A handheld microfluidic manifold for massively multiplexed CRISPR-based nucleic acid detection

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.