Amplified Single Base-Pair Mismatch Detection via Aggregation of Exonuclease-Sheared Gold Nanoparticles

Abstract

Single nucleotide polymorphism (SNP) detection is important for early diagnosis, clinical prognostics, and disease prevention, and a rapid and sensitive low-cost SNP detection assay would be valuable for resource-limited clinical settings. We present a simple platform that enables sensitive, naked-eye detection of SNPs with minimal reagent and equipment requirements at room temperature within 15 min. SNP detection is performed in a single tube with one set of DNA probe-modified gold nanoparticles (AuNPs), a single exonuclease (Exo III), and the target in question. Exo III’s apurinic endonucleolytic activity differentially processes hybrid duplexes between the AuNP-bound probe and DNA targets that are perfectly matched or contain a single-base mismatch. For perfectly matched targets, Exo III’s exonuclease activity facilitates a process of target recycling that rapidly shears DNA probes from the particles, generating an AuNP aggregation-induced color change, whereas no such change occurs for mismatched targets. This color change is easily observed with as little as 2 nM of target, 100-fold lower than the target concentration required for reliable naked eye observation with unmodified AuNPs in well-optimized reaction conditions. We further demonstrate that this system can effectively discriminate a range of different mismatches.

Gold Nanoparticles for In Vitro Diagnostics

“…An interesting finding by the authors is that the melting transition is extremely sharp, offering excellent selectivity of fully complementary DNA targets over those with one or more mismatches. 65 On the basis of a similar strategy, biorecognition element modified AuNPs have been successfully employed as colorimetric reporters for glucose, dopamine, glutathione (GSH), adenosine triphosphate (ATP), K + , etc. 16f The controlled loss of surface ligands can also be applied to construct colorimetric bioassays.…”

Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling

“…We have previously demonstrated that the apurinic endonuclease activity of Exo III can effectively enable EATR-mediated AuNP aggregation for rapid and sensitive detection of single-base-mismatched DNA. 6 Here, we showed that Exo III’s apurinic endonuclease activity could mediate CBSA-based EATR in solution as well as on particle surface. We thus believe that the highly active and robust apurinic endonuclease activity of Exo III should allow many DNA-based EATR sensor platforms to be applied to our CBSA-based EATR assays for detection of various small-molecule targets.…”

“…Thus, a substantial fluorescent signal can be generated by a single copy of the target though EATR, greatly decreasing the limit of detection. 3,5 Given that DNA can be easily modified with various signaling reporters 3,6–8 and that different nucleases can be utilized to process the target–probe duplex, 3,5,7,9 many EATR-based assays have been developed for DNA detection with ultrahigh sensitivity. 4 …”

Colorimetric biosensor using dual‐amplification of enzyme‐free reaction through universal hybridization chain reaction system

“…Nucleic acid test (NAT) is a technique for diagnosing genotypes by detecting the presence, quantity, and mutation of nucleotide sequences using the Watson‐Crick interaction and DNA replication. NAT can be utilized to detect single‐nucleotide polymorphism (SNP) specifically (Eom et al, ; Wu et al, ) or to detect microRNA (miRNA) using noninvasive sampling (Ge et al, ; Yang et al, ; Ying, Ju, Sun et al, ). Also, pathogens including virus can be detected for the prevention of infectious diseases (Guo et al, ; Yu et al, ).…”