(A) study on the development of point-of-caretesting devices for pathogen and biomarkers detection

다국어 초록 (Multilingual Abstract)

As the demand for personalized healthcare and rapid diagnostics continues to grow, the development of integrated point-of-care testing (POCT) platforms capable of performing real-time, on-site analysis has become increasingly critical. This thesis focuses on the design and implementation of two advanced POCT systems for both indirect and direct health monitoring, addressing key challenges associated with field-deployable diagnostics.
For indirect health monitoring, which includes the detection of pathogens in food matrices, a tunable magnetic capturing cartridge (TMCC) was developed for the rapid and quantitative detection of Staphylococcus aureus from large-volume food samples without the need for time-consuming enrichment steps. Magnetophoretic concentration was employed to process large sample volumes, while a dielectrophoresis-based separation device was utilized to remove unbound probes and food particles, thereby improving detection accuracy. The TMCC enabled efficient bacterial capture and imaging for quantitative analysis using a custom portable fluorescence reader. A sandwich immunoassay format was employed to ensure high specificity and sensitivity. The platform demonstrated superior performance compared to standard methods.
For direct health monitoring, a self-driven microfluidic immunoassay cartridge was developed to enable fully integrated, pump-free biomarker detection using a compact, capillary flow-based design. All reagents required for the immunoassay were preloaded into the cartridge, allowing sample-to-answer operation without external equipment. Magnetic nanoparticles were employed as a movable substrate, and quantum dots were used as fluorescent tags to enhance sensitivity and signal amplification. A dedicated washing zone was incorporated to improve the precision of target quantification by removing the unbound probes, and the use of a glass fiber membrane for probe loading simplified fabrication and ensured assay reproducibility.
By addressing critical limitations in current POCT systems, such as insufficient sensitivity, external actuation requirements, reagent instability, and matrix interference, this work presents robust, field-ready solutions for both clinical diagnostics and food safety monitoring. The integrated platforms developed in this study represent a significant step toward achieving portable, low-cost, and highly accurate POCT systems, opening new possibilities for widespread deployment in preventive healthcare and real-time monitoring applications.

국문 초록 (Abstract)

개인 맞춤형 의료 및 신속 진단에 대한 수요가 계속 증가함에 따라, 실시간 현장 분석이 가능한 통합 현장 현시 검사(POCT) 플랫폼의 개발이 점점 더 중요해지고 있습니다. 본 논문은 간접 및 직접 건강 모니터링을 위한 두 가지 첨단 POCT 시스템의 설계 및 구현에 초점을 맞추고 있으며, 현장 배치 가능 진단과 관련된 주요 과제를 해결합니다.
식품 매트릭스 내 병원균 검출을 포함하는 간접 건강 모니터링을 위해, 시간이 많이 소요되는 농축 단계 없이 대용량 식품 샘플에서 황색포도상구균을 신속하고 정량적으로 검출하기 위한 가변 자기 포획 카트리지(TMCC)가 개발되었습니다. 자기영동 농축법은 대용량 샘플을 처리하는 데 사용되었으며, 유전영동 기반 분리 장치는 결합되지 않은 프로브와 식품 입자를 제거하여 검출 정확도를 향상시키는 데 활용되었습니다. TMCC는 맞춤형 휴대용 형광 판독기를 사용하여 정량 분석을 위한 효율적인 박테리아 포획 및 이미징을 가능하게 했습니다. 높은 특이도와 민감도를 보장하기 위해 샌드위치 면역분석법 형식이 사용되었습니다. 이 플랫폼은 표준 방법과 비교하여 우수한 성능을 보여주었습니다.
직접 건강 모니터링을 위해, 소형의 모세관 유동 기반 설계를 사용하여 완전히 통합된 펌프 없는 바이오마커 검출을 가능하게 하는 자가 구동 미세유체 면역분석 카트리지가 개발되었습니다. 면역분석에 필요한 모든 시약은 카트리지에 미리 탑재되어 외부 장비 없이 시료 주입부터 결과 분석까지의 작동을 가능하게 합니다. 자기 나노입자는 이동 가능한 기질로 사용되었고, 양자점은 민감도와 신호 증폭을 향상시키기 위한 형광 태그로 사용되었습니다. 결합되지 않은 프로브를 제거하여 표적 정량화의 정밀도를 향상시키기 위해 전용 세척 구역이 통합되었으며, 프로브 로딩을 위한 유리 섬유 멤브레인의 사용은 제작을 단순화하고 분석 재현성을 보장했습니다.
불충분한 민감도, 외부 구동 장치 요구사항, 시약 불안정성 및 매트릭스 간섭과 같은 현재 POCT 시스템의 주요 한계를 해결함으로써, 본 연구는 임상 진단 및 식품 안전 모니터링 모두를 위한 견고하고 현장 적용 가능한 솔루션을 제시합니다. 본 연구에서 개발된 통합 플랫폼은 휴대 가능하고 저렴하며 매우 정확한 POCT 시스템을 구현하는 데 있어 중요한 진전을 나타내며, 예방적 의료 및 실시간 모니터링 분야에서의 광범위한 배치를 위한 새로운 가능성을 엽니다.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1.1 Point-of-care testing Technology (POCT) 1
• 1.2 POCT Device Architecture and Integration 5
• 1.2.1 Lab-on-a-chip Systems 5
• 1.2.2 Microfluidics-based POCT 5
• 1.2.3 Integrated POC Cartridges 7
• 1.3 Detection Techniques in POCT Devices 10
• 1.3.1 Electrochemical Sensing 10
• 1.3.2 Colorimetric Assays 10
• 1.3.3 Chemiluminescence Assays 10
• 1.3.4 Fluorescence-Based Detection 11
• 1.3.5 Digital fluorescence-based POCT 11
• 1.4 Detection Modalities in POCT: Direct and Indirect health monitoring 14
• 1.4.1 Direct health monitoring 14
• 1.4.2 Indirect health monitoring 14
• 1.5 Immunoassay and its integration in POCT 17
• 1.5.1 Direct immunoassay 17
• 1.5.2 Indirect immunoassay 17
• 1.5.3 Competitive immunoassay 17
• 1.5.4 Sandwich immunoassay 18
• 1.6 Integration of nanoparticles in POCT 18
• 1.6.1 Properties and benefits of NPs in POCT 19
• 1.6.2 Fluorescence-based NPs in POCT 20
• 1.6.3 Magnetic NPs in POCT 20
• 1.7 Literature review 21
• 1.7.1 Use of POCT in indirect health monitoring 21
• 1.7.2 Use of POCT in direct health monitoring 22
• 1.8 Shortcomings of current platforms 25
• 1.9 Overview of the thesis 27
• Chapter 2. Culture-Free Quantification of Bacteria Using Digital Fluorescence Imaging in a Tunable Magnetic Capturing Cartridge for Onsite Food Testing 29
• Abstract 29
• 2.1 Introduction 31
• 2.2 Experimental Section 36
• 2.2.1 Chemical and Sample Preparation 36
• 2.2.2 Sample preparation for imaging and counting 36
• 2.2.3 Materials, design, and fabrication of the TMCC 38
• 2.2.4 Digital fluorescence imaging system hardware and software 39
• 2.3 Results and Discussion 40
• 2.3.1 Design of the TMCC and bacterial detection strategy 40
• 2.3.2 Control of capture time in the TMCC 44
• 2.3.3 Design and fabrication of an automated, highly portable fluorescence reader 53
• 2.3.4 Statistical evaluation and accuracy of the developed system 56
• 2.3.5 Operational efficiency of bacterial detection from real food samples 62
• 2.4 Summary 65
• Chapter 3. A self-driven microfluidic immunoassay cartridge with magnetic nanoparticles for high-precision fluorescence-based quantification 67
• Abstract 67
• 3.1 Introduction 68
• 3.2 Experimental Section 72
• 3.2.1 Chemical and reagents 72
• 3.2.2 Probe modifications 72
• 3.2.3 GFM treatment for loading of Abs 73
• 3.2.4 Fabrication of SDMIC 74
• 3.2.5 Dimensions of the SDMIC 78
• 3.2.6 Digital imaging and post-processing 78
• 3.2.7 COMSOL simulation 78
• 3.2.8 Machine Learning 79
• 3.3 Results and Discussion 81
• 3.3.1 Design and working principle of SDMIC 81
• 3.3.2 Concept of washing and bubble trapping on SDMIC 84
• 3.3.3 Fabrication of SDMIC 89
• 3.3.4 Characterization of flow time, release of MNPs, and capturing efficiency in the artridge 92
• 3.3.5 Proof of concept of off-the-cartridge measurements 97
• 3.3.6 Optimization and quantification of different CRP concentrations in the cartridge 104
• 3.3.7 Integration of Machine Learning into the SDMIC platform 110
• 3.4 Summary 116
• Chapter 4. Conclusion and Future Perspectives 117
• References 120
• 논문요약 161