Hybrid Integrated Microfluidic Device for Sample Preparation and qPCR on an EWD Platform

Abstract

The molecular diagnosis of blood infections is challenging because pathogens exist in the bloodstream in very low concentrations. Consequently, such sparse target populations require DNA extraction and purification from large volume biofluids. This work utilizes the advances in microfluidic technologies to demonstrate the nucleic acid purification - qPCR sequence detection workflow for this application. Immiscible Phase Filtration (IPF) for nucleic acid purification and Electrowetting-on Dielectric (EWD) droplet actuation are combined on a hybrid microfluidic device that translates from large volume sample-to-small-volume analysis. After IPF reduces the sample volume from a milliliter-sized lysate to a microliter-sized eluent, EWD can be used to automatically prepare the PCR mixture. This step begins with transporting droplets of the PCR reagents to mix with the eluent droplets. Suitable assays, microfluidic stages, and auxiliary systems are described. The extent of purification obtained per IPF wash, and hence the number of washes needed for uninhibited qPCR, are determined via on- chip UV absorbance. The performance of on-chip qPCR, particularly the copy number to threshold cycle correlation, is characterized. Lastly, the above developments accumulate to an experiment that includes the following on-chip steps: DNA purification by IPF, PCR mixture preparation via EWD, and target quantification using qPCR - thereby demonstrating the core procedures in the proposed approach.