The accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest. In this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed. Based on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 1010 copies/μL, (1.53 ± 0.10) × 1010 copies/μL and (1.70 ± 0.11) × 1010 copies/μL respectively.

Pb(II) can cause serious damaging effects to human health, and thus, the study of Pb²⁺ detection methods to sensitively and selectively monitor Pb(II) pollution has significant importance. In this work, we have developed a label-free fluorescence sensing strategy based on a Pb(II) DNAzyme cleavage and the ThT/G-quadruplex complex. In the presence of Pb(II), a G-rich tail was cut and released from the substrate strand, which then would form a G-quadruplex structure by combination with ThT dye. The fluorescence signal increase was then measured for sensitive Pb(II) quantification with a limit of detection of 0.06 nM. Our sensor also demonstrated high selectivity against six different metal ions, which is very important for the analysis of complex samples.

We report here a practical application of a multiplexed electrochemical DNA sensor for highly specific single-nucleotide polymorphism (SNP) detection. In this work, a 16-electrode array was applied with an oligonucleotide-incorporated nonfouling surfaces (ONS) on each electrode for the resistance of unspecific absorption. The fully matched target DNA templated the ligation between the capture probe assembled on gold electrodes and the tandem signal probe with a biotin moiety, which could be transduced to peroxidase-based catalyzed amperometric signals. A mutant site (T93G) in uidA gene of E. coli was analyzed in PCR amplicons. 10% percentage of single mismatched mutant gene was detected, which clearly proved the selectivity of the multiplexed electrochemical DNA biosensor when practically applied.

PolyA-TDF enables PCR-free qualitative detection of drug-resistant genes, integrating biosensor recognition and probe immobilization via strong adsorption to gold.