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

Journal: ACS sensors
PMID: 38602267

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.

Authors

  • Mei Fang
    College of Biosystems Engineering and Food Science, State Key Laboratory of Fluid Power and Mechatronic Systems, Zhejiang University, Hangzhou 310058, China.
  • Yiru Wang
    College of Biosystems Engineering and Food Science, State Key Laboratory of Fluid Power and Mechatronic Systems, Zhejiang University, Hangzhou 310058, China.
  • Tao Yang
    The First Clinical Medical College, The Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China.
  • Jing Zhang
    MOEMIL Laboratory, School of Optoelectronic Information, University of Electronic Science and Technology of China, Chengdu, China.
  • Hanry Yu
    Department of Physiology, The Institute for Digital Medicine (WisDM), Yong Loo Lin School of Medicine, MD9-04-11, 2 Medical Drive, Singapore, 117593, Singapore; Institute of Bioengineering and Bioimaging, A*STAR, The Nanos, #06-01, 31 Biopolis Way, Singapore, 138669, Singapore; CAMP, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, Level 4 Enterprise Wing, Singapore, 138602, Singapore; Mechanobiology Institute, National University of Singapore, T-Lab, #05-01, 5A Engineering Drive 1, Singapore, 117411, Singapore; Ants Innovate Pte. Ltd., 7 Temasek Boulevard #12-07, Suntec Tower One, Singapore, 038987, Singapore; Lead Contact, Singapore. Electronic address: phsyuh@nus.edu.sg.
  • Zisheng Luo
    College of Biosystems Engineering and Food Science, State Key Laboratory of Fluid Power and Mechatronic Systems, Zhejiang University, Hangzhou 310058, China.
  • Bin Su
    Department of Chemistry, Cleveland State University, USA.
  • Xingyu Lin
    College of Biosystems Engineering and Food Science, State Key Laboratory of Fluid Power and Mechatronic Systems, Zhejiang University, Hangzhou 310058, China.

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