Kramer Sijbren, Song Ryungeun, Huang Yujia, Hong Soonwoo, Motlani Ibrahim, Stone Howard A, Myhrvold Cameron
Department of Molecular Biology, Princeton University, Princeton, New Jersey, 08544, USA.
Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, 08544, USA.
bioRxiv. 2025 Aug 12:2025.08.11.669785. doi: 10.1101/2025.08.11.669785.
Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. During the COVID-19 pandemic, we developed Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based technology for nucleic acid detection. Here, we introduce microfluidic SHINE (mSHINE) which enables >100-plex nucleic acid detection using a highly portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized SHINE reagents and a microscopic stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. A subsequent sealing step allows for >100 SHINE reactions to proceed independently and in parallel. We demonstrate that mSHINE has equal sensitivity to SHINE, allowing for highly multiplexed pathogen detection in ≤ 1 hour. In addition, mSHINE can detect single-nucleotide variants, including mutations associated with drug susceptibility. mSHINE shifts the paradigm of laboratory-based multiplexed nucleic acid testing, greatly benefiting patients and public health.
核酸检测的多重方法在实验室环境之外的部署极具挑战性。相反，可现场部署的方法仅限于低水平的多重检测。在新冠疫情期间，我们开发了用于疫情防控的感染简易检测技术（SHINE），这是一种基于CRISPR的灵敏且可部署的核酸检测技术。在此，我们介绍微流控SHINE（mSHINE），它使用高度便携的微流控通道实现了100重以上的核酸检测。该通道将稀释后的样本导入各个反应室，每个反应室都含有冻干的SHINE试剂以及用于混合的微型搅拌棒或珠子。样本可以通过手动注射器加载，极大地简化了检测过程。随后的密封步骤允许100多个SHINE反应独立且并行地进行。我们证明mSHINE与SHINE具有同等的灵敏度，能够在1小时内实现高度多重的病原体检测。此外，mSHINE可以检测单核苷酸变体,包括与药物敏感性相关的突变。mSHINE改变了基于实验室的多重核酸检测模式，极大地造福了患者和公众健康。
