Naked-Eye Detection of LAMP-Produced Nucleic Acids in Saliva Using Chitosan-Capped AuNPs in a Single-Tube Assay.

Loop-mediated isothermal amplification (LAMP) is a low-technology molecular assay that is highly adaptable to point-of-care (POC) applications. However, achieving sensitive naked-eye detection of the amplified target in a crude sample is challenging. Herein, we report a simple yet highly efficient and sensitive methodology for the colorimetric visualization of a single target copy in saliva using chitosan-capped gold nanoparticles (Chit-AuNPs) synthesized via a green chemistry approach. The presence or absence of free Chit in the Chit-AuNPs solution was shown to affect LAMP colorimetric detection oppositely: the observed stabilization in the negative samples and aggregation in the positive samples in the presence of free Chit were reversed in the case of neat Chit-AuNPs. The mechanism of the two assays was investigated and attributed to electrostatic and depletion effects exerted between the Chit-AuNPs, free Chit, and the solution components. The developed contamination-free, one-tube assay successfully amplified and detected down to 1-5 cfu of and 10 copies of SARS-CoV-2 per reaction (25 μL) used, respectively, as model DNA and RNA targets in the presence of 20% saliva, making the method suitable for POC applications. Compared to the commonly used pH-sensitive dyes, Chit-AuNPs are shown to have an enhanced sensitivity toward naked-eye colorimetric observation owing to the direct detection of DNA amplicons. Thus, this is a simple, highly sensitive, fast, and versatile naked-eye detection methodology that could be coupled to any LAMP or RT-LAMP assay, avoiding the need of using complicated sample pretreatments and/or AuNPs long and laborious functionalization processes.