Single-Step Nucleic Acid Assay for On-Site Molecular Diagnostics

Nucleic acid biosensors often require ultrasensitivity due to the low abundance of target in biofluids. In this context, Quantitative polymerase chain reaction (qPCR) is widely regarded as the gold standard. However, such ultrasensitivity resulted in requiring skilled experts and equipment for sample preparation and nucleic acid amplification, restricting its application to centralized labs. Recent advancements have focused on simplifying and streamlining the procedures within the devices, enabling frequent and decentralized testing. However, the current approach requires expensive equipment with complex designs, which still poses a hurdle for point-of-care testing. In this study, we suggest a nature-inspired CRISPR-based system design as an sample preparation-free and enzymatic amplification-free approach to facilitate point-of-care testing. Since the whole assay occurs in a single-step reaction, the detection was very rapid while maintaining the high sensitivity of CRISPR-based detection (sub-femtomolar). Moreover, the assay operates at room temperature. These advancements significantly reduce assay complexity and allow for a straightforward design of a smartphone-based fluorescence reader, enhancing its accessibility and distribution.