Quantitative polymerase chain reaction (qPCR) renders profiling of genes of interest less time-consuming and cost-effective. Recently, multiplex profiling of miRNAs has enabled identifying or investigating predominant miRNAs for various diseases such as cancers and neurodegenerative diseases. Conventional multiplex qPCR technologies mostly use colorimetric measurements in solution phase, yet not only suffer from limited multiplexing capacity but also require target-screening processes due to non-specific binding between targets and primers. Here, we present hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease (AD). Our methodology promises two key advantages compared with the conventional solution-based PCR: 1) nearly no non-specific crosstalks between targets and primers, and 2) practically valuable multiplexing by spatial encoding within a single microchamber. Specifically, we immobilized hydrogel microposts (~ 400 µm in diameter) within commercially available polycarbonate PCR chips by multi-step ultraviolet (UV, 365 nm) exposure. We optimized this photoimmobilization for thermal cycles of PCR as well. Acrylated forward primers incorporated in polyethylene glycol diacrylate (PEGDA) posts played a crucial role to confine fluorescent signal of cDNA amplification within the PEGDA hydrogel. To demonstrate the potential of our platform, we successfully verified multiplex detection of five miRNAs, which were reported to be highly correlated with AD, from a complex buffer of human plasma.

定量聚合酶链反应（qPCR）使得感兴趣的基因谱分析耗时且成本低廉。最近，对miRNA进行多重分析已能够鉴定或研究各种疾病（例如癌症和神经退行性疾病）中的主要miRNA。传统的多重qPCR技术大多在溶液相中使用比色法测量，但不仅受多重能力的限制，而且由于靶标和引物之间的非特异性结合，还需要进行靶标筛选过程。在这里，我们提出基于水凝胶微柱的qPCR用于与阿尔茨海默氏病（AD）相关的miRNA的多重检测。与传统的基于溶液的PCR相比，我们的方法具有两个关键优势：1）靶标和引物之间几乎没有非特异性串扰，2）在单个微腔内通过空间编码进行实用上有价值的复用。具体来说，我们通过多步紫外线（UV，365 nm）曝光，将水凝胶微柱（直径约400 µm）固定在市售的聚碳酸酯PCR芯片中。我们还针对PCR的热循环优化了这种光固定化。纳入聚乙二醇二丙烯酸酯（PEGDA）柱中的丙烯酸正向引物在限制PEGDA水凝胶中cDNA扩增的荧光信号方面起着至关重要的作用。为了证明我们平台的潜力，我们成功地从人血浆的复杂缓冲液中成功验证了五个与miRNA高度相关的miRNA的多重检测。我们通过多步紫外线（UV，365 nm）曝光，将水凝胶微柱（直径约400 µm）固定在市售的聚碳酸酯PCR芯片中。我们还针对PCR的热循环优化了这种光固定化。纳入聚乙二醇二丙烯酸酯（PEGDA）柱中的丙烯酸正向引物在限制PEGDA水凝胶中cDNA扩增的荧光信号方面起着至关重要的作用。为了证明我们平台的潜力，我们成功地从人血浆的复杂缓冲液中成功验证了五个与miRNA高度相关的miRNA的多重检测。我们通过多步紫外线（UV，365 nm）曝光，将水凝胶微柱（直径约400 µm）固定在市售的聚碳酸酯PCR芯片中。我们还针对PCR的热循环优化了这种光固定化。纳入聚乙二醇二丙烯酸酯（PEGDA）柱中的丙烯酸正向引物在限制PEGDA水凝胶中cDNA扩增的荧光信号方面起着至关重要的作用。为了证明我们平台的潜力，我们成功地从人血浆的复杂缓冲液中成功验证了五个与miRNA高度相关的miRNA的多重检测。纳入聚乙二醇二丙烯酸酯（PEGDA）柱中的丙烯酸正向引物在限制PEGDA水凝胶中cDNA扩增的荧光信号方面起着至关重要的作用。为了证明我们平台的潜力，我们成功地从人血浆的复杂缓冲液中成功验证了五个与miRNA高度相关的miRNA的多重检测。纳入聚乙二醇二丙烯酸酯（PEGDA）柱中的丙烯酸正向引物在限制PEGDA水凝胶中cDNA扩增的荧光信号方面起着至关重要的作用。为了证明我们平台的潜力，我们成功地从人血浆的复杂缓冲液中成功验证了五个与miRNA高度相关的miRNA的多重检测。