Switchable inhibitory behavior of divalent magnesium ion in DNA hybridization-based gene quantification.

Contrary to the understanding that divalent cations only result in under-estimation of gene quantification via DNA hybridization-based assays, we have discovered that Mg2+ could cause either under or over-estimation at different concentrations. Its switchable inhibitory behavior is likely due to its rigid first solvation (hydrated) shell and hence it is inclined to form non-direct binding with DNA. At low concentrations, it caused under-estimation by occupying the hybridization sites. At high concentrations, it caused probe, signaling and target DNA to aggregate non-specifically via Coulomb forces. By quantifying target DNAs at a range of Mg2+ concentrations using a gene quantification assay (NanoGene assay), a Mg2+ inflection concentration of ∼10-3 M was observed for both target ssDNA and dsDNA. Field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FT-IR) were employed to observe Mg2+-induced non-specific binding in the complexes that mimicked the presence of target DNA. Together with two other divalent cations Ca2+ and Cu2+, they were further examined via zeta potential measurements as well as NanoGene assay. This study revealed the importance of Mg2+ in achieving accurate gene quantification. Through a better mechanistic understanding of this phenomenon, it will be possible to develop strategies to mitigate the impact of Mg2+ on DNA hybridization-based gene quantification.

A simple reagent-less approach using electrical discharge as a substitution for chelating agent in addressing genomic assay inhibition by divalent cations.

Electrical discharge treatment was shown to be a viable substitution for chelating agent in genomic assays. Divalent cation Mg2+ inhibits the performance of DNA hybridization based genomic assays by binding to the DNA and disrupting DNA hybridization. Until now, chelating agents such as ethylenediaminetetraacetic acid (EDTA) was the only option to address the presence of Mg2+ in samples. However, EDTA is a well-known environmental contaminant. In this work, we successfully employed electrical discharge instead of EDTA to render Mg2+ insipid. Its preliminary efficacy was first observed via circular dichroism (CD) and zeta potential analyses. After electrical discharge treatment, the reduction in CD shift at 280 nm was significant for samples with 10-3 and 10-8 M Mg2+. The zeta potential of Mg2+ laden samples were also restored from -4.71 ± 1.38 to -20.59 ± 6.37 mV after electrical discharge treatment. Both CD shift and change in zeta potential suggested that 2 min of electrical discharge treatment could prevent Mg2+ from binding to DNA. The complete efficacy of electrical discharge treatment was demonstrated with the performance recovery (within ∼15% of the control) of a genomic assay variant (NanoGene assay) while analyzing Mg2+ laden samples (10-5-10-3 M). Assuming 10 million samples are analyzed annually, the proposed electrical discharge treatment (∼50 mW per sample) would allow us to trade environmental contamination by ∼50 kg of hazardous EDTA with a single 250 W STC (standard test conditions) solar panel.

A multi-functional reagent suitable for 1-step rapid DNA intercalation fluorescence-based screening of total bacteria in drinking water.

The prerequisites for rapid screening of total bacteria in drinking water are low detection limit and convenience. Inspired by commercial adenosine 5'-triphosphate (ATP) based total bacterial detection kits, we pursued likewise convenience but with much lower detection limit. Existing intercalation fluorescence-based techniques employ multiple reagents to permeate the cell membrane and intercalate dye into the DNA in discrete sequential steps. A simple multi-functional reagent is proposed to do the same within one step. Surfactants (TritonX and SDS), and intercalating dyes (SYBR green, SYBR gold) were examined for their mutual compatibility and augmented with EDTA. Evaluation was performed with Gram negative Escherichia coli K12 (E. coli K12) and Gram positive Bacillus subtilis (B. subtilis) at serial dilution ratios from 10-6 to 10-2. Comparison was made with absorbance (600 nm) measurements and a commercial ATP kit. Using charge integrated photodetection, the proposed 1-step reagent achieved an LOD (1.00 × 10-6, B. subtilis) that is two orders of magnitude lower than that of ATP kit (LOD = 1.06× 10-4). This means it could detect minute quantity of total bacteria that is otherwise undetected by the ATP kit.

Clustered Detection of Eleven Phthalic Acid Esters by Fluorescence of Graphene Quantum Dots Displaced from Gold Nanoparticles.

A gold nanoparticle-quenched graphene quantum dot-based aptasensor was developed to perform clustered detection of 11 phthalic acid esters (PAEs). The binding of the target PAEs to the aptasensor frees the graphene quantum dots that are otherwise quenched by the carrier gold nanoparticle. The resultant fluorescence upon excitation is proportional to the number of freed graphene quantum dots and hence the target PAE concentration. The synthesis of the proposed aptasensor was first verified step-by-step via FT-IR measurement, scanning electron microscopy, and fluorescence measurement. Selectivity was evaluated for individual and combined target PAEs and compared against seven non-PAE endocrine disrupting compounds. The proposed aptasensor successfully quantified 11 PAEs in test samples with varying concentrations of 0.001-50 ng PAEs/mL and demonstrated a limit of detection of ∼4 pg./mL. Finally, the AuNP-gQD aptasensor was employed to detect multiple combinations of commonly regulated PAEs (DBP, DIBP, DEHP, and BBP). The recovery (%) for all four PAEs combination in environmentally relevant concentrations of 0.5, 1, 5, and 10 ng/mL were ∼100%.