Electrochemical Detection Of microRNA Research Articles

Self-Powered DNAzyme Walker Enables Dual-Mode Biosensor Construction for Electrochemiluminescence and Electrochemical Detection of MicroRNA.

Herein, an electrochemiluminescence (ECL) and electrochemical (EC) dual-mode biosensor platform with a self-powered DNAzyme walking machine was established for accurate and sensitive detection of miRNA-21. By employing a magnesium ion (Mn2+)-dependent DNAzyme cleavage cycling reaction, the walking machine was built by assembling DNAzyme walking strands and ferrocene (Fc)-labeled substrate strands on the Au nanoparticles and graphitic carbon nitride nanosheet (g-C3N4 NS)-covered electrode. The DNAzyme walking strand was first prohibited by a blocker strand. After the addition of target miRNA-21 and Mn2+, the DNAzyme walker could be activated and produce autonomous movements along the electrode track fueled by Mn2+-dependent DNAzyme-catalyzed substrate cleavage without additional energy supply. Notably, each walking step resulted in the cleavage of a substrate strand and the release of a Fc-labeled DNA strand fragment, allowing us to acquire an extreme ECL signal recovery of g-C3N4 inhibited by Fc. Meanwhile, numerous Fc-labeled DNA fragments escaped from the surface of the electrode, directly producing an obvious decrease in the square wave voltammetry (SWV) signal from Fc on the same sensing platform. This work not only avoided difficultly assembling various signal indicators but also significantly improved the sensitivity through using self-powered DNAzyme-walker amplification. Moreover, the proposed design employed the same reaction to produce two signal output modes, which could eliminate the interference from diverse reactive pathways on the outcome to mutually improve the accuracy. Therefore, the dual-mode miRNA-21 biosensor exhibited wide detection ranges of 100 aM to 100 nM with low detection limits of 54.3 and 78.6 aM by ECL and SWV modes, respectively, which provided an efficient and universal biosensing approach with extensive applications in early disease diagnosis and bioanalysis.

Preconcentration, Release and Electrochemical Detection of microRNA on Microfluidics

Introduction One of the challenges of the 21st in medical diagnosis lies in the identification of specific biomarkers for the detection of infectious or cancerous diseases before the first symptoms appear and thus treat the patients effectively.After the discovery of their implication in cancer pathologies [1] at early stage, micro ribonucleic acids (miRNA) became promising candidates for this application due to their rapid, precise and reliable diagnosis. These miRNAs, constituted by around 20 nucleotides, are highly involved in post-transcriptional regulation mechanisms capable of inducing the extinction or expression of certain gens and their presence in body fluids (plasma, urine, saliva...) is a sign of important perturbations [2].However, in order to detect the in-vivo change of molecular expression levels (under femtomolar [3]) current techniques rely in chemical amplification stages of a specific target to be detected usually done by Polymerase Chain Reaction (PCR). The main drawbacks of PCR are an increase in the entire protocol time from miRNA extraction in biological sample to its detection (optical or electrical), as well as nonspecific sequence amplification, especially for short sequences as in miRNAs [4].The approach developed and patented by our laboratory [5] proposes an alternative to the use of chemical amplification techniques. This alternative is based in the preconcentration of the circulating miRNA, their localized release and detection. During the preconcentration stage, functionalized magnetic nanoparticles (NPMs) capture the circulating miRNAs and then release them, via magnetic hyperthermia. Once released, the miRNAs are detected by functionalized gold microelectrodes embedded in a microfluidic chip [6].In this communication we will present the results obtained combining the localized release of the miRNA and their electrochemical detection. Method The used nanoparticles have a diameter of 80 nm and they are formed by a maghemite core protected by a silica shell functionalized with –NH2 groups which allow a covalent coupling with modified RNA probe.The microelectrode pairs used to detect the hybridation of the previously released miRNA are formed by a 30 µm width by 300 µm length working gold microelectrode and a 30 µm width by 2 mm length gold counter-electrode patterned on a glass substrate by a lift-off process.The microfluidic channels are microfabricated by molding dimethylpolysiloxane (PDMS) onto a SU-8 (from MicroChem) mold 50-micron thickness with the pattern of the channels. PDMS is spin coated on the mold to obtain a thickness of 500 µm which allows to keep the total thickness of the microfluidic device under 1mm after the bonding of the glass substrate and the PDMS microchannels.Functionalization of the microelectrodes is done by introducing a solution of DNA probes modified with a thiol termination in 0.5 M NaCl. Then the channel is rinsed with 0.5 M NaCl to stabilize the probes.The magnetic field needed for the magnetic hyperthermia is generated by a power amplifier that provides the current to the tank circuit formed by a capacitor and an air-gapped ferrite with a certain number of turns. The microfluidic device is placed in the air-gap of the ferrite to maximize the amplitude of the applied alternating magnetic fieldElectrochemical measurements are performed using a commercial potentiostat connected to the microfluidic device through a dedicated chip holder. Electrochemical measurements are performed in 0.5 M NaCl as supporting electrolyte containing a 3 mM FeIIICN6 3-/ FeIICN6 4- redox couple, and 10-8 M methylene blue as redox intercalant. Results and Conclusions The measured impedance between a working microelectrode and the counter-electrode before and after the hybridation of the miRNA released by the magnetic nanoparticles after being subjected to an alternating magnetic field as previously described [5].In this presentation we will present impedance measurements before hybridation that shows a high electron charge transfer resistance (Rct) since grafted DNA probe (single strands) inhibits the redox complex diffusion towards the electrodes. After the miRNA hybridization on microelectrodes (double strand), the electron charge transfer resistance decreases related to the intercalation of MB in DNA duplex which acts as an electron charge mediator between the redox and the microelectrode. Indeed, Rct decrease highlights the success of the hybridization step after it release from magnetic hyperthermia runs.The proposed methodology, based in the combination of magnetic hyperthermia and electrochemical detection, has shown to be effective to locally release the recruited miRNA and detect them by electrochemical means. In addition, the whole process is performed in a microfluidic device offering the advantages of this devices: batch processing, low sample volume, portability, etc.

Electrochemical Detection of Micro-RNAs As Potential Cancer Biomarker

MicroRNAs (miRNAs) are single-stranded RNA transcripts, typically of 19–23 nucleotides in length. Recently, miRNAs have been identified as important biomarkers that hold promise in early cancer diagnosis. Accumulative evidences have proved that abnormal expression of miRNAs is closely related with the occurrence, diagnosis and treatment of cancer [1]. Conventional methods of miRNA detection have some intrinsic drawbacks including the need for tedious and complex procedures, requirement of large amount of samples, highly skilled experts and high-end equipment. To address these shortcomings as well as to provide multiplexing capability, different types of electrochemical cancer biosensors have been developed. Electrochemical biosensors have been widely paid attention due to their merits including high sensitivity, specificity, low-cost, and multiplexing [2]. Herein, a label-free and sensitive biosensor was developed for miRNA-21 detection at femtomolar concentrations. A glassy carbon electrode (GCE) was first modified with carbon nanotube (CNT) which serves as platform for immobilization of probe DNA as well as providing higher surface area. Capture DNA probes were immobilized onto the surface of CNT modified electrode using an established molecular tethering method [3,4]. Electrochemical analyses confirmed the surface modification, immobilization, and hybridization between probe DNA and the target miRNA. Besides, different variables such as probe concentration and hybridization time were investigated and optimized to find the optimal experimental conditions for miRNA detection. The obtained results demonstrated that the proposed biosensor exhibits excellent analytical properties including high sensitivity, low detection limit, selectivity, and reproducibility for miRNA detection. References Lan H, Lu H, Wang X, Jin H (2015) MicroRNAs as potential biomarkers in cancer: opportunities and challenges. BioMed research international 2015Kilic T, Erdem A, Ozsoz M, Carrara S (2018) MicroRNA biosensors: Opportunities and challenges among conventional and commercially available techniques. Biosensors and Bioelectronics 99:525-546Ramasamy RP, Luckarift HR, Ivnitski DM, Atanassov PB, Johnson GR (2010) High electrocatalytic activity of tethered multicopper oxidase–carbon nanotube conjugates. Chemical Communications 46 (33):6045-6047Zhou Y, Fang Y, Ramasamy RP (2019) Non-covalent functionalization of carbon nanotubes for electrochemical biosensor development. Sensors 19 (2):392

Electrochemical detection of microRNAs by graphene oxide modified disposable graphite electrodes

Graphene oxide (GO) modified disposable pencil graphite electrodes (PGEs) were developed for the first time in the present study for miRNA detection by measuring the guanine oxidation signal. GO modified PGEs were used as solid phase to immobilize amino linked DNA probe and then the complementary target RNA sequence (miRNA-34a) was recognized. The electrochemical behaviour of the GO modified electrodes was investigated by using electrochemical impedance spectroscopy (EIS) and microscopic characterization was performed by scanning electron microscopy (SEM). The optimization studies and detection of hybridization between miRNA-34a target and its complementary DNA probe was performed by differential pulse voltammetry (DPV). The selectivity of our assay was tested in the presence of non-complementary miRNA sequence. A good discrimination could be achieved compared to the results obtained with the full match hybridization of probe with its RNA target miRNA-34a target against other miRNA sequences; miRNA-16 and miRNA-155.

Electrochemical Detection of MicroRNAs via Gap Hybridization Assay

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.