ABSTRACT
The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.
Subject(s)
Microfluidics , Nucleic Acid Amplification Techniques , DNA/genetics , RNA/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A sheathless interface making use of a porous tip has been used for coupling capillary electrophoresis and electrospray ionization mass spectrometry. First, effective flow rates using the interface have been characterized. It was found that the interface is capable of generating a stable spray with flow rates ranging from below 10 nL/min to >340 nL/min, enabling its use in either the mass or concentration-sensitive region of the electrospray process. Subsequently, by analyzing peptide mixtures of increasing complexity, we have demonstrated that this platform provides exquisite sensitivity enabling the detection of very low amounts of materials with very high resolving power. Transient isotachophoresis (t-ITP) can also be integrated with this setup to increase the mass loading of the system while maintaining peak efficiency and resolution. Concentration limits of detection in the subnanomolar or nanomolar range can be achieved with or without t-ITP, respectively. The application of a vacuum at the inlet of the separation capillary further allowed the peak capacity of the system to be improved while also enhancing its efficiency. As a final step in this study, it was demonstrated that the intrinsic properties of the interface allows the use of coated noncharged surfaces so that very high peak capacities can be achieved.
