Point-Of-Care System for Automated Genotyping: Monitoring Infectious
Abstract: Complete integration of the sample processing pipeline into a single system is of particular interest for potential point-of-care applications, especially in the area of in vitro diagnostics. Microfluidics is a promising technology platform as it can provide automated sample handling and reagent delivery as well as timely results with minimal operator intervention. We will discuss the development of a microfluidic system for DNA analyses capable of detecting single nucleotide polymorphisms (SNPs) in a package incorporating all of the instrument peripherals, including an ultrasensitive fluorescence imager. The entire DNA processing is carried out using a polymer-based microfluidic card, which consists of operational steps for cell sorting, cell lysis, solid-phase extraction of DNA, PCR amplification, discrimination reactions for identifying SNPs and readout using a low-density microarray. The microfluidic is manufactured as a 3-dimensional stack of 2 modules; a polycarbonate, PC, module used for sample and reagent loading, cell lysis, solid-phase extraction, PCR, and a ligase detection reaction; and a poly(methyl methacrylate), PMMA, module used for detection of the ligation products using microarray readout fitted with a monolithic laser coupling prism and air-embedded waveguide. Successful hybridization events indicative of the presence of a SNP was signaled by a fluorescence signature that was sampled using evanescent excitation generated from laser light launched into the air-embedded waveguide. As an application example, we will present results on the detection of mycobacterium tuberculosis (Mtb). Mtb is a global health threat responsible for approximately 2 million deaths annually. The rising problem of multi-drug resistant Mtb (MDR-Mtb) resulting from Mtb strains that fail to respond to first-line drugs such as rifampicin and isoniazid creates a formidable challenge in formulating global plans to eradicate this disease. Less than 5% of new MDR-Mtb cases occurring each year are diagnosed and treated in a timely fashion due to the length (~1 month) and complexity of traditional cell culture in vitro diagnostics. SNPs in the rpoB gene of Mtb’s were analyzed using our integrated system for determining their drug susceptibility directly from sputum samples. Results could be secured in less than 30 min compared to >12 h using conventional bench-top instrumentation.