Naked-Eye Detection of LAMP-Produced Nucleic Acids in Saliva using Chitosan-capped AuNPs in a Single-Tube Assay

Abstract

Loop-mediated isothermal amplification (LAMP) is a low-technology molecular assay 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 was 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 Salmonella and 10 copies of SARS-CoV-2 per reaction (25 uL) 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 the 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.