核酸盘培养:基于无标记和裸眼的机器学习水凝胶数字环介导等温扩增。

Nucleic Acid Plate Culture: Label-Free and Naked-Eye-Based Digital Loop-Mediated Isothermal Amplification in Hydrogel with Machine Learning.

机构信息

College of Biosystems Engineering and Food Science, State Key Laboratory of Fluid Power and Mechatronic Systems, Zhejiang University, Hangzhou 310058, China.

Institute of Analytical Chemistry, Department of Chemistry, Zhejiang University, Hangzhou 310058, China.

出版信息

ACS Sens. 2024 Apr 26;9(4):2010-2019. doi: 10.1021/acssensors.3c02807. Epub 2024 Apr 11.

Abstract

Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system (, 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.

摘要

数字核酸扩增能够实现单分子的绝对定量。然而,由于数字系统中的超小反应体积(,短光路),大多数数字系统仅限于荧光信号,而无标记和肉眼读取仍然具有挑战性。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。纳米限域环介导等温扩增与细菌培养一样简单,通过使用聚丙烯酰胺(PAM)水凝胶作为扩增基质来进行。PAM 水凝胶中离子聚合物链的纳米限域可以显著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。与在水溶液中相比,增强光散射特性的水凝胶中捕获的 MPP 可以用肉眼清晰地看到,形成白色“菌落”斑点,可以以无标记和无仪器的方式进行简单计数。MPP 还可以用智能手机拍摄,并通过机器学习算法自动计数,从而实现对各种实际样本中抗生素耐药病原体的绝对定量。

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