An Integrated Array-based Microfluidic Device for Parallel Loop-Mediated Isothermal Amplification (LAMP)

Abstract

Nucleic-based acid technology (NAT) is a reliable and well-established method in molecular diagnosis for the detection of bacterial infection. Specifically, PCR (polymerase chain reaction) is the most popular technique to amplify the number of DNA or RNA copies in the sample. However, due to the thermal cycles in the PCR method, advanced equipment and technologies are required to precisely control the temperature during the cycles. To overcome this limitation, isothermal amplification methods have been developed which function at constant temperatures and help reduce the need for state-of-the-art machines to perform the amplification. Among isothermal amplification methods, LAMP (loop mediated isothermal amplification) has demonstrated robustness and sensitivity compared to PCR. Additionally, microfluidic lab-on-a-chip (LOC) technology can facilitate the intensive processes which have been used traditionally in laboratories by automating the required procedures, reducing the volume of the reagents and minimizing the cost and the time of experiments. Although many microfluidic LOC devices have been developed in order to be used in resource poor settings, there is still a need for a simple setup which is inexpensive, accurate and can be performed without the need for a trained technician. In this thesis, a disposable microfluidic device was developed which is capable of performing high-throughput DNA amplification by using a simple segmentation method in order to digitize the sample into multiple micro-wells. Moreover, design and fabrication of a disposable, inexpensive flexible heater which is an inevitable part of the setup using a direct write process was introduced in order to provide the required energy for the LAMP reaction. Parallel real-time DNA amplification with limit of detection down to few copies per micro-well in less than an hour was illustrated. Using E. coli 0157, it was demonstrated that the detection time of E.coli can be as quick as 11 to 55 minutes with sample concentrations varying from 700,000 copies/micro-well (11 minutes), 70,000 copies/micro-well (18 minutes), 700 copies/micro-well (31 minutes), 7 copies/micro-well (40 minutes) and 0.07 copies/micro-well (55 minutes). Finally, the capability of the device for on chip reagent storage up to 3 days without using any coating methods was illustrated.