Digital polymerase chain reaction system for point-of-care testing of infectious disease
- 주제어 (키워드) Polymerase chain reaction , Point-of-care testing , Plasemonic heating , Multiplexed detection , Pandemic disease
- 발행기관 서강대학교 일반대학원
- 지도교수 정봉근
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 박사
- 학과 및 전공 일반대학원 기계공학과
- 실제URI http://www.dcollection.net/handler/sogang/000000076702
- UCI I804:11029-000000076702
- 본문언어 영어
- 저작권 서강대학교 논문은 저작권 보호를 받습니다.
초록
The proposed thesis aims to establish a digital polymerase chain reaction (dPCR) system for on-site testing of infectious disease. The dPCR has been attracting a great deal of attention in the biomedical field, as it offers an absolute quantification of target molecules with excellent specificity. Despite the advancements in dPCR technology, its practical application in point- of-care testing (POCT), particularly in resource-limited settings, remains a challenge. This research addresses this issue by conceiving a novel heating mechanism, leveraging the capabilities of the Internet of Things (IoT), and introducing an alternative amplification method. The initial objective of the research involves the design and optimization of a digital PCR system based on plasmonic heating. This innovative heating element, fabricated with a thin gold layer and a blue LED, facilitates efficient light-to-heat conversion. Due to its unique properties, the system outperforms the conventional PCR platforms in heating efficiency. Its potency is demonstrated through the successful detection of a wide range of DNA concentrations. Combined with polydimethylsiloxane (PDMS) microwell membrane, the system enables rapid amplification and precise quantification of the target DNA, achieving exceptional sensitivity and linearity. Additionally, the compactness and low power consumption of the system make it a potentially transformative tool for applications in resource-constrained settings. The second objective is to build an IoT-enabled dPCR system for effective on-site detection of infectious diseases. This highly integrated system performs the entire process of dPCR analysis, involving sample digitization, DNA amplification, fluorescence detection, and quantitative analysis. Utilizing a self-priming microfluidic chip, the system is capable of detecting nine RNA groups related to three infectious diseases in a single operation. A custom-developed smartphone application, built on an IoT platform, facilitates automatic processing, data collection, and cloud storage. To further demonstrate its efficacy, the system is validated using SARS-CoV-2 RNA samples and the quantification performance is compared with a commercially available real-time PCR system. Lastly, a novel multiplexed droplet digital recombinase polymerase amplification method is proposed. Leveraging a droplet-based microfluidic chip, this method enables an accurate initiation of the amplification process and allows the assay to be completed within 30 minutes. The assay demonstrates a multiplexing capability, successfully detecting different types of human coronavirus with remarkable specificity.
more목차
I. Chapter 1. Introduction 1
A. Literature Review 1
B. Scope of Thesis 5
II. Chapter 2. Plasmonic heating-based portable digital PCR system for rapid detection of influenza virus 7
A. Introduction 7
B. Materials and Methods 10
C. Results and Discussion 14
D. Conclusion 32
III. Chapter 3. IoT-integrated multiplex digital PCR system for on-site diagnosis of pandemic viruses 34
A. Introduction 34
B. Materials and Methods 37
C. Results and Discussion 41
D. Conclusion 60
IV. Chapter 4. Droplet-based digital isothermal amplification for quantitative detection of SARS-CoV-2 RNA 61
A. Introduction 61
B. Materials and Methods 64
C. Results and Discussion 68
D. Conclusion 84
References 85
