【研究成果】2012

研究成果42

Detection of influenza virus using a lateral flow immunoassay for amplified DNA by a microfluidic RT-PCR chip
Naoki Nagatani, Keiichiro Yamanaka, Hiromi Ushijima, Ritsuko Koketsu, Tadahiro Sasaki, Kazuyoshi Ikuta, Masato Saito, Toshiro Miyahara and Eiichi Tamiya
Analyst, 2012, 137, 3422

Influenza virus RNA was amplified by a continuous-flow polydimethylsiloxane microfluidic RT-PCR chip within 15–20 min. The amplified influenza virus RNA was observed with the naked eye, as the red color at the test line, using a lateral flow immunoassay within 1 min. The amplified DNA from influenza virus RNA, using a fluorescein isothiocyanate　(FITC)　labelled primer and biotinylated primer, by the microfluidic continuous-flow　reverse transcription PCR (RT-PCR)　chip was detected by　amplified DNA detection lateral flow immunoassay　(ADLFIA). The sensitivity of　ADLFIA is comparable to that of agarose gel electrophoresis. The detection by　ADLIFA needs only the naked eye.　 ADLFIA will be appended to a conjugate pad containing a dried　excess volume of biotin antibody-labelled gold nanoparticles for　a one-step assay. The conjugate pad appending ADLFIA can integrate　easily into the solution exuding from the outlet of the chip. The　integration enables the easy detection by ADLFIA without a mixing　step.　We are continuing our effort towards a battery powered portable　PCR chip, and a battery powered portable PCR chip with ADLFIA　has the potential to give the portable system a diagnostic test.

Schematic representation of the detection of the influenza virus using the continuous-flow RT-PCR chip and ADLFIA. Influenza virus RNA　(target RNA) is amplified by the continuous-flow RT-PCR chip (52 x 76 mm2) using an FITC labelled and biotinylated primer set. The amplified　DNA is　mixed with biotin antibodies labelled with gold nanoparticles. The size of the ADLFIA strip is 4 x 40 mm2. FITC antibody and mouse IgG antibody are　immobilized on the test and control line, respectively. The mixture is absorbed for ADLFIA, the amplified DNA complex with biotin antibody-labelled　gold nanoparticles is captured by the FITC antibody at the test line. Finally, a red color appears as the result of accumulation of gold nanoparticles.