Colorimetric detection-based centrifugal microfluidic platforms for molecular diagnostics

Abstract

Increasing attention to well-being and healthcare has fueled the development of innovative techniques for point-of-care testing of health-related factors. For example, enormous damage and casualties that are caused by out-breaks of contagious diseases related to pathogenic bacteria, viruses, and parasites have accelerated the industrial growth in the field of microfluidics-based molecular diagnostics. Recently, a number of microfluidic devices have been developed to fulfill the existent demands, and yet most of them remain incomplete in terms of accuracy, rapidity, portability and user-friendliness. As powerful alternatives to the previous devices, this thesis presents microfluidic devices which provide automated and multiplex foodborne pathogen detection in combination with a metal indicator-mediated colorimetric detection assay on, while taking advantages of centrifugal microfluidics which does not necessitate the use of external pumps, valves and complicated detection equip-ment for operation. Furthermore, by combining the centrifugal microdevices with solution-loading cartridges, real ‘full automation’ from sample handling to result-out was successfully accomplished. In Chapter 3, colorimetric loop-mediated isothermal amplification (LAMP) using Eriochrome Black T (EBT) for molecular diagnostics of foodborne pathogens is presented. Four kinds of pathogenic bacteria (Esche-richia coli O157:H7, Staphylococcus aureus, Salmonella Typhimurium and Vibrio parahaemolyticus) were selected as targets, and LAMP primer sets were designed to specifically amplify target genes under an isothermal condition. EBT, a magnesium ion indicator, was added in the reactors to facilitate colorimetric detection of LAMP products by the naked eye. EBT reveals different colors according to the concentration of magnesium ion in solutions. As the LAMP reaction proceeded, the color of the LAMP mixture turned from purple into sky blue, which validated the feasibility of using EBT as an indicator for LAMP reactions. Four target bacteria were successfully detected by the EBT-mediated colorimetric LAMP assay. In Chapter 4, a centrifugal LAMP microfluidic device for rapid, multiplex and colorimetric foodborne pathogen detection is presented. Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. Sequential loading and aliquoting of LAMP cocktails, primer mixtures, and DNA sample solutions were accomplished by optimized zig-zag-shaped microchannels and RPM control. Three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella Typhimurium and Vibrio parahaemolyticus) were targeted, and the amplicons of LAMP were detected by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7 was carried out, and the color change was observed with 380 copies of the target. The used primer set in the LAMP reaction was only specific to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive and high-throughput pathogen identification by the naked eye. The entire process was completed in 60 min. In Chapter 5, fully automated and colorimetric foodborne pathogen detection on an integrated cen-trifugal microfluidic device is described. All the processes for molecular diagnostics including sample pretreatment, DNA amplification and amplicon detection were integrated on a single disc. Silica microbeads incorporated in the disc enabled extraction and purification of bacterial genomic DNA from bacteria-contaminated milk samples. Four kinds of foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Vibrio parahaemolyticus and Listeria monocytogenes) were targeted and LAMP was performed to amplify the specific genes of the targets. Colorimetric detection mediated by EBT confirmed the results of the LAMP reactions with the color change of the LAMP mixtures from purple to sky blue. The whole process was conducted in an automated manner using the lab-on-a-disc and a miniaturized rotary instrument equipped with three heating blocks. This work demonstrated that a milk sample contaminated with foodborne pathogens can be automatically ana-lyzed on the centrifugal disc even at the 10 bacterial cell level in 65 min. These advanced centrifugal microsys-tems would provide an ideal genetic analysis platform that can be used even in resource-limited environments. In Chapter 6, a centrifugal microfluidic device was combined with solution-loading cartridges to ac-complish fully automated and user-friendly pathogen diagnostic system. Basically, this device could provide all experimental processes of molecular diagnostics including silica bead-based DNA extraction, isothermal DNA amplification by LAMP and EBT-mediated colorimetric detection. In addition, 3D-printed solution-loading car-tridges were employed to increase the sample handling capacity of the device and simplify the operation process. Since the solution-loading cartridge contains all essential components for molecular diagnostics and supplies them to the microdevice, the difficulty during the step of sample loading and injection into the device was dramatically simplified and user-friendly. We have successfully demonstrated that four kinds of foodborne pathogenic bacteria can be detected within 65 min based on the proposed microdevice, even when only $10^2$ of bacterial cells existed. This brand-new device would be a powerful tool in the field of point-of-care molecular diagnos-tics owing to total integration, automatic operation, and easy interpretation of the colorimetric results.