Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system (i.e., short light path), most digital systems are limited to fluorescence signals, while label-free and naked-eye readout remain challenging. In this work, we report a digital nucleic acid plate culture method for label-free, ultrasimple, and naked-eye nucleic acid analysis. As simple as the bacteria culture, the nanoconfined digital loop-mediated isothermal amplification was performed by using polyacrylamide (PAM) hydrogel as the amplification matrix. The nanoconfinement of PAM hydrogel with an ionic polymer chain can remarkably accelerate the amplification of target nucleic acids and the growth of inorganic byproducts, namely, magnesium pyrophosphate particles (MPPs). Compared to that in aqueous solutions, MPPs trapped in the hydrogel with enhanced light scattering characteristics are clearly visible to the naked eye, forming white “colony” spots that can be simply counted in a label-free and instrument-free manner. The MPPs can also be photographed by a smartphone and automatically counted by a machine-learning algorithm to realize the absolute quantification of antibiotic-resistant pathogens in diverse real samples.

中文翻译：

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核酸平板培养：利用机器学习在水凝胶中进行无标记、基于肉眼的数字环路介导的等温扩增

数字核酸扩增能够对单个分子进行绝对定量。然而，由于数字系统中的反应体积超小（即光路短），大多数数字系统仅限于荧光信号，而无标记和肉眼读出仍然具有挑战性。在这项工作中，我们报告了一种用于无标记、超简单和肉眼核酸分析的数字核酸平板培养方法。与细菌培养一样简单，使用聚丙烯酰胺（PAM）水凝胶作为扩增基质进行纳米限制数字环介导的等温扩增。具有离子聚合物链的 PAM 水凝胶的纳米限制可以显着加速目标核酸的扩增和无机副产物，即焦磷酸镁颗粒（MPP）的生长。与水溶液中的MPP相比，捕获在水凝胶中的MPP具有增强的光散射特性，肉眼清晰可见，形成白色“菌落”斑点，可以通过无标记和无仪器的方式简单地计数。 MPP还可以通过智能手机拍照并通过机器学习算法自动计数，以实现对不同真实样本中抗生素耐药病原体的绝对定量。