Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
Institute of Applied Physics, Vienna University of Technology, 1040 Vienna, Austria.
Biosens Bioelectron. 2016 Apr 15;78:1-6. doi: 10.1016/j.bios.2015.11.013. Epub 2015 Nov 10.
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 1h on-chip hybridization.
在这里，我们报告了一种用于转录和扩增自由检测 DNA 和 RNA 分子的设备的开发，其检测下限可达zepto-mole 范围。带有内置微阵列的微流控芯片用于处理纳升级样品体积。通过双重杂交方法实现了目标分子的特异性染色和固定，从而避免了由于逆转录和 PCR 扩增等酶促过程引起的偏差。因此，目标分子通过预杂交与互补的 Cy5 标记探针间接标记。每个目标分子的剩余单链部分随后可以与微阵列的互补捕获探针杂交。因此，发生了靶介导的标记 DNA 固定。通过超灵敏荧光读出，可以检测到与微阵列杂交的所有分子。最小化的样品体积和单分子检测的组合在芯片上杂交 1 小时后，对靶 DNA 的检测限为 39 fM（35.4 nl 测定体积中 831 个分子），对靶 RNA 的检测限为 16 fM（338 个分子）。
