Real-time multiplexed PCR using surface enhanced Raman spectroscopy in a thermoplastic chip

Surface enhanced Raman spectroscopy (SERS) has the potential to enable point-of-care sensing across the spectrum of chemical and biological analytes. In diagnostic assays, SERS has been demonstrated to increase the multiplexing density while reducing the burden of fluorescence hardware. One particular application of interest is the use of SERS to provide a multiplexed optical read-out following polymerase chain reaction (PCR). To date, however, the reported PCR-SERS assays require endpoint mixing with a plasmonic nanoparticle solution for detection, thus adding manual steps and preventing real-time, quantitative PCR. In this work, we detail a real-time PCR-SERS thermoplastic microsystem that allows simultaneous nucleic acid amplification and product separation into a SERS-active silver colloid for real-time detection. Specifically, a laser cut thermoplastic fluidic chip has been devised to utilize a dialysis membrane capable of isolating a PCR reaction from the silver colloid. As the reaction progresses, a Raman-reporter-labeled DNA probe is degraded, liberating the reporter from probe DNA, allowing passage across the size-restricting dialysis membrane into the SERS-active colloid, where the accumulating reporter can be measured in real time. Here, we demonstrate that this system is capable of real-time and single-well multiplexed readout of a PCR reaction to simultaneously detect two biomarker genes for methicillin-resistant S. aureus (MRSA).