Electrochemical Detection of MicroRNAs via Gap Hybridization Assay

Abstract

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable

“…Electrochemical Biochip Analysis of mRNA Levels-Electrochemical biochip analysis of mRNA levels was performed as described previously (22,23). Capture oligonucleotides were as follows: securin, ACGGTTCCCGAAGGCACATTCTCATTTT-TTTTT (3Ј-thiol); and separase, TCTGCCCCCGAAGGGGAC-GTCCTATTTTTTTTT (3Ј-thiol).…”

“…The integrity of ribosomal RNA was confirmed by agarose gel electrophoresis, and the quantity and purity of total RNA were spectrophotometrically confirmed. Deviating from the described procedure for microRNA analysis (22), total RNA was fragmented in the presence of 30 mM magnesium acetate, 100 mM potassium acetate, and 40 mM Tris-HCl (pH 8.1) at 95°C for 15 min before hybridization. Fragmented RNA in 450 mM NaCl, 0.025% Tween 20, 1 mg/ml BSA, 25 mM EDTA, 30 mM NaH 2 PO 4 (pH 7.4), 1 g of each complementary RNA probe, and 0.2 M esterase 2-detector oligodeoxynucleotide conjugate was applied to each electrode.…”

“…The enzyme substrate p-aminophenyl butyrate was delivered through the flow chamber at a flow rate of 250 l/min. After the flow was stopped, the esterase 2 activity was measured as a change in current intensity (dI) per time (dt) within the first 5 s (22,23).…”

Microfluidic Chips for Point‐of‐Care Immunodiagnostics

“…Proteins can be detected in picomolar concentrations but the process requires incubation and many fl uid manipulation steps. Interestingly, by patterning enzymelabeled receptors directly on working electrodes analytes such as glucose, lactate and oxygen, [ 244 , 245 ] nucleic acid [ 246 ] and protein [ 247 , 248 ] can be detected electrochemically in one step.…”

Electrochemical Detection of miRNAs

“…One relevant example is the use of the thermostable reporter enzyme esterase 2 (ETS2) from Alicyclobacillus acidocaldarius. [37] EST2 offers the possibility of site specific modification with oligonucleotide probes and was found to be superior to common enzyme labels such as alkaline phosphatase and horseradish peroxidase (HRP) [38].…”