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

Abstract

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. Here, we introduce Scalable On-site Nucleic Acid Testing Architecture (SONATA), enabling >100-chamber reaction partitioning for multiplexed nucleic acid amplification and detection with a portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized reagents and a small stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. We demonstrate the integration of the platform with Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based detection technology. We show that deployed with SONATA, SHINE retains its sensitivity, enabling highly multiplexed pathogen detection in [≤] 1 hour. In addition, we demonstrate the detection of single-nucleotide variants, including mutations associated with drug susceptibility.