Recycling Amplification Research Articles

Integrating CRISPR-Cas12a with a crRNA-Mediated Catalytic Network for the Development of a Modular and Sensitive Aptasensor.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated trans-cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a trans-cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 μM (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 μM. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics.

Versatile Electrochemical Biosensor Based on the Target-Controlled Capture and Release of DNA Nanotubes for the Ultrasensitive Detection of Multiplexed Biomarkers.

Herein, an ultrasensitive and versatile electrochemical biosensor was developed through the target-controlled capture and release of signal probe-loaded DNA nanotube for the ultrasensitive detection of two different types of cancer-related biomarkers, microRNA-21 (miRNA-21) and glutathione (GSH). In this system, target 1 (miRNA-21) first triggered duplex-specific nuclease (DSN)-assisted recycle amplification to generate numerous disulfide-linked DNA strands (DL), which could effectively capture DNA nanotube to immobilize methylene blue (MB) to produce remarkable electrochemical signals and achieve the ultrasensitive detection of miRNA-21 with a detection limit down to 32.6 aM. Furthermore, in the presence of target 2 (GSH), the electrochemical signal was significantly reduced by a thiol-disulfide bond exchange reaction on DL to release MB-immobilized DNA nanotubes away from the sensing interface, which enabled the sensitive analysis of GSH with a detection limit of 0.379 nM. Impressively, this strategy could achieve ultrasensitive detection of different types of biomarkers to prominently lessen false-positive responses from the current sensing methods toward a single biomarker or the same type of biomarker and remarkably heighten the accuracy and precision of early cancer diagnosis. Meanwhile, the proposed electrochemical biosensor made it possible to realize the regenerative analysis of targets over four times without extra fuel, which could conspicuously improve the analytical efficiency compared with that of traditional biosensing assays. As a result, this study might open up novel insights to design a versatile and multifunctional sensing platform and encourage deeper exploration for detecting different types of biomarkers in the fields of early disease diagnosis and biochemical research.

Nuclease-propelled target dual-recycling amplification strategy integrated with cascaded sensitization effect of ZnO/CuInS2/Ag2Se photoactive structures for lab-on-paper photoelectrochemical microRNA bioassay

Herein, a new paper-based photoelectrochemical (PEC) biosensor was proposed for microRNA determination based on the cascaded sensitization effect of ZnO/CuInS2/Ag2Se photoactive structures and nuclease-propelled target dual-recycling amplification strategy. Concretely, the paper-based ZnO sensitized with CuInS2 were served as a signal generator and a substrate for immobilizing hairpin DNA (H2). Ag2Se quantum dots (QDs), labeling at the terminal of probe DNA (pDNA), served as the sensitization agents. When target microRNA existed, duplex-specific nuclease activated the first recycling amplification procedure, resulting in the release of numerous converted DNA sequences (cDNA). Afterward, the second target recycling amplification procedure was initiated upon the H2-immobilized electrode was incubated with cDNA and lambda exonuclease, which made massive short DNA fragments free from H2. Consequently, a small quantity of target miRNA-141 was converted into abundant short DNA fragments, which could further hybridize with Ag2Se QDs labelled pDNA, activating the cascaded sensitization effect of ZnO/CuInS2/Ag2Se photoactive structures and giving great signal enhancement. Benefiting from the considerable signal amplification enabled by nuclease-assisted target dual-recycling amplification strategy and cascaded photosensitive structures, the proposed biosensor realized the ultrasensitive detection of miRNA-141 with great accuracy and selectivity, which rendered a prospective paradigm to develop high-performance microfluidic paper analytical devices.

Methylation-blocked cascade strand displacement amplification for rapid and sensitive fluorescence detection of DNA methyltransferase activity

DNA methylation catalyzed by DNA adenine methylation methyltransferase (Dam MTase) is strongly connected with a variety of biological processes, hence, monitoring Dam MTase activity is of great importance. Here, we developed a rapid and sensitive fluorescence sensing strategy for the detection of Dam MTase activity based on methylation-blocked enzymatic recycling amplification. In this fluorescence sensing system, Dam MTase-induced methylation blocked the subsequent reactions. In contrast, in the absence of Dam MTase, the unmethylated probe initiated the cascade strand displacement amplification for significant signal amplification. Under optimized conditions, this method has a lower detection limit of 0.67 U/mL and a shorter assay time (90 min) compared with previously reported similar methodologies.

A microfluidic-based SERS biosensor with multifunctional nanosurface immobilized nanoparticles for sensitive detection of MicroRNA

Developing sensitive and miniaturized biosensors for the detection of microRNAs (miRNAs) is highly desirable due to their association with early cancer diagnosis and prognosis. Here, a new microfluidic-based biosensor, combined with multifunctional nanosurface and DSN-assisted target recycle amplification strategy, is designed for the detection of miRNA-21. The design of nanosurface includes gold nanoparticles on porous anodic aluminum oxide (AAO) for surface enhanced Raman scattering (SERS) substrate, AuMBA@Ag core-shell nanoparticles for SERS nanotags and single-stranded DNA (ssDNA) in between for miRNA capture and nanotags immobilization. When the target miRNA is present near the nanosurface, it will be captured by ssDNA via hybridization reaction. Then, triggered by the DSN-assisted target recycle process, the freshly formed DNA/miRNA heteroduplexes are cleaved by DSN enzyme into DNA fragments and single-strand miRNA. The SERS nanotags are also dissociated from the nanosurface, leading to decrease of SERS signal. The cleaved target miRNA can be captured and SERS nanotags are released again in the next cycle, resulting in amplification of detection signal. To improve the accuracy of this biosensor, the functionalized AAO membrane is subdivided into two groups - AAO/Au array linked with encoded core-shell SERS nanotags acting as a reactor and primary detector and AAO/Au@Ag array serving as a collector and secondary detector for the dissociative SERS nanotags from the reactor. The decrease of SERS signal in primary detector and increase of signal in secondary detector ensures the accuracy and it is called dual-SERS detection strategy. The detection of miRNA-21 can be achieved with only 30 μL sample and 10 μL enzyme and a wide linear range of 10 fM∼10 nM is obtained. In addition, the microfluidic dual-SERS detection strategy can greatly reduce the possibility of false positive or false negative in single detection mode and it can be applied to the simultaneous detection of multiple miRNAs via integrating different probes.

Graphene-oxide-based bioassay for the fluorometric determination of agrC gene transcription in methicillin-resistant Staphylococcus aureus that uses nicking-enzyme-assisted target recycling and a hybridization chain reaction

Herein, we report the development of a graphene-oxide-based (GO-based) fluorescent bioassay for determining agrC gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). The design is based on nicking-enzyme-assisted (Nb.BbvcI-assisted) target recycling amplification (NATR) and a hybridization chain reaction (HCR). The system consists of a helper probe (HP), a molecular beacon (MB) probe, four hairpins, and endonuclease Nb.BbvcI, which plays a role in target recycling and signal amplification. In the absence of the target, all of the carboxyfluorescein-labeled (FAM-labeled) hairpins are adsorbed through π-stacking interactions onto the surface of GO, resulting in FAM signal quenching. When the target is added, three nucleic acid chains hybridize together to form a triple complex that is recognized by Nb.BbvCI. The MB probe is then cleaved by Nb.BbvCI to generate an HP/target complex and two new DNA fragments; the former is hybridized to another MB probe and enters the next round of reaction. The two newly reproduced DNA fragments induce a HCR with the assistance of hairpins 1–4 to create double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and generate strong fluorescence at excitation/emission wavelengths of 480/514 nm. Importantly, synergy between FAM and the dsDNA-SYBR Green I duplex structure led to significantly amplified fluorescence and enhanced sensitivity. The bioassay showed a detection limit of 7.5 fM toward the target and a good linearity in the 10 fM to 100 pM range. The developed method was applied to monitor biofilm formation and study the mechanism of drug action, with satisfactory results obtained.

A novel split-type photoelectrochemical biosensor based on double-strand specific nuclease-assisted cycle amplification coupled with plasmonic Ag enhanced BiVO4 performance for sensitive detection of microRNA-155

A novel split-type and signal-on photoelectrochemical (PEC) biosensor was proposed based on double-strand specific nuclease (DSN)-assisted recycling amplification coupled with photodeposited Ag enhanced BiVO4 photoanode for sensitive detection of microRNA (miRNA)− 155. In the presence of target miRNA, the capture DNA (cDNA) modified with silver nanocube（AgNC）would hybridize with the miRNA, then DSN-assisted cycle amplification was triggered, leading to the release of AgNCs. The released AgNCs were acidolyzed and photodeposited on the BiVO4 electrode. Compared with the BiVO4 electrode, an enhanced photocurrent could be achieved at this obtained BiVO4/Ag electrode. And the morphology change from worm-like nanoparticles to nanosheets after Ag photodeposition was observed for the first time. This PEC signal enhancement was mainly contributed to the synergistic action of localized surface plasmon resonance (LSPR) effect of Ag, Schottky junction formed between BiVO4 and Ag, and the formation of BiVO4 nanosheets with larger specific surface area. The PEC signal enhancement was positively related to the miRNA-155 concentration, so this method could be used to sensitively and selectively detect miRNA-155. A limit of detection of 8 aM could be achieved. This strategy does not require layer-by-layer modification of the electrode, so it is easy to operate, and owns good stability.

Biomineralization-inspired magnetic nanoflowers for sensitive miRNA detection based on exonuclease-assisted target recycling amplification.

Biomineralization-inspired magnetic hybrid nanoflowers were prepared facilely, and capture probes were easily immobilized on the obtained nanoflowers without tedious processing. Based on the magnetic hybrid nanoflowers and exonuclease-assisted target recycling amplification, a fluorescence miRNA sensor was fabricated. The presence of target miRNA leads to the formation of the double-strand structure, which would then be selectively digested by the exonuclease and increase fluorescence intensity. The target miRNA can be released for recycling and signal amplification. Under optimized reaction conditions, the hybrid nanoflower-based miRNA sensor had a broad detection range from 0.001nM to 100nM and a limit of detection of 0.23pM (S/N = 3). The sensitive detection of miRNA in serum was also achieved with recoveries from 94.3% to 116.1%. This work provides a new insight into the fabrication of bioconjugated materials and shows great potential in miRNA sensing.

Modulating the Fluorescence of Silver Nanoclusters Wrapped in DNA Hairpin Loops via Confined Strand Displacement and Transient Concatenate Ligation for Amplifiable Biosensing.

It is intriguing to modulate the fluorescence emission of DNA-scaffolded silver nanoclusters (AgNCs) via confined strand displacement and transient concatenate ligation for amplifiable biosensing of a DNA segment related to SARS-CoV-2 (s2DNA). Herein, three stem-loop structural hairpins for signaling, recognizing, and assisting are designed to assemble a variant three-way DNA device (3WDD) with the aid of two linkers, in which orange-emitting AgNC (oAgNC) is stably clustered and populated in the closed loop of a hairpin reporter. The presence of s2DNA initiates the toehold-mediated strand displacement that is confined in this 3WDD for repeatable recycling amplification, outputting numerous hybrid DNA-duplex conformers that are implemented for a transient "head-tail-head" tandem ligation one by one. As a result, the oAgNC-hosted hairpin loops are quickly opened in loose coil motifs, bringing a significant fluorescence decay of multiple clusters dependent on s2DNA. Demonstrations and understanding of the tunable spectral performance of a hairpin loop-wrapped AgNC via switching 3WDD conformation would be highly beneficial to open a new avenue for applicable biosensing, bioanalysis, or clinical diagnostics.

Fuel-Powered DNA Nanomachines for Biosensing and Cancer Therapy.

Inspired by biological motors, various artificial nanomachines and DNA motors have been fabricated to manipulate their motion on the nanometer scale, due to the high predictability and programmability of Watson-Crick base pairing. Among them, a fuel-powered DNA nanomachine is essentially a toehold-mediated strand exchange reaction and its driving force mainly comes from the hybridization of fuel DNA. It can be initiated by an input strand and automatically operate with the assistance of fuel DNA, realizing input strand recycle and signal amplification. Meanwhile, it is used as a carrier to load various drugs, such as Dox, siRNA and photosensitizers. Due to its excellent cellular penetrability and biocompatibility, fuel-powered DNA nanomachine usually enables operation inside living systems to execute all kinds of specific tasks according to the well-designed DNA strand displacement reaction, such as biomarker imaging, DNA computing and cancer theranostic applications. Therefore, as a catalytic amplification strategy and intelligent drug release platform, fuel-powered DNA nanomachine has attracted widespread attention in biosensors and cancer therapy. Hence, we present a Review on the design mechanism of fuel-powered DNA nanomachines and recent research advances in bioimaging and cancer therapy. It is hoped that this Review will provide the constructive direction for the design of fuel-powered DNA nanomachines and their applications in biological analysis and cancer treatment.

Redox-Responsive Breakup of a Nucleic Acids@CoOOH Nanocomplex Triggering Cascade Recycling Amplification for Sensitive Sensing of Alkaline Phosphatase.

Alkaline phosphatase (ALP), an essential hydrolase with crucial roles in living organisms, has widely been regarded as a biomarker for various human diseases in clinical diagnoses. Herein, taking advantage of cobalt oxyhydroxide (CoOOH) nanoflakes and nonenzymatic cascade recycling amplification (CRA), a highly sensitive and label-free fluorescence biosensing strategy for the determination of ALP activity is introduced. In our design, ALP can promote the dephosphorylation of l-ascorbic acid 2-phosphate (AAP) to reduce ascorbic acid (AA), which is then able to decompose CoOOH in a nucleic acids@CoOOH nanocomplex into Co2+ cofactors. Further, enzyme-free CRA was rapidly initiated by integrating DNAzyme recycling amplification and catalytic hairpin assembly, resulting in the generation of an abundance of G-quadruplex structure-contained DNA duplexes. In the presence of thioflavin T (ThT), analytical target ALP was converted in an amplified and activatable fluorescence signal. The experimental results show that this method can be applied for the quantitative analysis of ALP activity with a low detection limit of 0.027 mU/mL. Moreover, this developed biosensing approach exhibits excellent specificity, and the evaluation of ALP activity in the complex human serum samples was successfully realized, indicating that it can afford a reliable, robust, and cost-effective nanoplatform for an ALP-based clinical diagnosis and for biomedical research.

Target Deoxyribonucleic Acid-Recycled Lighting-Up Amplifiable Ratiometric Fluorescence Biosensing of Bicolor Silver Nanoclusters Hosted in a Switchable Deoxyribonucleic Acid Construct.

Ratiometric assays of label-free dual-signaling reporters with enzyme-free amplification are intriguing yet challenging. Herein, yellow- and red-silver nanocluster (yH-AgNC and rH-AgNC) acting as bicolor ratiometric emitters are guided to site-specifically cluster in two template signaling hairpins (yH and rH), respectively, and originally, both of them are almost non-fluorescent. The predesigned complement tethered in yH is recognizable to a DNA trigger (TOC) related to SARS-CoV-2. With the help of an enhancer strand (G15E) tethering G-rich bases (G15) and a linker strand (LS), a switchable DNA construct is assembled via their complementary hybridizing with yH and rH, in which the harbored yH-AgNC close to G15 is lighted-up. Upon introducing TOC, its affinity ligating with yH is further implemented to unfold rH and induce the DNA construct switching into closed conformation, causing TOC-repeatable recycling amplification through competitive strand displacement. Consequently, the harbored rH-AgNC is also placed adjacent to G15 for turning on its red fluorescence, while the yH-AgNC is retainable. As demonstrated, the intensity ratio dependent on varying TOC is reliable with high sensitivity down to 0.27 pM. By lighting-up dual-cluster emitters using one G15 enhancer, it would be promising to exploit a simpler ratiometric biosensing format for bioassays or clinical theranostics.

A new ratiometric electrochemical aptasensor for the sensitive detection of carcinoembryonic antigen based on exonuclease I-assisted target recycling and hybridization chain reaction amplification strategy

In this work, a novel ratiometric electrochemical aptasensor was designed on the basis of dual-amplification mechanism by using exonuclease I (Exo I)-assisted target recycling and hybridization chain reaction (HCR). The thiol-modified capture DNA (Cp-DNA) was fixed on the surface of the gold electrode (AuE) modified with gold nanoparticles (AuNPs) through Au-S bonds. The ferrocene (Fc)-labeled DNA (Fc-DNA) hybridized with Cp-DNA to form Fc-DNA/Cp-DNA duplex. The presence of carcinoembryonic antigen (CEA) led to the dissociation of Fc-DNA from Fc-DNA/Cp-DNA duplex and initiated Exo Ⅰ-assisted target recycling to digest Fc-DNA, so numerous Fc left away from the electrode surface and resulting in the “signal off” of Fc. Moreover, Cp-DNA was exposed to bind trigger DNA and initiate HCR with the presence of H1 and H2 probes, resulting in the intercalating of numerous methylene blue (MB) molecules and the “signal-on” of MB. This dual signal strategy significantly amplified the decrease of Fc signal and the increase of MB signal, achieving a linear range of 1 pg mL−1 to 100 ng mL−1 with a detection limit of 0.479 pg mL−1 (S/N = 3). Besides, the developed aptasensor exhibited high specificity, good stability and reproducibility. More importantly, the aptasensor had been applied in detection of CEA in diluted human serum and displayed satisfactory anti-interference property. By changing the corresponding aptamer sequences, the designed strategy can be employed to detect other targets, which holds promising application in clinical diagnosis.

Integration of exonuclease III-assisted recycling amplification and multi-site enzyme polymerization labeling for sensitive detection of p53 gene

• A sensitive biosensor for p53 gene detection was proposed and characterized. • The parameters of ExoIII amplification were optimized to ameliorate the non-specific reaction. • sTDN provided accurate enzyme loading and well-organized spatial arrangement for multi-site enzymatic labeling. • Compared with exogenous nanomaterials, sTDN possessed higher yield, better biocompatibility and stability. • The integration of the ExoIII amplification with multi-site enzymatic labeling enhanced the biosensor's sensitivity. • With low detection limit, the modified biosensor exhibited acceptable stability and reproducibility. Protein p53 induces cell growth arrest, differentiation, apoptosis and DNA repair. More than 50% of malignant tumors produce mutations in the p53 gene. Hence, p53 sequence-specific analysis helps in early cancer diagnosis, increasing the treatment success rate. The single nucleic acid amplification method limits sensitivity, so we innovatively integrated exonuclease III (ExoIII)-assisted target recycling amplification with multi-site enzyme polymerization labeling. The integration dramatically improved the biosensor’s sensitivity. Given the nonspecific reaction of ExoIII, we optimized the ExoIII amplification system to reduce false positives and background current. The single-strand DNA produced in the ExoIII system served as the bridge between the electrode and signal DNA tetrahedron (sTDN). Moreover, sTDN provided accurate enzyme loading and well-organized spatial arrangement for multi-site enzymatic labeling. Compared with single-stranded signal probes, sTDN modified by terminal transferase provided higher catalytic efficiency. Under the optimal experimental conditions, the cathodic current exhibited a logarithmic relation over 1.0–5.0 × 10 6 fM p53, with a detection limit of 0.47 fM. Applying this biosensor has successfully detected p53 in serum samples, providing a potential tool for cancer diagnosis and treatment.

A universal constructing method for high performance DNA biosensors based on the optimized photoelectrode material and dual recycling amplification

Many effective strategies for photoelectrochemical DNA sensors have been developed in the past decades. Most of them focused on the detection performance including sensitivity, limit of detection and linear range, but few on the pivotal roles of photoelectrode materials and the universality for strategies. Herein, the photoelectrode material was optimized by density functional theory for improving the comprehensive performances of DNA biosensor for the first time. And the universal strategies, based on dual recycling amplification, was discussed by different nucleic acid sequences. After systematical optimization of detection conditions, a large linear range of 10-16 to 10-6 M, high sensitivity of 5.66 μA·cm−2·dec-1 and low limit of detection of 10-18 M (a gene copy per microliter) were achieved for the detection of DNA sequences from the I27L gene. Finally, detection of DNA sequences from Orientia tsutsugamushi was used to prove the universality of the novel method.

A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 gene via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies.

A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 (ORAOV1) gene was constructed via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. Capture DNA with a sulfhydryl group at its 3' terminus was modified onto the surface of a bare gold electrode via an Au-S bond. Assisted DNA hybridized with basal DNA to form hybrid DNA in advance, and ORAOV1 gene hybridized continuously with such a hybrid DNA from the other terminus to construct intact double-stranded DNA. Exonuclease III digested basal DNA in such intact double-stranded DNA specifically, and both ORAOV1 gene and assisted DNA were released into solution. ORAOV1 gene induced another intact double-stranded DNA digestion for target recycling, while assisted DNA hybridized with the capture DNA to form double-stranded DNA on the modified electrode surface. Unhybridized capture DNA on the modified electrode surface was hydrolyzed by RecJf exonuclease to reduce the background electrochemical signal. The 3' terminus of double-stranded DNA on the modified electrode surface was prolongated to be guanine-rich oligonucleotides under the catalysis of terminal deoxynucleotidyl transferase. In the presence of K+ ions, hemin adsorbed onto guanine-rich oligonucleotides to construct a G-quadruplex/hemin complex with a large steric hindrance effect to efficiently avoid the charge transfer of the [Fe(CN)6]3-/4- probe toward the electrode surface. The electrochemical impedance value was increased significantly after the addition of ORAOV1 gene via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. The electrochemical impedance value was linearly related to the logarithmic concentration of ORAOV1 gene in the range from 0.05 fM to 20 pM, and the detection limit of ORAOV1 gene was low to 0.019 fM. This biosensor was used to detect ORAOV1 gene in complicated human saliva samples with satisfactory results.

A Carbon Nanocages-Mediated Fluorescent Aptasensor for Aflatoxin B1 Detection Based on T7 Exonuclease Double Recycling Amplification

A Carbon Nanocages-Mediated Fluorescent Aptasensor for Aflatoxin B1 Detection Based on T7 Exonuclease Double Recycling Amplification

A signal-on electrochemical DNA biosensor based on exonuclease III-assisted recycling amplification.

DNA electrochemical detection technology has attracted tremendous interest in recent years. However, a facile and sensitive method for the detection of the disease indicators or genes is still waiting. Herein, we constructed a signal-on electrochemical platform for detecting the manganese superoxide dismutase (MnSOD) gene by incorporating a redox electrochemical signal probe (methylene blue) and exonuclease III-assisted target recycling signal amplification strategy. The sensor was prepared by self-assembly of a capture DNA probe of thiol-modified on GCE with gold electrodeposition. In the presence of target DNA, the exonuclease III can cleave the duplexes formed by the target DNA and the redox-labeled hairpin probes, release the target DNA and produce a residual sequence. The target DNA can continue to hybridize with the hairpin probe for the next cycle of amplification. The residual sequence hybridized with the surface-immobilized capture probes on AuNPs-modified GCE to generate a significantly amplified redox current. In particular, the redox current value of the resultant sensor showed a linear relationship with MnSOD gene concentration in the range of 1-104 pM with the detection limit as low as 0.3 pM. Furthermore, the sensor has excellent specificity and can distinguish single-base mismatch from perfectly matched target DNA. The sensor is fast in operation, and simple in design for detecting different DNA sequences or DNA identification by selecting the appropriate probe sequence, thus shedding light on a good promising application when encountering disease outbreaks or for the early clinical diagnosis of gene-related diseases.

Ladder-Like DNA Nanostructure-Mediated Cascade Catalytic Nanomachine for Construction of Ultrasensitive Biosensors.

Herein, a novel two-dimensional ladder-like DNA nanostructure (LDN)-mediated cascade catalytic nanomachine (LDN-CCN) with a higher catalytic efficiency beyond those of a conventional one-dimensional hybrid chain reaction (HCR) nanostructure-mediated CCN was constructed and applied to design an electrochemical biosensing platform with first-rank performance for ultrasensitive detection of target miRNA-21. First, output DNA S1' and S2' were produced through the DNAzyme recycle amplification when the target miRNA-21 existed. Then, the controllable LDN-CCN was constructed on S1-S2 modified electrodes by the subsequent reaction triggered by S1' and S2' with H1-AuNPs, H2, H3-AuNPs, and H4 with the assistance of K+ and hemin, in which the hemin/G-quadruplexes could produce a prominent electrochemical signal response to the substrate glucose. The best performance of cascade catalysis was acquired when the distance of Au nanoparticles (glucose oxidase-like activity) modified on H1 and H3 and hemin/G-quadruplexes (peroxidase-like activity) formed by the sticky ends of H2 and H4 was roughly 9 nm (27 bp) in LDN-CCN. The constructed electrochemical platform realized the sensitive detection of target miRNA-21 with the linear range from 100 aM to 10 nM and with a detection limit as low as 48.5 aM, which provided novel insights to explore the new functional DNA nanostructure and well-performing mimic enzyme cascade catalytic platforms for applications in biological fields and early diagnosis of diseases.

Portable electrochemical biosensor based on laser-induced graphene and MnO2 switch-bridged DNA signal amplification for sensitive detection of pesticide

Developing portable, quantitative, and user-friendly analytical tools for sensitive pesticide assay is of significant importance for guaranteeing food safety. Herein, a novel electrochemical biosensor was constructed by integrating laser-induced graphene (LIG) electrode on polyimide (PI) foil and MnO2 nanosheets loaded on the paper for point-of-care test (POCT) of organophosphorus (OPs) residues. The principle of this biosensor relied on acetylcholinesterase (AChE)-catalyzed hydrolytic product-triggered disintegration of MnO2 nanosheets for releasing assistant DNA to initiate nicking enzyme-aided recycling amplification. In the presence of OPs, the activity of AChE was inhibited and could not initiate the cleavage of the electroactive molecules-labeled hairpin probe on the electrode, resulting in the maintenance of the electrochemical response to realize a “sign-on” determination of OPs. The proposed biosensor exhibited satisfactory analytical performance for OPs assay with a linear range from 3 to 4000 ng/mL and a limit of detection down to 1.2 ng/mL. Moreover, the biosensor was useful for evaluating the residual level of pesticides in the vegetables. Therefore, this novel biosensor holds great promise for OPs assay and opens a new avenue on the development of higher-performance POCT device for sensing applications in the environment and food safety fields.