We report on the development of a microfluidic multiplexing technology for highly parallelized sample analysis via quantitative polymerase chain reaction (PCR) in an array of 96 nanoliter-scale microcavities made from silicon. This PCR array technology features fully automatable aliquoting microfluidics, a robust sample compartmentalization up to temperatures of 95 °C, and an application-specific prestorage of reagents within the 25 nl microcavities. The here presented hybrid silicon–polymer microfluidic chip allows both a rapid thermal cycling of the liquid compartments and a real-time fluorescence read-out for a tracking of the individual amplification reactions taking place inside the microcavities. We demonstrate that the technology provides very low reagent carryover of prestored reagents < 6 × 10⁻² and a cross talk rate < 1 × 10⁻³ per PCR cycle, which facilitate a multi-targeted sample analysis via geometric multiplexing. Furthermore, we apply this PCR array technology to introduce a novel digital PCR-based DNA quantification method: by taking the assay-specific amplification characteristics like the limit of detection into account, the method allows for an absolute gene target quantification by means of a statistical analysis of the amplification results.

我们报告了微流体多重技术的开发，该技术可通过定量聚合酶链式反应 (PCR) 在硅制成的 96 纳升级微腔阵列中进行高度并行的样品分析。该 PCR 阵列技术具有全自动等分微流体、高达 95 °C 温度的强大样品区室以及 25 nl 微腔内特定应用试剂的预储存。这里提出的混合硅聚合物微流控芯片允许液体隔室的快速热循环和实时荧光读数，以跟踪微腔内发生的各个扩增反应。我们证明，该技术在每个 PCR 循环中提供预存试剂 < 6 × 10 ^-2的极低试剂残留和串扰率 < 1 × 10 ^-3 ，这有助于通过几何多重进行多目标样品分析。此外，我们应用这种 PCR 阵列技术引入了一种新型的基于数字 PCR 的 DNA 定量方法：通过考虑特定于检测的扩增特性（如检测限），该方法允许通过统计方法进行绝对基因目标定量。分析扩增结果。