Duplex-specific Nuclease Assisted Target Recycling Research Articles

A glucose/O2 biofuel cell as self-powered sensor for ultrasensitive microRNA detection based on CRISPR-Cas12a cleavage and duplex-specific nuclease-assisted target recycling

The development of effective strategies for the construction of enzymatic biofuel cell based self-powered biosensors is important. Herein, we report ultrasensitive self-powered sensing of microRNA by integrating a glucose/O2 biofuel cell with CRISPR-Cas12a-based cleavage and duplex-specific nuclease (DSN) assisted target recycling. For the construction of the bioanode, glucose oxidase (GOD) is immobilized on Au nanoparticles-graphene nanosheets nanocomposite modified electrode by using a short single-stranded DNA as a bridge. The target miRNA-155 can initiate DSN-assisted target recycling and CRISPR-Cas12a cleavage toward ssDNA, leading to the release of GOD from the bioanode. Because of the decreased loading capacity of GOD on the bioanode at increasing miRNA-155 concentration, the short-circuit current of the glucose/O2 biofuel cell decreases, enabling self-powered sensing of miRNA-155. The self-powered biosensor features a wide dynamic range for detecting miRNA-155 from 10−16 to 10−10 M, with an ultralow detection limit of 23 aM. This work provides a highly sensitive self-powered biosensing platform for miRNA detection.

A Glucose/O2 Biofuel Cell as Self-Powered Sensor for Ultrasensitive Microrna Detection Based on Crispr-Cas12a Cleavage and Duplex-Specific Nuclease-Assisted Target Recycling

A Glucose/O2 Biofuel Cell as Self-Powered Sensor for Ultrasensitive Microrna Detection Based on Crispr-Cas12a Cleavage and Duplex-Specific Nuclease-Assisted Target Recycling

A dual-signaling electrochemical ratiometric strategy combining “signal-off” and “signal-on” approaches for detection of MicroRNAs

A dual-signaling electrochemical ratio metric strategy was developed for detection microRNA-18a based on the duplex-specific nuclease-assisted target recycling and electrochemical atom transfer radical polymerization signal amplification. In the presence of target microRNA, RNA/DNA duplexes are formed, which become the substrate of the duplex-specific nuclease-assisted target recycling. Hence only the DNA strand is cleaved by duplex-specific nuclease enzyme, resulting in the throw away of methylene blue (MB) from the electrode (signal off) accompanied by releasing of target microRNA, which can be recycled in the next hybridization. The remaining piece of capture DNAs on the electrode surface hybridize with the Azide labeled-signal DNAs. “Click reactions” were carried out between 3-Butynyl-2-bromoisobutyrate and Azide to initiate the electrochemical atom transfer radical polymerization reaction. This process could bring a great number of ferrocenylmethyl methacrylate (FMMA) on the surface of electrode (signal on). The IFMMA/IMB value was proportionate to the microRNA-18a concentration and measured by square wave voltammetry.The promising potential of the proposed biosensor in clinical analyses was exhibited by its remarkable features such as strong performance, high specificity, agreeable storage stability, and notable selectivity in real sample evaluation with no pretreatment or amplification. Finally, our biosensing method offers such an application to be used for the early clinical diagnosis of Pancreatic Cancer.

Ultrasensitive electrochemical microRNA-21 biosensor coupling with carboxylate-reduced graphene oxide-based signal-enhancing and duplex-specific nuclease-assisted target recycling

An electrochemical biosensor for ultrasensitive detection of microRNA-21 (miRNA-21) coupling with carboxylate-reduced graphene oxide (COOH-rGO)-based signal-enhancing and duplex-specific nuclease (DSN)-assisted target recycling. Capture DNA (cDNA) was self-assembled on the surface of gold electrode (GE) and hybridized with the target miRNA-21 to form cDNA/miRNA-21 hybrids. COOH-rGO was further aggregated on cDNA/miRNA-21 hybrids through π − π stacking interaction, leading to a accumulation of electroactive methylene blue (MB) molecules and a significant differential pulse voltammetry (DPV) response of MB with peak potential of −0.28 V (vs. Ag/AgCl). At the present of DSN, DSN digested the cDNA/miRNA-21 hybrids and released miRNA-21 to hybridize with another cDNA, resulting in a significant decrease of the length of cDNA sequence. The amount of MB on the electrode surface decreased dramatically, following with the decrease of the DPV signal of MB. Under optimal experimental conditions, this approach displayed an ultrahigh sensitivity (with a 0.01 fM detection limit) and excellent selectivity among similar miRNAs sequences even with single-base difference. This biosensor further exhibited excellent applicability for target miRNA-21 in human serum samples with satisfactory results.

An ultrasensitive electrochemical biosensor for detection of microRNA-21 based on redox reaction of ascorbic acid/iodine and duplex-specific nuclease assisted target recycling

A novel electrochemical biosensor was developed based on multiwall carbon nanotubes/graphene oxide nanoribbons (MWCNTs/GONRs) for sensitive analysis of microRNA-21. Signal-amplified strategy was achieved by duplex-specific nuclease assisted target recycling and alkaline phosphatase-induced redox reactions. At the fabrication process of the sensor, ssDNA capture probes were immobilized on the surface of the MWCNTs@GONRs/AuNPs modified electrode through the Au-S bond, and the streptavidin-conjugated alkaline phosphatase (SA-ALP) was attached to the end of the probe. In the absence of miRNA-21, SA-ALP catalysed the conversion of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA), triggered a redox reaction under iodine, producing a marked electrochemical response. When miRNA-21 was hybridized to the capture probe, the duplex would be cleaved by the duplex-specific nuclease (DSN), causing the electrochemical signals being significantly decreased as a result of SA-ALP detached from the electrode surface. Under the optimized conditions, our biosensor showed satisfactory sensitivity (detection limit, 0.034 fM), excellent selectivity and good accuracy (recoveries, 77.4–120.2%; RSD, 5.2–7.3%) after systematic evaluations. The proposed approach was applied to detect miRNA-21 from human serum samples, which indicated that it was reliable and could be widely used as an effective tool for rapid detection of the target in serums.

MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification

DNA technology based bio-responsive nanomaterials have been widely studied as promising tools for biomedical applications. Gold nanoparticles (AuNPs) and graphene oxide (GO) sheets are representative zero- and two-dimensional nanomaterials that have long been combined with DNA technology for point-of-care diagnostics. Herein, a cascade amplification system based on duplex-specific nuclease (DSN)-assisted target recycling and electrocatalytic water-splitting is demonstrated for the detection of microRNA. Target microRNAs can form DNA: RNA heteroduplexes with DNA probes on the surface of AuNPs, which can be hydrolyzed by DSN. MicroRNAs are preserved during the reaction and released into the suspension for the digestion of multiple DNA probes. After the DSN-based reaction, AuNPs are collected and mixed with GO to form AuNP/GO nanocomposite on an electrode for the following electrocatalytic amplification. The utilization of AuNP/GO nanocomposite offers large surface area, exceptional affinity to water molecules, and facilitated mass diffusion for the water-splitting reaction. For let-7b detection, the proposed biosensor achieved a limit detection of 1.5 fM in 80 min with a linear detection range of approximately four orders of magnitude. Moreover, it has the capability of discriminating non-target microRNAs containing even single-nucleotide mismatches, thus holding considerable potential for clinical diagnostics.

Ultra-sensitive detection of microRNA-21 based on duplex-specific nuclease-assisted target recycling and horseradish peroxidase cascading signal amplification

The detection and analysis of microRNA (miRNA) can provide critical information for biomedical research and clinical diagnosis. In this paper, we constructed a simple method to achieve ultra-sensitive and rapid detection of miRNA based on the double-specific nuclease (DSN) assisting target recovery and horseradish peroxidase (HRP) cascading signal amplification. In the existence of target microRNA-21 (miR-21), the DSN enzyme specifically cut the single-stranded DNA (ssDNA) hybridized with the target miRNA but still kept the target miRNA intact, so there were more free HRP released from the polystyrene microparticles-ssDNA-HRP conjugates (PS-ssDNA-HRP) because of the target recycle and signal amplification. The free HRP in the supernatant were collected to react with the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The color and absorbance changes of HRP-TMB system further resulted in qualitative screening by naked eyes and quantitative detection by the microplate reader with the detection limit as low as 3.1 fM. This assay protocol not only showed high sensitivity and selectivity, but also well distinguished the difference between the miRNA family members, as well as the simple operation, fast detection, naked eye qualitative and microplate reader quantification. It has great application value in the biomedical research and clinical diagnosis.

On-Particle Rolling Circle Amplification-Based Core-Satellite Magnetic Superstructures for MicroRNA Detection.

Benefiting from the specially tailored properties of the building blocks as well as of the scaffolds, DNA-assembled core-satellite superstructures have gained increasing interest in drug delivery, imaging, and biosensing. The load of satellites plays a vital role in core-satellite superstructures, and it determines the signal intensity in response to a biological/physical stimulation/actuation. Herein, for the first time, we utilize on-particle rolling circle amplification (RCA) to prepare rapidly responsive core-satellite magnetic superstructures with a high load of magnetic nanoparticle (MNP) satellites. Combined with duplex-specific nuclease-assisted target recycling, the proposed magnetic superstructures hold great promise in sensitive and rapid microRNA detection. The long single-stranded DNA produced by RCA serving as the scaffold of the core-satellite superstructure can be hydrolyzed by duplex-specific nuclease in the presence of target microRNA, resulting in a release of MNPs that can be quantified in an optomagnetic sensor. The proposed biosensor has a simple mix-separate-measure strategy. For let-7b detection, the proposed biosensor offers a wide linear detection range of approximately 5 orders of magnitude with a detection sensitivity of 1 fM. Moreover, it has the capability to discriminate single-nucleotide mismatches and to detect let-7b in cell extracts and serum, thus showing considerable potential for clinical applications.

Nanopore-Based Electrochemiluminescence for Detection of MicroRNAs via Duplex-Specific Nuclease-Assisted Target Recycling.

In this study, we proposed a nanopore-based electrochemiluminescence (ECL) sensor combined with duplex-specific nuclease (DSN)-assisted target recycling amplification to detect microRNAs. Because of the synergetic effect of electrostatic repulsion and volume exclusion of gold nanoparticle-labeled DNA capture (DNA-Au NPs) to the negatively charged luminol anion probe, the DNA-Au NP-modified anodized aluminum oxide (AAO) nanopore electrode exhibited high ECL decline in comparison with the bare AAO electrode. Upon the introduction of DSN and target microRNA, the specific DNA-RNA binding and enzyme cleaving could trigger the detachment of capture DNA from the membrane surface, resulting in uncapping of AAO and an increased ECL signal. For comparison, positively charged Ru(bpy)32+ was used as the ECL probe instead of luminol. Because the electrostatic attraction effect between DNA and Ru(bpy)32+ is partially offset by the volume exclusion effect of Au NPs, the AAO electrode modified with only DNA capture is more suitable for the Ru(bpy)32+ case. In our experiment, the case of negatively charged luminol combined with the synergetic effect of electrostatic repulsion and volume exclusion of DNA-Au NPs provides a quantitative readout proportional to the target microRNA concentration in the range of 1.0 fM to 1.0 nM, with a lower detection limit of 1 fM.

Optomagnetic Detection of MicroRNA Based on Duplex-Specific Nuclease-Assisted Target Recycling and Multilayer Core-Satellite Magnetic Superstructures.

Superstructural assembly of magnetic nanoparticles enables approaches to biosensing by combining specially tailored properties of superstructures and the particular advantages associated with a magnetic or optomagnetic read-out such as low background signal, easy manipulation, cost-efficiency, and potential for bioresponsive multiplexing. Herein, we demonstrate a sensitive and rapid miRNA detection method based on optomagnetic read-out, duplex-specific nuclease (DSN)-assisted target recycling, and the use of multilayer core-satellite magnetic superstructures. Triggered by the presence of target miRNA and DSN-assisted target recycling, the core-satellite magnetic superstructures release their "satellites" to the suspension, which subsequently can be quantified accurately in a low-cost and user-friendly optomagnetic setup. Target miRNAs are preserved in the cleaving reaction and can thereby trigger more cleavage and release of "satellites". For singleplex detection of let-7b, a linear detection range between 10 fM and 10 nM was observed, and a detection limit of 4.8 fM was obtained within a total assay time of 70 min. Multiplexing was achieved by releasing nanoparticles of different sizes in the presence of different miRNAs. The proposed method also has the advantages of single-nucleotide mismatch discrimination and the ability of quantification in a clinical sample matrix, thus holding great promise for miRNA routine multiplex diagnostics.

Sensitive detection of microRNAs by duplex specific nuclease-assisted target recycling and pyrene excimer switching.

Herein, we develop a simple fluorescence method for the sensitive detection of microRNAs based on duplex specific nuclease-assisted target recycling and pyrene excimer switching. This method is extremely sensitive with a detection limit of 0.58 fM, and it can be applied to absolutely detect endogenous microRNAs in cancer cells, holding great potential for clinical diagnosis and biomedical research studies.

An immobilization-free electrochemical impedance biosensor based on duplex-specific nuclease assisted target recycling for amplified detection of microRNA.

An immobilization-free electrochemical impedance biosensor for microRNA detection was developed in this work, which was based on both the duplex-specific nuclease assisted target recycling (DSNATR) and capture probes (Cps) enriched from the solution to electrode surface via magnetic beads (MBs). In the absence of miR-21, Cps cannot be hydrolyzed due to the low activity of duplex-specific nuclease (DSN) against ssDNA. Therefore, the intact Cps could be attached to the surface of magnetic glass carbon electrode (MGCE), resulting in a compact negatively charged layer as well as a large charge-transfer resistance. While in the presence of miR-21, it hybridized with Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzed the target-binding part of the Cp while liberating the intact miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. In this way, a single miR-21 was able to trigger the permanent hydrolysis of multiple Cps. Finally, all Cps were digested. Thus, the negatively charged layer could not be formed, resulting in a small charge-transfer resistance. By employing the above strategy, the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with a detection limit of 60aM. Meanwhile, the method showed little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human serum samples of breast cancer patients.

Label-free microRNA detection based on terbium and duplex-specific nuclease assisted target recycling

In this paper, we describe a novel label-free fluorescence method for microRNA-21 (miR-21) detection based on terbium (Tb(3+)) and duplex-specific nuclease (DSN) assisted target recycling. Capture probes (Cps), containing a target-binding part and a signal-output part, are immobilized on magnetic beads (MBs). In the presence of the target miR-21, it hybridizes with the target-binding part of a Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzes the target-binding part of the Cp while liberating the intact target miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. Eventually, through magnetic separation, only the signal-output part of the Cp could remain in solution and function as a signalling flare to increase the fluorescence intensity of Tb(3+) dramatically. By employing the above strategy, this approach can gain an amplified fluorescent signal and detect as low as 8 fM miR-21 under the optimized conditions. Moreover, due to the high selectivity of DSN, the method shows little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human cell lysate samples of breast cancer patients.

Integration of DNA bio-gates and duplex-specific nuclease signal amplification: towards electrochemiluminescence detection of survivin mRNA.

Using the DNA bio-gate and duplex-specific nuclease assisted target recycling, a facile electrochemiluminescence assay was developed for the sensitive detection of survivin mRNA.

An ultrasensitive label-free electrochemical biosensor for microRNA-21 detection based on a 2'-O-methyl modified DNAzyme and duplex-specific nuclease assisted target recycling.

Based on a highly efficient 2'-O-methyl modified G-quadruplex-hemin DNAzyme and duplex-specific nuclease (DSN) assisted target recycling, a novel label-free electrochemical biosensor for microRNA-21 (miR-21) detection is developed here. By employing the strategy, this DNA biosensor can detect as low as 8 aM miR-21 and exhibits high discrimination ability even against a single-base mismatch.