Mimicking a Cellular Crowding Environment for Enzyme-Free Paper-Based Nucleic Acid Tests at the Point of Care

Point of care (PoC) nucleic acid amplification tests (NAATs) are a cornerstone of public health, providing the earliest and most accurate diagnostic method for many communicable diseases in the same location where the patient receives treatment. Communicable diseases, such as human immunodeficiency virus (HIV), disproportionately impact low-resource communities where NAATs are often unobtainable due to the resource-intensive enzymes that drive the tests. Enzyme-free nucleic acid detection methods, such as hybridization chain reaction (HCR), use DNA secondary structures for self-driven amplification schemes, producing large DNA nanostructures, capable of single-molecule detection <i>in cellulo</i>. These thermodynamically driven DNA-based tests have struggled to penetrate the PoC diagnostic field due to their inadequate limits of detection or complex workflows. Here, we present a proof-of-concept NAAT that combines HCR-based amplification of a target nucleic acid sequence with paper-based nucleic acid filtration and enrichment capable of detecting sub-pM levels of synthetic DNA. We reconstruct the favorable hybridization conditions of an <i>in cellulo</i> reaction <i>in vitro</i> by incubating HCR in an evaporating, microvolume environment containing poly(ethylene glycol) as a crowding agent. We demonstrate that the kinetics and thermodynamics of DNA-DNA and DNA-RNA hybridization is enhanced by the dynamic evaporating environment and inclusion of crowding agents, bringing HCR closer to meeting PoC NAAT needs.