Reinholt, Sarah J. and Behrent, Arne and Greene, Cassandra and Kalfe, Ayten and Baeumner, Antje J. (2014) Isolation and Amplification of mRNA within a Simple Microfluidic Lab on a Chip. ANALYTICAL CHEMISTRY, 86 (1). pp. 849-856. ISSN 0003-2700, 1520-6882
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The major modules for realizing molecular biological assays in a micrototal analysis system (mu TAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)(25), and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 1015 fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | SEQUENCE-BASED AMPLIFICATION; CRYPTOSPORIDIUM-PARVUM; NUCLEIC-ACIDS; ANALYSIS SYSTEMS; PMMA-MICROCHIP; DNA; PURIFICATION; DEVICE; BIOSENSOR; DIAGNOSTICS; |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institut für Analytische Chemie, Chemo- und Biosensorik > Chemo- und Biosensorik (Prof. Antje J. Bäumner, formerly Prof. Wolfbeis) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 28 Nov 2019 10:31 |
| Last Modified: | 28 Nov 2019 10:31 |
| URI: | https://pred.uni-regensburg.de/id/eprint/10831 |
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