Next-generation pathogen detection: Exploring the power of nucleic acid amplification-free biosensors

Accurate, rapid, cost-effective, and point-of-care pathogen identification is crucial for public health safety and encompasses disease prevention, food safety, and environmental governance. Despite the current prevalent detection methods that enable the inspection of various pathogens, certain limitations necessitate resolution. For instance, the plate culture method is time- and labor-intensive. Conversely, antibody- and large-equipment-based detection methods expedite the detection. However, their advancement has been constrained by the availability of antibodies and the inconvenience of deploying large equipment for point-of-care testing (POCT). The development of nucleic acid amplification technology can address the aforementioned issues to a certain extent. However, challenges such as intricate primer design and dependency on thermal cycling persist. Therefore, nucleic acid amplification-free biosensors have garnered significant attention. Biosensors are broadly categorized based on the following tools: nanomaterials-based nucleic acid amplification-free biosensors, clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated nuclease (CRISPR/Cas)-based nucleic acid amplification-free biosensors, and nucleic acid amplification-free biosensors comprising nanomaterials and CRISPR/Cas. This review not only introduces biosensor performance and CRISPR/Cas applications, but also systematically elucidates the working principles and specific applications of nanomaterials and CRISPR/Cas in optimizing nucleic acid amplification-free detection systems, while judiciously categorizing them based on signal output modalities. Moreover, it discusses the challenges and prospects of nucleic acid amplification-free biosensors and provides valuable insights for further research.