Multiplex Analysis System Integrated with Air Sampler and DC Impedance-based Microfluidic Cytometer

다국어 초록 (Multilingual Abstract)

This dissertation is a study on the development of multiplex analytical platform equipped with air sampling device and microfluidic cytometer. There are the increasing demands for chemical analysis in everyday life and various field. In particular, multiplex assay has become important for point-of-care testing and biosurveillance because it enables a rapid, low-cost, and reliable quantification. In this work, I will demonstrate the use of a microfluidic cytometry platform based on DC impedance to conduct multiplex immunoassays and combine with air sampling.
First, DC impedance-based flow cytometry using virus-tethered gold microspheres was utilized for multiplex immunoassays. For common use of bead-based multiplex immunoassays, enhanced sensitivity, effective prevention of non-specific adsorption and miniaturization of the detection device are required. In this work, we have conducted multiplex immunoassay applications employing virus-tethered gold microspheres and a DC impedance-based microfluidic cytometer. The merits of virus-tethered gold microspheres are excellent prevention of non-specific adsorption in biological fluid and signal enhancement arising from the large quantity of antibody loading on each virus. Using these merits, a microfluidic chip-based flow cytometer detected DC impedance and fluorescence signals of virus-tethered gold microspheres for detection and quantification of biomarkers. This platform successfully realized multiplex immunoassays involving four biomarkers: cardiac troponin I (cTnI), prostate specific antigen (PSA), creatine kinase MB (CK-MB), and myoglobin in intact human sera, enhancing sensitivity by up to 5.7-fold compared to the gold microspheres without virus. Constructive integration between filamentous virus-tethered gold microspheres and use of a microfluidic cytometer suggests a promising strategy for multiplex immunoassay development based on bead-based immunoassays.
Second, DC impedance-based microfluidic cytometer was utilized for bioaerosol detection using wet-cyclone air sampler. We present a bioaerosol detection system consisting of a DC impedance based microfluidic cytometer and wet cyclone air sampler. Microbeads and bacteria E. coli are uniformly dispersed into an air chamber and collected by wet-cyclone air sampler. The collected liquid sample is transferred to the microfluidic cytometer using syringe pump and valve. Then, the microfluidic cytometer measured size and concentration of collected particles. The microfluidic cytometer was validated by analyzing the diameters of microbeads ranging from 0.96 to 2.95 µm and concentrations of microbeads ranging from 1 × 103 mL-1 to 1 × 107 mL-1. Simultaneous measurement of DC impedance and fluorescence using microfluidic cytometer implemented detection of collected particles and E. coli, demonstrating capability to measure concentration of particle similar to commercial instrument FACS. From these results, the detection strategy of airborne particle using microfluidic cytometer is established for biosurveillance.
The developed components show that the microfluidic-based cytometry platform is capable of multiplexing and measuring aerosols of bacteria by combination with air sampler. The developed components based on DC impedance microfluidic cytometry will be the cornerstone of the integrated total analytical platform with other microfluidic system.

목차 (Table of Contents)

• 1. Introduction & Background 1
• 1.1 Multiplex assay 1
• 1.2 Biosurveillance 2
• 1.3 Micro total analysis system (μTAS) 3
• 1.4 Multiplex Suspension Array 5
• 1.5 Flow Cytometry 6
• 1.6 Microfluidics-based Flow Cytometer 9
• 1.7 DC impedance-based microfluidic cytometer 10
• 1.8 Goals of study 13
• 2. Multiplex Immunoassays using Virus-Tethered Gold Microspheres by DC Impedance-based Flow Cytometry 14
• 2.1 Introduction 14
• 2.2. Experimental 18
• 2.2.1. Reagents and Apparatus 18
• 2.2.2. Production of filamentous phages with acceptor peptide (AP) 19
• 2.2.3. Optimization of azide conjugation 20
• 2.2.4. DBCO conjugation on microbead 21
• 2.2.5. Surface modification of Au-layered microspheres 21
• 2.2.6. Immunoassay 21
• 2.2.7. Fluorescence microscopy 22
• 2.2.8. Fabrication of a microfluidic chip 22
• 2.2.9. Chip-based microfluidic immunoassay 23
• 2.3. Result and Discussion 25
• 2.3.1. Characterization of virus-tethered Au-layered microspheres 25
• 2.3.2. Detection of specificity using DC impedance-based microfluidic cytometry 30
• 2.3.3. Signal-enhancement in virus-bead based immunoassay 35
• 2.3.4. Feasibility of multiplex immunoassay using Virus-Tethered Gold Microspheres by DC Impedance-based Microfluidic Cytometry 38
• 2.4. Conclusion 41
• 3. DC impedance-based microfluidic cytometer for bioaerosol detection using wet-cyclone air sampler 43
• 3.1. Introduction 43
• 3.1.1 Bioaerosol 43
• 3.1.2 Air sampling methods 44
• 3.1.3 Detection method of bioaerosols 44
• 3.1.4 Goal of study 45
• 3.2. Experimental 46
• 3.2.1. Bioaerosol generation and chamber 46
• 3.2.2. Airborne bacteria collection setup 47
• 3.2.3 Numerical analysis method 49
• 3.2.4. Fabrication of a microfluidic cytometer 50
• 3.2.5. Sample preparation 51
• 3.2.6. Microfluidic cytometry 51
• 3.3. Result and Discussion 53
• 3.3.1 Bioaerosol sampling operation 53
• 3.3.2 Device operation 58
• 3.3.3 Analysis of Microbead Suspension and Bacteria Suspension 60
• 3.3.4 Quantification of aerosolized microbead and bacteria 65
• 3.4. Conclusion 69
• 4. Summary and Perspective 70
• References 72
• 국문초록 80