Mimicking a Cellular\nCrowding Environment for Enzyme-Free\nPaper-Based Nucleic Acid Tests at the Point of Care

Point of care (PoC) nucleic acid amplification tests\n(NAATs) are\na cornerstone of public health, providing the earliest and most accurate\ndiagnostic method for many communicable diseases in the same location\nwhere the patient receives treatment. Communicable diseases, such\nas human immunodeficiency virus (HIV), disproportionately impact low-resource\ncommunities where NAATs are often unobtainable due to the resource-intensive\nenzymes that drive the tests. Enzyme-free nucleic acid detection methods,\nsuch as hybridization chain reaction (HCR), use DNA secondary structures\nfor self-driven amplification schemes, producing large DNA nanostructures,\ncapable of single-molecule detection <i>in cellulo</i>.\nThese thermodynamically driven DNA-based tests have struggled to penetrate\nthe PoC diagnostic field due to their inadequate limits of detection\nor complex workflows. Here, we present a proof-of-concept NAAT that\ncombines HCR-based amplification of a target nucleic acid sequence\nwith paper-based nucleic acid filtration and enrichment capable of\ndetecting sub-pM levels of synthetic DNA. We reconstruct the favorable\nhybridization conditions of an <i>in cellulo</i> reaction <i>in vitro</i> by incubating HCR in an evaporating, microvolume\nenvironment containing poly(ethylene glycol) as a crowding agent.\nWe demonstrate that the kinetics and thermodynamics of DNA–DNA\nand DNA–RNA hybridization is enhanced by the dynamic evaporating\nenvironment and inclusion of crowding agents, bringing HCR closer\nto meeting PoC NAAT needs.