低丰度、小尺寸和序列相似性使得微小RNA (miRNA) 的检测具有挑战性，特别是在真实样本中，由于更丰富分子的干扰，量化弱表达的miRNA可能会变得困难。标准的定量逆转录聚合酶链反应 (qRT-PCR) 需要多个步骤、热循环和昂贵的酶反应，这些反应可能会对结果产生负面影响。在这里，我们介绍一种基于微胶粒子结合分子信标 (MB) 的直接、精确、无酶测定方法，能够光学检测真实样本中的低丰度miRNA。我们使用 qRT-PCR 作为参考技术来验证微胶测定法的适用性。作为一个相关案例，我们选择了miR-103-3p，这是一种对于乳腺癌有价值的诊断生物标记物，在血清样本和MCF7细胞中都可以使用。结果，微胶测定法可以在室温下的单步操作中定量miRNA分子，用时1小时(相比于qRT-PCR的4小时)，不需要互补DNA合成、扩增或昂贵试剂。微胶测定法展示了飞摩尔级别的灵敏度、单核苷酸特异性以及广泛的线性范围(102-107 fM)(比qRT-PCR更宽)，具有低样品消耗(2 μL)和优秀的线性(R2= 0.98)。为了测试微胶测定法在真实样本中的选择性，考虑到MCF7细胞，研究了另外8种miRNA池在miRNA 103-3p方面的相对调节。在这样复杂的环境中，微胶测定法能够选择性地检测到miRNA靶点，主要是由于MB高级稳定性和特异性以及微胶的高抗污染性能造成的。这些结果显示了微胶测定法在真实样本中检测miRNA的可靠性。版权所有©2023年作者。由Elsevier B.V.出版。保留所有权利。

Low abundance, small size, and sequence similarities render microRNA (miRNAs) detection challenging, particularly in real samples, where quantifying weakly expressed miRNAs can be arduous due to interference of more abundant molecules. The standard quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires multiple steps, thermal cycles, and costly enzymatic reactions that can negatively affect results. Here we present a direct, precise, enzyme-free assay based on microgels particles conjugating molecular beacons (MB) capable of optically detecting low abundant miRNAs in real samples. We validate the applicability of microgels assay using qRT-PCR as a reference technology. As a relevant case, we chose miR-103-3p, a valuable diagnostic biomarker for breast cancer, both in serum samples and MCF7 cells. As a result, microgels assay quantifies miRNA molecules at room temperature in a single step, 1 h (vs. 4 hrs for qRT-PCR) without complementary DNA synthesis, amplification, or expensive reagents. Microgels assay exhibits femtomolar sensitivity, single nucleotide specificity, and a wide linear range (102-107 fM) (wider than qRT-PCR), with low sample consumption (2 μL) and excellent linearity (R2= 0.98). To test the selectivity of the microgel assay in real samples, MCF7 cells were considered where the pool of 8 other miRNAs were further upregulated with respect to miRNA 103-3p. In such complex environments, microgels assay selectively detects the miRNA target, mainly due to MB advanced stability and specificity as well as high microgel antifouling properties. These results show the reliability of microgels assay to detect miRNAs in real samples.Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.