Colorimetric detection of single base-pair mismatches based on the interactions of PNA and PNA/DNA complexes with unmodified gold nanoparticles

Abstract

Rapid and sensitive single nucleotide polymorphisms (SNPs) genotyping is of particular important for early diagnosis, prevention, and treatment of specific human diseases. A simple and low-cost SNP detection method would be valuable for routine analysis in resource-limited settings. Here, we demonstrated a novel and convenient gold nanoparticle (AuNPs) based colorimetric approach for efficient screening of SNPs at room temperature without instrumentation. SNP detection is performed in a single tube with one set of unmodified AuNPs, a label-free peptide nucleic acid (PNA) probe, a single exonuclease (S1 nuclease), and the target to be tested. S1 nuclease could digest DNAs in DNA/PNA duplexes involving a mismatch into small fragments, while DNAs in the fully-matched DNA/PNA duplexes can be effectively protected by PNA from enzymatic degradation. This difference could be easily discriminated by color changes associated with gold aggregation. PNA oligomers can induce immediate AuNP aggregation even in the presence of nucleoside monophosphates (dNMPs), the digestion products of DNA. Whereas PNA/DNA duplexes can effectively stabilize unmodified AuNPs, and the stabilization effect of PNA/DNA is better than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Without the need of precise temperature control and extra salt addition, SNPs are detected with a detection limit of 2.3 nM in cell lysate. Moreover, this system can effectively discriminate a range of different mismatches even in spiked cell lysate, demonstrate the potential use of this biosensor for biological samples.