Facile surface modification of gold nanoparticles for closed-tube colorimetric detection of isothermal DNA amplification

Abstract

Gold nanoparticles (AuNPs) have attracted considerable interest for solution-phase colorimetric DNA detection. Two oligonucleotide-modified AuNP probes (each being complementary to half of a target sequence) are typically included. The two AuNP probes are dispersed in the absence of the target (appear red) while cross-linked/aggregated by the target (turn purple). The limit of detection (LOD) is in the nanomolar range, which is not practical for direct target detection (attomolar level). In view of this, efforts have been devoted to their integration with enzymatic DNA amplification. Despite the resulting high sensitivity, there are two main issues that limit their widespread use. The first one is associated with oligonucleotide-modified AuNPs that their preparation is laborious, time-consuming, and expensive. The second one is the high risk of carryover contamination. This is caused by the fact that the incorporation of AuNPs into an amplification reaction mixture would result in enzyme inhibition, thereby necessitating post-amplification open-tube addition of AuNPs. Our group developed two new AuNP probes, featuring facile and low-cost preparation, for closed-tube loop-mediated isothermal amplification (LAMP) assay: (1) 11-mercaptoundecanoic acid-modified AuNPs (MUA–AuNPs); (2) thiolated poly(ethylene glycol) and MUA co-modified AuNPs (PEG/MUA–AuNPs). In essence, magnesium ion (enzyme cofactor) in a negative sample (absence of the target) as well as magnesium pyrophosphate (LAMP reaction by-product) in a positive sample (presence of the target) resulted in different behaviors (dispersion, aggregation, or precipitation) of the AuNP probes. Our assay platforms possessed the advantages of cost-effective probe, simple temperature control, and ultrasensitive detection. They are readily applicable to decentralized DNA testing for various applications such as medical diagnostics, food safety control, and environmental surveillance.