SEMI-INTEGRATED SILICON-BASED MICRO QPCR DEVICE FOR RAPID ON-SITE DETECTION OF PATHOGENES

Infectious diseases, such as the recent case of coronavirus disease in 2019, have brought the prospect of point-of-care (POC) diagnostic testing into the spotlight. Rapid, accurate, low-cost, and easy-to-use testing in the field could stop epidemics before they develop into full-blown pandemics. For pathogen detection, polymerase chain reaction (PCR) is widely used and has been considered the gold standard in diagnostics. PCRs for POCs is still a research field in its infancy, therefore support for related research activities is essential. The main goal of this project is to develop a “Semi-integrated Silicon-based micro-qPCR device” for rapid on-site detection of pathogens. The research will focus on the development of a low thermal loss miniature and a time-responsive innovative ""double bridge"" structure of the thermal-cycler (thermocycler). Other key components of the PCR system such as the microfluidic chip for the enrichment of the analytical sample, the reaction chamber chip, and dedicated control electronics for thermocycler temperature control in connection with the fluorescence detection will be built around the designed thermocycler in such a way that the above-mentioned criteria of POC PCR analytical systems are met. In addition, we will study solutions to implement an optimal optical fluorescence detection system and develop control electronics and software to display the PCR test result on a PC or smartphone platform. In Sample Collection and Preparation, we will investigate two strategies for purifying and concentrating NA from samples to minimal elution volume employed for analysis in a microPCR device, i.e. the silica-coated magnetic beads and silicon/PDMS modified micropillars in a microfluidic chip. We will also investigate the most appropriate strategy for flow control in the microchannel of the designed microfluidic chip, with the priority of building the chip with passive microfluidic components. Our starting criteria for developing a reaction chamber are that it should be as small as possible to present minimal thermal load for thermocycler and that it should be disposable and cheap. Therefore, we propose the development of a small reaction chamber chip with a volume of a few mm3 built on a thin glass substrate, with a virtual reaction chamber based on hydrophobic/oleophobic surfaces, and, as an alternative, with a PDMS reservoir. Within that, we propose to develop a reaction chamber on a single chip as well as on a large glass substrate, which will enable the simultaneous fabrication of many reaction chamber chips, which is of great importance for the use of disposable reaction chambers.    Furthermore, we will include fluorescence detection in the system in order to validate the efficiency of the project-developed micro qPCR components and the system.  The enrichment efficiency of the sample and its nucleic acid amplification (NAA) in the developing qPCR will be evaluated by observing the fluorescence under a fluorescence microscope, with an additional imaging camera, and in a customized commercial fluorescence detection system, based on a single optical fiber system or another system whose optical part of the fluorescent detector will fit the thermocycler. The conducted work will ultimately lead to the development of an autonomous, portable qPCR system built on an innovative thermocycler with the following performance characteristics: droplet sample volume <1 µl, sensitivity of one copy, a dynamic range of 10 decades, temperature range from 5°C to 100 °C with accuracy ± 0.2 °C, heating and cooling rate between 10°C/s and 20 °C/s, which will enable PCR amplification of the sample in less than 20 min.