Integrated microdevice of reverse transcription-polymerase chain reaction with colorimetric immunochromatographic detection for rapid gene expression analysis of influenza A H1N1 virus - PubMed Skip to main page content
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. 2012 Mar 15;33(1):88-94.
doi: 10.1016/j.bios.2011.12.024. Epub 2012 Jan 8.

Integrated microdevice of reverse transcription-polymerase chain reaction with colorimetric immunochromatographic detection for rapid gene expression analysis of influenza A H1N1 virus

Affiliations

Integrated microdevice of reverse transcription-polymerase chain reaction with colorimetric immunochromatographic detection for rapid gene expression analysis of influenza A H1N1 virus

Yong Tae Kim et al. Biosens Bioelectron. .

Abstract

An integrated microdevice of a reverse transcription-polymerase chain reaction (RT-PCR) reactor and an immunochromatographic strip was constructed for colorimetric detection of gene expression of influenza A virus subtype H1N1. An RT-PCR cocktail, which included Texas Red-labeled primers, dNTP including biotin-labeled dUTP, and RNA templates of influenza A H1N1 virus, was filled in the PCR chamber through the micropump, and the RT-PCR was performed to amplify the target H1 gene (102 bp). The resultant amplicons bearing biotin moieties and Texas Red haptens were subsequently eluted to the immunochromatographic strip, in which they were first conjugated with the gold nanoparticle labeled anti-hapten antibody in the conjugation pad, and then captured on the streptavidin coated test line through the biotin-streptavidin interaction. By observing a violet color in the test line which was derived from the gold nanoparticle, we confirmed the H1N1 target virus. The entire process on the integrated microdevice consisting of a micropump, a 2 μL PCR chamber, and an immunochromatographic strip was carried out on the portable genetic analyzer within 2.5h, enabling on-site colorimetric pathogen identification with detection sensitivity of 14.1 pg RNA templates.

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Figures

Fig. 1
Fig. 1
(A) The structure of an ICS. The reaction mechanism on the ICS: (B) The RT-PCR product bearing the biotin and Texas Red hapten was loaded in the conjugate pad to be linked with Au NP labeled anti-hapten antibodies. (C) The Au NP labeled RT-PCR amplicons were moved to the nitrocellulose membrane by adding a buffer in the buffer loading pad. (D) The product was captured by streptavidin on the test line, while the excess of the Au NP labeled anti-hapten antibodies were immobilized on the anti-mouse IgG in the control line.
Fig. 2
Fig. 2
(A) Exploded view of five layers for constructing an integrated RT-PCR-ICS microdevice: from bottom to top, an ICS, a RTD wafer, a channel wafer, a monolithic PDMS membrane, and a glass manifold. (B) Schematics of an integrated RT-PCR-ICS microdevice: a pneumatic micropump (orange), a PCR chamber (green), a microfabricated RTD (yellow), a PDMS microvalve (violet), and an ICS (grey). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3
Fig. 3
(A) Digital image of the integrated RT-PCR-ICS microdevice on the portable genetic analyzer. (B) Schematics of the portable genetic analyzer which consists of power supplies, a diaphragm pump, solenoid valves, and an electronic control board. (C) Thermal cycling profile with cooling ramping rate of 5.7 °C/s and heating ramping rate of 4.4 °C/s.
Fig. 4
Fig. 4
Optimization of an ICS based colorimetric detection by tuning the length of the spacer between biotin and dUTP and the concentration ratio of dTTP and biotin–dUTP. The carbon number of the spacer was (A) 11, (B) 16, and (C) 18. The ratio of dTTP:biotin–dUTP was 1:0, 2:1, 4:1 and 8:1.
Fig. 5
Fig. 5
Detection sensitivity comparsion between the ICS (top panel) and the agarose gel electrophoresis (bottom panel) by using the serially diluted viral RNA templates from 1.72 ng to 17.2 fg. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 6
Fig. 6
On-chip RT-PCR reaction and ICS based colorimetric detection on the portable genetic analyzer for H1N1 virus identification by using RNA templates of 1.41 ng, 0.141 ng, 0.0141 ng and 0 ng from left to right. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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References

    1. Beyor N., Seo T.S., Liu P., Mathies R.A. Biomed. Microdevices. 2008;10:909–917. - PubMed
    1. Beyor N., Yi L., Seo T.S., Mathies R.A. Anal. Chem. 2009;81:3523–3528. - PMC - PubMed
    1. Blazej R.G., Kumaresan P., Mathies R.A. Proc. Natl. Acad. Sci. U.S.A. 2006;103:7240–7245. - PMC - PubMed
    1. Blažková M., Javurková B., Fuka L., Rauch P. Biosens. Bioelectron. 2011;26:2828–2834. - PubMed
    1. Chen D., Mauk M., Qiu X., Liu C., Kim J., Pamprasad S., Ongagna S., Abrams W.R., Malamud D., Corstjens P.L.A.M., Bau H.H. Biomed. Microdevices. 2010;12:705–723. - PMC - PubMed

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