2.6 Measurement of Impedance 73
• 3.2.7 Measurement of Cyclic Voltammetry 73
• 3.2.8 Selectivity and Stability of the Biosensor 74
• 3.2.9 Extraction of DNA and Polymerase Chain Reaction (PCR) 74
• 3.2.10 Statistical Evaluation 75
• 3.3 Results and discussion 75
• 3.3.1 Characterization of 6-MHA on Au-ISWEs surface 75
• 3.3.2 Reaction of Biosensors 76
• 3.3.3 Electrochemical Evaluation of the Biosensor Platform Interface 76
• 3.3.4 Analytical Performance of the Biosensor in Response to S. aureus 78
• 3.4. Conclusion 82
• 3.5 Supporting Information 84
• References 86
• CHAPTER 4. Impedimetric biosensor for Gram-positive bacteria utilizing vancomycin-coated silica nanoparticles on a gold nanocluster-deposited electrode 90
• 4.1 Introduction 90
• 4.2 Experiment 93
• 4.2.1 Materials and Reagents 93
• 4.2.2 Construction of the Au-PCB Electrode 94
• 4.2.3 Preparing and Characterizing SiNPs-VAN 94
• 4.2.4 Construction of the Biosensor Platform 95
• 4.2.5 Preparation of Bacteria 96
• 4.2.6 Electrochemical Assessment 96
• 4.2.7 Extraction of DNA and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) 97
• 4.3 Results and discussion 98
• 4.3.1 Characterization of 6-MHA on Au-ISWEs surface 98
• 4.3.2 SiNPs-VAN characterization 100
• 4.3.3 Gram-Positive Bacteria Electrochemical Characterization and Analytical Performance of the Biosensor 101
• 4.3.4 Evaluation of Performance in Environmental Samples 105
• 4.4. Conclusion 109
• 4.5 Supporting Information 110
• References 114
• CHAPTER 5. Innovative High-throughput Electrochemical Detection of Gram-positive Bacteria Utilizing AuNPs@Ti3C2Tz Functionalized with Sandwich Peptides 118
• 5.1 Introduction 118
• 5.2 Experiment 120
• 5.2.1 Materials and Reagents. 120
• 5.2.2 Techniques for Characterization 121
• 5.2.3 Preparation of Bacteria 121
• 5.2.4 Flow Cytometric Analysis for Peptide Screening 122
• 5.2.5 Electrochemical Measurements 122
• 5.2.6 Synthesis of Ti3C2Tz 123
• 5.2.7 Development of an Electrochemical Biosensor for Bacteria Utilizing Sandwich Peptide on 16-GDEs 123
• 5.2.8 Multiplex Detection in Freshwater 125
• 5.2.9 DNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) 125
• 5.2.10 Statistical Analysis 126
• 5.3 Results and discussion 126
• 5.3.1 Characterization of the Nanocomposites 126
• 5.3.2 Evaluation of a Related Pair of Peptides and Validation 128
• 5.3.3 Development of a Sandwich-type Affinity-based Electrochemical Biosensor Utilizing a Cognate Pair of Peptides 130
• 5.3.4. High-throughput Method of Gram-positive Bacteria in Environmental Sample 132
• 5.4 Conclusion 134
• 5.5 Supporting Information 136
• References 145
• CHAPTER 6. Reusable and Functionalized VAN-PDA@AuNPs Interdigitated Electrodes for Capacitive Detection of Gram- positive Bacteria in Plasma 152
• 6.1 Introduction 152
• 6.2 Experiment 155
• 6.2.1 Chemicals and Reagents 155
• 6.2.2 Bacteria Preparation 155
• 6.2.3 Electrochemical Characterization of IDEs 156
• 6.2.4 Extraction of DNA, applying Quantitative Reverse Transcription Polymerase Chain Reaction 156
• 6.2.5 Preparation of VAN-PDA@AuNPs 157
• 6.2.6 Spectroscopic/Microscopic Study of Nanocomposite Characterization 158
• 6.2.7 Preparation of the Bacteria Capacitive Biosensor 158
• 6.2.8 Plasma Separation using a Dual-gradient Janus Membrane 159
• 6.2.9 Regenerating VF-IDE Surfaces for Reusable Gram-positive Bacteria Detection 160
• 6.2.10 Statistical Analysis 160
• 6.3 Results and discussion 161
• 6.3.1 Characterization of the Nanocomposites 161
• 6.3.2 Electrochemical Behavior 164
• 6.3.3 Development of a Capacitive Biosensor for Analytical Performance of the Nanocomposite in Gram-positive Bacteria Detection 167
• 6.3.4 Capacitive Detection of S. aureus, B. cereus or M. luteus in Plasma Sample 171
• 6.3.5 Regenerating VF-IDE Surfaces for Reusable Gram-positive Bacteria Detection 173
• 6.4. Conclusion 177
• CHAPTER 1. Introduction 1
• 1.1 The needs of electrochemical biosensor: Advanced strategies for biomarkers and pathogen applications 1
• 1.1.1 The history of electrochemical biosensor 2
• 1.1.2 Principle of electrochemical chemical methods 5
• 1.1.3 Label-based electrochemical detection 8
• 1.1.4 Label-free electrochemical detection 9
• 1.1.5 Specific clinical and pathogen detection needs 10
• 1.2 Inhibition electrochemical biosensor scenario 12
• 1.3 Explore the next generation: Micro/Nano electrode array sensors - Pushing the boundaries of fabrication techniques and bioanalysis applications 13
• 1.4 Recent advances and trends on electrochemical detection 16
• 1.4.1 Fabrication of electrodes 16
• 1.4.2 Nanomaterials 20
• 1.4.3 Computational biology and chemistry 21
• 1.5 Thesis outlines and research aim 24
• 1.5.1 Research aims 24
• 1.5.2 Thesis outlines 25
• References 28
• CHAPTER 2. Electrochemical impedance biosensor for the label-free assessment of plasma P-tau181 concentrations for precise clinical diagnosis of mild cognitive impairment and Alzheimer's disease 35
• 2.1 Introduction 35
• 2.2 Experiment 37
• 2.2.1 Examination of participants for the acquisition of human clinical plasma specimens 37
• 2.2.2 Chemicals and Materials 39
• 2.2.3 Functionalization of the label-free electrochemical impedance biosensor for the selective detection of the p-tau181 biomarker 40
• 2.2.4 Preparation of p-tau181 biomarker detection and quantification of plasma clinical samples 40
• 2.2.5 Statistical Evaluation 41
• 2.3 Results and discussion 42
• 2.3.1 Characterization of the fabrication of an electrochemical impedance-based biosensor utilizing EIS and CV 42
• 2.3.2 P-tau 181 biomarker detection: calibration curve and dissociation constant 44
• 2.3.3. Impedance-based biosensor's selectivity and interference test 47
• 2.3.4. Identification of individuals with healthy controls, patients with MCI, and AD using the impedance detection of p-tau181 in human clinical plasma samples 49
• 2.3.5. Assessment of p-tau 181 in human plasma clinical specimens with an impedance-based biosensor to differentiate between patients with AD, MCI, and healthy control subjects 51
• 2.3.6. Comparative analysis of the developed electrochemical impedance-based biosensor with participants' demographic characteristics for clinical disease stage identification 52
• 2.4. Conclusion 54
• 2.5 Supporting Information 55
• References 61
• CHAPTER 3. Ultrasensitive and Fast Staphylococcus aureus Detection Utilizing a Gold-Interdigitated Single-Wave-Shaped Electrode (Au-ISWE) Electrochemical Biosensor 67
• 3.1 Introduction 67
• 3.2 Experiment 70
• 3.2.1 Materials and Reagents 70
• 3.2.2 Instrumentation 70
• 3.2.3 Construction of the Au-ISWEs 71
• 3.2.4 Preparation of bacteria 71
• 3.2.5 Construction of the Biosensor Platform 72
• 3.2.6 Measurement of Impedance 73
• 3.2.7 Measurement of Cyclic Voltammetry 73
• 3.2.8 Selectivity and Stability of the Biosensor 74
• 3.2.9 Extraction of DNA and Polymerase Chain Reaction (PCR) 74
• 3.2.10 Statistical Evaluation 75
• 3.3 Results and discussion 75
• 3.3.1 Characterization of 6-MHA on Au-ISWEs surface 75
• 3.3.2 Reaction of Biosensors 76
• 3.3.3 Electrochemical Evaluation of the Biosensor Platform Interface 76
• 3.3.4 Analytical Performance of the Biosensor in Response to S. aureus 78
• 3.4. Conclusion 82
• 3.5 Supporting Information 84
• References 86
• CHAPTER 4. Impedimetric biosensor for Gram-positive bacteria utilizing vancomycin-coated silica nanoparticles on a gold nanocluster-deposited electrode 90
• 4.1 Introduction 90
• 4.2 Experiment 93
• 4.2.1 Materials and Reagents 93
• 4.2.2 Construction of the Au-PCB Electrode 94
• 4.2.3 Preparing and Characterizing SiNPs-VAN 94
• 4.2.4 Construction of the Biosensor Platform 95
• 4.2.5 Preparation of Bacteria 96
• 4.2.6 Electrochemical Assessment 96
• 4.2.7 Extraction of DNA and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) 97
• 4.3 Results and discussion 98
• 4.3.1 Characterization of 6-MHA on Au-ISWEs surface 98
• 4.3.2 SiNPs-VAN characterization 100
• 4.3.3 Gram-Positive Bacteria Electrochemical Characterization and Analytical Performance of the Biosensor 101
• 4.3.4 Evaluation of Performance in Environmental Samples 105
• 4.4. Conclusion 109
• 4.5 Supporting Information 110
• References 114
• CHAPTER 5. Innovative High-throughput Electrochemical Detection of Gram-positive Bacteria Utilizing AuNPs@Ti3C2Tz Functionalized with Sandwich Peptides 118
• 5.1 Introduction 118
• 5.2 Experiment 120
• 5.2.1 Materials and Reagents 120
• 5.2.2 Techniques for Characterization 121
• 5.2.3 Preparation of Bacteria 121
• 5.2.4 Flow Cytometric Analysis for Peptide Screening 122
• 5.2.5 Electrochemical Measurements 122
• 5.2.6 Synthesis of Ti3C2Tz 123
• 5.2.7 Development of an Electrochemical Biosensor for Bacteria Utilizing Sandwich Peptide on 16-GDEs 123
• 5.2.8 Multiplex Detection in Freshwater 125
• 5.2.9 DNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) 125
• 5.2.10 Statistical Analysis 126
• 5.3 Results and discussion 126
• 5.3.1 Characterization of the Nanocomposites 126
• 5.3.2 Evaluation of a Related Pair of Peptides and Validation 128
• 5.3.3 Development of a Sandwich-type Affinity-based Electrochemical Biosensor Utilizing a Cognate Pair of Peptides 130
• 5.3.4. High-throughput Method of Gram-positive Bacteria in Environmental Sample 132
• 5.4 Conclusion 134
• 5.5 Supporting Information 136
• References 145
• CHAPTER 6. Reusable and Functionalized VAN-PDA@AuNPs Interdigitated Electrodes for Capacitive Detection of Gram-positive Bacteria in Plasma 152
• 6.1 Introduction 152
• 6.2 Experiment 155
• 6.2.1 Chemicals and Reagents 155
• 6.2.2 Bacteria Preparation 155
• 6.2.3 Electrochemical Characterization of IDEs 156
• 6.2.4 Extraction of DNA, applying Quantitative Reverse Transcription Polymerase Chain Reaction 156
• 6.2.5 Preparation of VAN-PDA@AuNPs 157
• 6.2.6 Spectroscopic/Microscopic Study of Nanocomposite Characterization 158
• 6.2.7 Preparation of the Bacteria Capacitive Biosensor 158
• 6.2.8 Plasma Separation using a Dual-gradient Janus Membrane 159
• 6.2.9 Regenerating VF-IDE Surfaces for Reusable Gram-positive Bacteria Detection 160
• 6.2.10 Statistical Analysis 160
• 6.3 Results and discussion 161
• 6.3.1 Characterization of the Nanocomposites 161
• 6.3.2 Electrochemical Behavior 164
• 6.3.3 Development of a Capacitive Biosensor for Analytical Performance of the Nanocomposite in Gram-positive Bacteria Detection 167
• 6.3.4 Capacitive Detection of S. aureus, B. cereus or M. luteus in Plasma Sample 171
• 6.3.5 Regenerating VF-IDE Surfaces for Reusable Gram-positive Bacteria Detection 173
• 6.4. Conclusion 177
• 6.5 Supporting Information 179
• References 186
• Chapter 7. Bacterial Cancer Therapy: Future Perspectives 193
• 7.1 Introduction 193
• 7.1.1 Overview of the Current Cancer Landscape 193
• 7.1.2 Overview of Bacteria as Biomaterials for Cancer Therapy 195
• 7.2. Why are bacteria considered innovative biomaterials for cancer therapy 198
• 7.2.1 Strategies of Potential Wild-Type Bacteria for Cancer Therapeutic Effects 198
• 7.2.2 The prominence of modified microorganisms in cancer therapy 202
• 7.3. The investigation of modified bacterial cancer therapies 210
• 7.3.1 Integration with traditional therapy 211
• 7.3.2 Collaboration with alternative engineered therapies 212
• 7.3.3 Diagnostic and Monitoring Applications 213
• 7.3.4 Clinical Investigations 213
• 7.4 Prospective Opportunities and Challenges 215
• References 218
• Chapter 8. Conclusions and Future Perspectives 228
• 8.1 Conclusions 228
• 8.2 Future perspective 232
• 8.2.1 Advancing microfluidic systems for complex sample analysis 232
• 8.2.2 Microfluidic lab-on-a-chip systems for point-of-care diagnostics 233
• 8.2.3 Enhancing automation and scalability through microfluidic systems 233
• 8.2.4 Customized electrodes and microfluidic integration 234
• 8.2.5 Expanding the frontiers of point-of-care and environmental monitoring 234
• 국문초록 236
• Acknowledgments 238