A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules

作者：Mayr Reinhard; Haider Michaela; Thuenauer Roland; Haselgruebler Thomas; Schuetz Gerhard J; Sonnleitner Alois; Hesse Jan^*

来源：Biosensors and Bioelectronics, 2016, 78: 1-6.

DOI：10.1016/j.bios.2015.11.013

• Ultra-sensitive detection
• DNA
• RNA
• Microarray
• Microfluidic biochip
• Lab-on-a-chip

Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1 h on-chip hybridization.

• 出版日期2016-4-15