Selection of G-rich ssDNA aptamers for the detection of enterotoxins of the cholera toxin family.

Cholera and milder diarrheal disease are caused by Vibrio cholerae and enterotoxigenic Escherichia coli and are still a prominent public health concern. Evaluation of suspicious isolates is essential for the rapid containment of acute diarrhea outbreaks or prevention of epidemic cholera. Existing detection techniques require expensive equipment, trained personnel and are time-consuming. Antibody-based methods are also available, but cost and stability issues can limit their applications for point-of-care testing. This study focused on the selection of single stranded DNA aptamers as simpler, more stable and more cost-effective alternatives to antibodies for the co-detection of AB5 toxins secreted by enterobacteria causing acute diarrheal infections. Cholera toxin and Escherichia coli heat-labile enterotoxin, the key toxigenicity biomarkers of these bacteria, were immobilized on magnetic beads and were used in a SELEX-based selection strategy. This led to the enrichment of sequences with a high % GC content and a dominant G-rich motif as revealed by Next Generation Sequencing. Enriched sequences were confirmed to fold into G-quadruplex structures and the binding of one of the most abundant candidates to the two enterotoxins was confirmed. Ongoing work is focused on the development of monitoring tools for potential environmental surveillance of epidemic cholera and milder diarrheal disease.

Bismuth Film-Coated Gold Ultramicroelectrode Array for Simultaneous Quantification of Pb(II) and Cd(II) by Square Wave Anodic Stripping Voltammetry.

The widespread presence of heavy metals in drinking water sources arises as a major health concern, particularly in developing countries. The development of low-cost and reliable detection techniques is identified as a societal need to provide affordable water quality control. Herein, a bismuth film-coated gold ultramicroelectrode array (BF-UMEA) was used for the detection of Pb(II) and Cd(II) in water samples via square wave anodic stripping voltammetry (SWASV). Experimental parameters such as deposition time, Bi(III) concentration, acetate buffer concentration, pH, square wave frequency, amplitude, and step potential were all varied to determine their effects on the current peak intensities of the target metal ions. Ten-fold excess in the concentration of interferences was found to cause a decrease in the stripping peak areas of Cd(II) and Pb(II) in the following order of magnitude: benzene < NaCl < Ni(II) < Cu(II). Using Box-Behnken design, the optimum SWASV parameters that provided maximum current peak areas were 14.76 Hz (frequency), 50.10 mV (amplitude), and 8.76 mV (step potential). The limits of detection of the as-prepared BF-UMEA were 5 and 7 µg L-1 for Pb(II) and Cd(II), respectively. These results demonstrate the potential use of a BF-UMEA in SWASV for the trace quantification of Pb(II) and Cd(II) in water samples.

Aptamer Selection against a Trichomonas vaginalis Adhesion Protein for Diagnostic Applications.

Trichomoniasis, caused by Trichomonas vaginalis, is the leading nonviral sexually transmitted infection worldwide. We report the selection of a DNA aptamer against a T. vaginalis adhesion protein, AP65, using a microtiter plate-based in vitro combinatorial chemistry process termed systematic evolution of ligands by exponential enrichment. The enriched library pool was sequenced by next-generation sequencing, and several aptamer candidates with high affinity and specificity were identified. The aptamer with the highest affinity and specificity had a KD in the low nanomolar range, as confirmed by three different techniques: surface plasmon resonance, enzyme-linked aptamer assay, and biolayer interferometry. The selected aptamer was demonstrated to have a high specificity to the AP65 protein and to T. vaginalis cells with no cross-reactivity to other enteric and urogenital microorganisms. Current work is focused on the development of inexpensive and easy-to-use aptamer-based diagnostic assays for the reliable and rapid detection of T. vaginalis in vaginal swabs.