Protein Electrocatalysis for Direct Sensing of Circulating MicroRNAs

MicroRNAs (miRNAs) are potentially useful biomarkers for diagnosis, classification, and prognosis of many diseases, including cancer. Herein, we developed a protein-facilitated electrocatalytic quadroprobe sensor (Sens^PEQ) for detection of miRNA signature of chronic lymphocytic leukemia (CLL) in human serum. The developed signal-ON sensor provides a compatible combination of two DNA adaptor strands modified with four methylene blue molecules and electrocatalysis using glucose oxidase in order to enhance the overall signal gain. This enhanced sensitivity provided the response necessary to detect the low-abundant serum miRNAs without preamplification. The developed Sens^PEQ is exquisitely sensitive to subtle π-stack perturbations and capable of distinguishing single base mismatches in the target miRNA. Furthermore, the developed sensor was employed for profiling of three endogenous miRNAs characteristic to CLL, including hsa-miR-16-5p, hsa-miR-21-5p, and hsa-miR-150-5p in normal healthy serum, chronic lymphocytic leukemia Rai stage 1 (CLL-1), and stage 3 (CLL-3) sera, using a non-human cel-miR-39-3p as an internal standard. The sensor results were verified by conventional SYBR green-based quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis

Four-Way Junction Formation Promoting Ultrasensitive Electrochemical Detection of MicroRNA

MicroRNAs (miRNAs) represent a class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. Here, we endeavored to develop a DNA four-way junction based electrochemical sensor (4J-SENS) for ultrasensitive miRNA analysis. The developed sensor can be operated within the dynamic range from 10 aM to 1 fM and detect as low as 2 aM of miR-122 (∼36 molecules per sample), without PCR amplification. Furthermore, the 4J-SENS was employed to profile endogenouse hsa-miR-122 in healthy human and chronic lymphocyitc leukemia (CLL) patient serum, and the results were validated by qPCR analysis

Three-Mode Electrochemical Sensing of Ultralow MicroRNA Levels

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown great promise for cancer classification and prognosis. In this work, we developed a three-mode electrochemical sensor for detection and quantitation of ultralow levels of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modalities based on hybridization (H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS). The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30 μL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 μM. The HPD sensor is made on a commercially available gold nanoparticles-modified electrode and is suitable for analyzing multiple miRNAs on a single electrode. This three-mode sensor exhibits high selectivity and specificity and was used for sequential analysis of miR-32 and miR-122 on one electrode. In addition, the H-SENS can recognize miRNAs with different A/U and G/C content and distinguish between a fully matched miRNA and a miRNA comprising either a terminal or a middle single base mutation. Furthermore, the H- and P-SENS were successfully employed for direct detection and profiling of three endogenous miRNAs, including hsa-miR-21, hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results were validated by qPCR

Sodium bisulfite pyrosequencing revealed that developmental exposure to environmental contaminant mixtures does not affect DNA methylation of DNA repeats in Sprague-Dawley rats

<p>Hypomethylation of DNA repeats has been linked to diseases and cancer predisposition. Human studies suggest that higher blood concentrations of environmental contaminants (EC) correlate with levels of hypomethylation of DNA repeats in blood. The objective of this study was to examine the effect of in utero and/or lactational exposure to EC on the methylation of DNA repeats (LINE-1 and identifier element) in Sprague-Dawley rat pups at birth, at postnatal day (PND) 21, and in adulthood (PND78–86). From gestation day 0 to PND20, dams were exposed to a mixture “M” of polychlorinated biphenyls (PCB), pesticides, and methylmercury (MeHg), at 0.5 or 1 mg/kg/d (0.5M and M). At birth, some control (C) and M litters were cross-fostered to create the following in utero/postnatal exposure groups: C/C, M/C, C/M, M/M. Additional dams received 1.8 ng/kg/d of a mixture of aryl-hydrocarbon receptor (AhR) agonists (non-<i>ortho</i>-PCB, PC-dibenzodioxins, and PC-dibenzofurans) without or with 0.5M (0.5MAhR). Measurements of EC residue levels confirmed differences in their accumulation across treatments, age, and tissues. Although induction of hepatic detoxification enzyme activities (cytochrome P-450) demonstrated biological effects of treatments, the assessment of methylation in DNA repeats by sodium bisulfite pyrosequencing of liver, spleen, and thymus samples revealed no marked treatment-related effects but significant tissue- and age-related methylation differences. Further studies are required to determine whether absence of significant observable treatment effects on methylation of DNA repeats in the rat relate to tissue, strain, or species differences.</p