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Development of a biosensing system for the analysis of live microbial and animal cells

  • 이경원 (KYUNG WON LEE, 아주대학교 일반대학원)

초록/요약

As interest in human health continues to grow, there is an increasing demand for biosensing systems capable of analyzing living microbial and whole animal cells. These systems are being developed to provide a more comprehensive understanding of biological activity at the fundamental unit of life, with the goal of improving personalized healthcare. The biosensing process follows a sequence in which bioreceptor probes recognize target substances, convert them into a signal interpretable by humans through a transducer, and display the signal on a screen. The system can analyze small molecules, such as metabolites, nucleic acids, and proteins, as well as the basic units of life, including microorganisms and animal cells. However, while systems for measuring small molecules have been miniaturized and made user-friendly, systems for directly analyzing living cells still require specialized personnel and large equipment. To address this challenge, I developed a user-friendly biosensor capable of highly sensitive protein analysis, demonstrating the feasibility of sensing cell components using simple assay methods. Time-resolved fluorescence (TRF) technology was integrated with the lateral flow immunoassay method to enable highly sensitive analysis of cardiac troponin I, a protein component of cardiac cells, through a simple sensing process. Raspberry-type TRF microparticles were immobilized onto the lateral flow immunoassay strip as fluorescence donors to form the test and control lines with TRF properties. Gold nanorods, which have fluorescence quenching ability, were applied as fluorescence acceptors to induce the Förster resonance energy transfer (FRET) quenching effect. This approach leverages the TR-FRET quenching phenomenon induced by the antigen-antibody reaction between Raspberry-type TRF microparticles and GNRs while maintaining user-friendliness with lateral flow assay methods. The strip was analyzed using a newly developed biosensing platform capable of precisely detecting time-resolved fluorescence signals. The system demonstrated excellent performance, achieving a limit of detection (LOD) of 0.97 ng/mL for cardiac troponin I in serum samples. Next, a biosensing system for living microorganisms was developed using a retroreflective Janus microparticle and a specially designed sensing chip, enabling analysis with simple non-spectroscopic optics. This approach enhances accessibility, making the system suitable for use in inspection sites within the food distribution chain and other fields at risk of microorganism contamination. I designed the biosensing chip with divided areas: one where microorganisms are captured and one where they allow free movement. The motility of microorganisms affects their aggregation with retroreflective microparticles, which can be observed using non-spectroscopic optics. As the concentration of microorganisms in the sample increases, the difference between the two areas becomes more pronounced. I could detect microorganisms in food samples down to 1 CFU/mL by applying an algorithm to quantify this difference. Finally, the application of retroreflective Janus microparticles was expanded to track the dynamics of live animal cells, specifically focusing on the effects of arginyl-glycyl-aspartic acid (RGP) peptide variants, considered competitive drugs on integrin-mediated adhesion and migration. By combining 3D cell culture micropatterned substrates with a non-spectroscopic optical analysis system, the platform enabled long-term, noninvasive monitoring of cell positions and behaviors, providing a robust tool for evaluating therapeutic molecules. To control and, more precisely, track cell migration, I applied a micropatterned substrate that allowed cells to attach and move in a defined manner. I then developed a system that uses retroreflective particles, which can attach to the surface of cells, to track particle movement along the cell migration path. This system demonstrated that the degree of cell migration, influenced by the integrin affinity of the drug, could be monitored by analyzing the movement patterns using a custom algorithm. Based on these findings, I successfully developed versatile biosensing systems that utilize microparticles and optical setups to analyze cell component proteins, live microbial, and whole animal cells. These systems enhance the detection of various target substances at the fundamental unit of life and provide a more comprehensive understanding of biological activity.

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목차

Chapter I. General Introduction 1
1.1 Biosensor 2
1.2 The transducers of biosensors 5
1.3 The target analytes of biosensor 8
1.4 The demand of live microbial and animal cell biosensor 14
1.5 Objectives of thesis 15
1.6 References 18
Chapter II. Development of cell-composition protein biosensor by integrating raspberry-type fluorescence microparticles and lateral flow immunoassay 19
Abstract 20
2.1 Introduction 21
2.2 Experimental Section 24
2.2.1 Materials and apparatus 24
2.2.2 Preparation of Eu-SiNP 24
2.2.3 Characterization of Eu-SiNP 25
2.2.4 Preparation of protein-conjugated particles 26
2.2.5 Quenching test between Eu-SiNP and GNR 27
2.2.6 Synthesis and characterization of Eu-raspberry particle 28
2.2.7 Preparing the LFIA strip 28
2.2.8 Calibration study for cTnI sensing 29
2.3 Results and Discussion 31
2.3.1 Sensing principle of TR-FRET-based LFIA for cTnI detection 31
2.3.2 Characterization of the Eu-SiNP 32
2.3.3 Confirmation of quenching effect between Eu-SiNP and GNR 35
2.3.4 Design and confirmation of the Eu-raspberry particle 37
2.3.5 Confirmation of the developed LFIA system 40
2.3.6 Calibration study for cTnI determination 42
2.4 References 45
Chapter III. Non-spectroscopic microbial biosensor utilizing Janus-microparticle-induced aggregation phenomenon for signal registration 47
Abstract 48
3.1 Introduction 49
3.2 Experimental Section 52
3.2.1 Materials and apparatus 52
3.2.2 Salmonella sample preparation and enrichment 52
3.2.3 Fabrication of the sensing chip 53
3.2.4 Fabrication of the optical probes 55
3.2.5 Quantitative analysis of the live Salmonella cells 55
3.2.6 qPCR assay for performance comparison of the developed sensing chip 56
3.2.7 Application of the developed system for the spiked food samples 56
3.3 Results and Discussion 58
3.3.1 Principle of the RJP-based bacterial live cell quantification system 58
3.3.2 Result image analysis procedure 60
3.3.3 Performance of the developed sensing system 63
3.3.4 Feasibility of the developed sensing system 65
3.4 References 67
Chapter IV. Biosensor for whole animal cell monitoring using micropatterned culture substrates and retroreflective optical signaling 68
Abstract 69
4.1 Introduction 70
4.2 Experimental Section 74
4.2.1 Materials and apparatus 74
4.2.2 Fabrication of micropatterned 3D cell culture substrate 74
4.2.3 Preparation of non-spectroscopic optical probe 77
4.2.4 Cell viability test 77
4.2.5 Qualitative observation of integrin inhibition effect using RGD variations 78
4.2.6 Quantification study of integrin inhibition effect using various substances 80
4.3 Results and Discussions 81
4.3.1 Construction of substrate for cell adhesion and migration evaluation 81
4.3.2 Fabrication of non-spectroscopic optical probes for cell movement 83
4.3.3 Integration of culture substrate and non-spectroscopic optical system 86
4.3.4 Observation of cell adhesion and migration effect by RGD variations 89
4.3.5 Quantitative analysis of integrin inhibitory effects using image processing methodology 93
4.3.6 Selectivity test with cell-affecting substances 97
4.4 References 99
Chapter V. Conclusions and Perspectives 104
5.1 Conclusions 105
5.2 Perspectives 106
국문요약 107

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