A microbead-incorporated centrifugal sample pretreatment microdevice.

Abstract

In this study, we present a novel bead-incorporated centrifugal sample pretreatment microdevice to purify influenza A H3N2 viral RNA. Simple revolution per minute (RPM) control can lead to RNA capture on a bead-bed, and the sequential loading of a washing solution and an elution solution. Tetraethoxy orthosilicate (TEOS)-treated glass microbeads were utilized as a capture matrix. The sample pretreatment microdevice consists of four reservoirs for storing an RNA sample, a washing solution, an elution solution, and a collected sample, and they were merged at the microbead-bed microchannel. The washing solution reservoir and the elution solution reservoir were connected to the bead-bed microchannel through a capillary valve and a siphon channel, respectively. An RNA sample (a lysed influenza A H3N2 virus), a washing solution (70% ethanol) and an elution solution (water or a reverse transcription-polymerase chain reaction (RT-PCR) cocktail) were loaded into the designated reservoirs, and they were successively transported to the bead-bed by RPM control owing to the optimized channel design. Purified RNAs could be obtained in 440 s. Then, a target H3 gene was amplified by an off-chip based real-time RT-PCR to evaluate the capture efficiency of RNA on our proposed microdevice. 81% of RNAs were successfully captured and purified. Interestingly, the use of the RT-PCR cocktail itself as an elution solution resulted in a 76% capture yield. Furthermore, we successfully performed RNA purification from the clinical nasopharyngeal swabs to identify the subtype of the influenza A virus. This platform provides high potential for the direct integration of the sample pretreatment microdevice into the downstream micro-PCR unit to create a total genetic analysis microsystem.