A fully integrated slidable and valveless microdevice for mini y chromosome short tandem repeat genotyping

Abstract

A fully integrated slidable and valveless microsystem, which performs solid phase DNA extraction (SPE), micro-polymerase chain reaction (μPCR) and micro-capillary electrophoresis (μCE) coupled with a portable genetic analyser, has been developed for forensic genotyping. The use of a slidable chip (a 10×10 mm glass wafer), in which a 1 μL-volume of the PCR chamber was patterned at the center, does not necessitate any microvalves and external pneumatic actuators. The functional micro-units of SPE, PCR, and CE were individually fabricated on a single glass wafer by conventional photolithography. The integrated microdevice consists of three layers: from top to bottom, a slidable wafer, a channel wafer in which a SPE chamber, a weir structured microchannel, and CE microchannels were fabricated, and a Ti/Pt resistance temperature detector (RTD) wafer. The channel glass wafer and the RTD glass wafer were thermally bonded, and the slidable chip was placed on the designated functional unit and manually moved from one to another. Prior to the chip operation, silica beads were packed into the SPE chamber and the CE channel was filled with a linear polyacrylamide (LPA) gel. Whole human blood sample mixed with a lysis buffer was injected into the SPE unit for genomic DNA extraction. The purified DNA solution was mixed with a PCR cocktail which contained primer sets for typing amelogenin and four mini Y chromosomal short tandem repeat (STR) loci (DYS393, DYS389I/II, DYS390, and DYS439), and then the PCR-ready solution was loaded into the PCR chamber of a slidable chip. The slidable chip was moved to the PCR unit for thermal cycling to amplify STR loci, and then slid to the CE unit for the amplicon separation. Finally, the resultant amplicons were analysed by a miniaturized laser-induced fluorescence detector. Monoplex and multiplex detection of amelogenin and mini Y STR loci were successfully analysed on the integrated slidable SPE-μPCR-μCE microdevice by using 1 μL whole human blood within 60 min. Our proposal provides an advanced genetic analysis microsystem which is capable of point-of-care DNA testing with sample-in-answer-out capability, more importantly, without use of complicated microvalve, micropump, and microtubing systems for liquid transfer and consecutive operation of sample pretreatment, PCR, and CE.