Mimicking an in cellulo 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, such as HIV, in the same location the patient receives treatment. Communicable diseases 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 and capable of single molecule detection in cellulo. 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 in cellulo reaction in vitro 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.

Leveraging Arylboronic Acid - Cellulose Binding as a Versatile and Scalable Approach to Hydrophobic Patterning.

Paper-based analytical devices, or µPADs, have proven to be valuable bioanalytical tools for a broad range of applications. New methods for µPAD fabrication are needed, however, to facilitate their mass production at a competitive cost. To address this need, we report the use of a boronic acid-containing siloxane polymer (BorSilOx) for patterning hydrophobic barriers for µPADs. This material functions by covalently binding to hydroxyl groups in the paper substrate. It is compatible with inkjet printing or roll-to-roll (stamping) processes, as demonstrated here using three different deposition methods. BorSilOx is able to render a broad range of cellulosic materials (from paper towels to wood) hydrophobic, with contact angle measurements demonstrating superhydrophobicity in many cases. We further demonstrate the utility of the polymer in µPADs via assays for pH and glucose.