Closure of Loop-Mediated Isothermal Amplification Chamber on Lab-On-Chip Using Thermal-Responsive Valve

Abstract

Point-of-care tests for nucleic acids are important for the diagnosis and management of infectious and genetic diseases, biowarfare agents, and for drug research. Recent integration of loop-mediated isothermal amplification (LAMP) onto a lab-on-chip (LOC) platform allows sensitive and specific, detection of target DNA or RNA sequences for point-of-care (POC) applications. However, because of the high risk of contamination to LAMP products, robust and simple methods of hermetically sealing the reaction chamber are essential. In addition, having a method for isolation of nucleic acids at the level of the chip is more effective for POC applications because a laboratory setting is not required to complete the analysis. This report describes an integrated, polymer-based cassette which was designed for detection of DNA/RNA target-sequences by using LAMP and a solid-state nucleic acid purification membrane. The LAMP chamber seals using a self-actuated thermalresponsive valve made from expandable microspheres suspended in Polydimethylsiloxane (PDMS). A flow-through, Flinders Technology Associates membrane (Whatman FT A®) was installed on the cassette with the expectation of providing isolation and purification of nucleic acids. The cassette was designed, fabricated and the efficacy of the valve to seal the LAMP chamber was investigated. The valve underwent expansion and held pressures of233 +/- 5.0 kPa without signs of leakage. A portable, battery-powered, polyimide-based thin film heater, placed outside the cassette, provided thermal control. In addition, a LAMP primer set was designed for the serotonin receptor 5HTRIA promoter gene using Primer Explorer V 4 software (http:/ /primerexplorer.jp/elamp4.0.0/index.html) for LAMP detection of neurotransmitter serotonin. The integrated, portable LAMP cassette will be attractive in global health-care challenges, mainly in resource-poor locations, where the availability of laboratory equipment and/or trained personal is restricted.