A disposable, integrated loop-mediated isothermal amplification cassette with thermally actuated valves
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An inexpensive, disposable, integrated, polymer-based cassette for loop-mediated isothermal amplification (LAMP) of target nucleic acids was designed, fabricated, and tested. The LAMP chamber was equipped with single-use, thermally actuated valves made with a composite consisting of a mixture of PDMS and expandable microspheres. The effect of the composite composition on its expansion was investigated, and the valve’s performance was evaluated. In its closed state, the valve can hold pressures as high as 200 kPa without any significant leakage. Both the LAMP chamber and the valves were actuated with thin film heaters. The utility of the cassette was demonstrated by carrying out LAMP of Escherichia coli DNA target and reverse transcribed loop meditated isothermal amplification (RT-LAMP) of RNA targets. The amplicons were detected in real time with a portable, compact detector. The system was capable of detecting as few as 10 target molecules per sample in well under 1 h. The portable, integrated cassette system described here is particularly suited for applications at the point of care and in resource-poor countries, where funds and trained personnel are in short supply.
KeywordsIntegrated microfluidic cassette Loop-mediated isothermal amplification (LAMP) Thermally actuated PDMS valves Escherichia coli Nucleic acid detection Real-time fluorescence quantification Expandable microsphere Thermal actuation
The work was supported by NIH/NIDCR Grant U01DE017855. Professor Abhay Vats from the Children’s Hospital of Pittsburgh provided useful advice and gifted template and primers for the Escherichia coli LAMP. Ms. Amanda Wynne Dyson machined the LAMP cassettes.
- Hatano B, Maki T, Obara T, Fukumoto H, Hagisawa K, Matsushita Y, Okutani A, Bazartseren B, Inoue S, Sata T, Katano H (2010) LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism. Jpn J Infect Dis 63(1):36–40Google Scholar
- Liu C, Geva E, Mauk M, Qiu X, Abrams WR, Malamud D, Curtis K, Owen M, Bau HH (2011) An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases. Analyst. doi: 10.1039/C1AN00007A
- Lutz S, Weber P, Focke M, Faltin B, Hoffmann J, Müller C, Mark D, Roth G, Munday P, Armes N, Piepenburg O, Zengerle R, von Stetten F (2010) Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA). Lab Chip 10(7):887–893CrossRefGoogle Scholar
- Mark JE (1999) Polymer data handbook. Oxford University Press, New YorkGoogle Scholar