Exonuclease-assisted Target Recycling Research Articles

A Highly Sensitive Electrochemical Aptasensor for Kanamycin: Leveraging RecJf Exonuclease-Assisted Target Recycling and Hybridization Chain Reaction Signal Amplification

A Highly Sensitive Electrochemical Aptasensor for Kanamycin: Leveraging RecJf Exonuclease-Assisted Target Recycling and Hybridization Chain Reaction Signal Amplification

CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output.

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.

Ultrasensitive Ochratoxin A Detection in Cereal Products Using a Fluorescent Aptasensor Based on RecJf Exonuclease-Assisted Target Recycling.

Ochratoxin A (OTA) is a mycotoxin widely found in foodstuffs such as cereal grains. It greatly threatens human health owing to its strong toxicity and high stability. Aptasensors have emerged as promising tools for the analysis of small molecule contaminants. Nucleic-acid-based signal amplification enables detectable signals to be obtained from aptasensors. However, this strategy often requires the use of complex primers or multiple enzymes, entailing problems such as complex system instability. Herein, we propose a fluorescent aptasensor for the ultrasensitive detection of OTA in cereal products, with signal amplification through RecJf exonuclease-assisted target recycling. The aptamer/fluorescein-labeled complementary DNA (cDNA-FAM) duplex was effectively used as the target-recognition unit as well as the potential substrate for RecJf exonuclease cleavage. When the target invaded the aptamer-cDNA-FAM duplex to release cDNA-FAM, RecJf exonuclease could cleave the aptamer bonded with the target and release the target. Thus, the target-triggered cleavage cycling would continuously generate cDNA-FAM as a signaling group, specifically amplifying the response signal. The proposed exonuclease-assisted fluorescent aptasensor exhibited a good linear relationship with OTA concentration in the range from 1 pg/mL to 10 ng/mL with an ultralow limit of detection (6.2 ng/kg of cereal). The analytical method showed that recoveries of the cereal samples ranged from 83.7 to 109.3% with a repeatability relative standard deviation below 8%. Importantly, the proposed strategy is expected to become a common detection model because it can be adapted for other targets by replacing the aptamer. Thus, this model can guide the development of facile approaches for point-of-care testing applications.

Electrochemical aptasensor based on DNA-templated copper nanoparticles and RecJf exonuclease-assisted target recycling for lipopolysaccharide detection.

An electrochemical aptasensor for detecting lipopolysaccharides (LPS) was fabricated based on DNA-templated copper nanoparticles (DNA-CuNPs) and RecJf exonuclease-assisted target recycling. The DNA-CuNPs were synthesized on a double-stranded DNA template generated through the hybridization of the LPS aptamer and its complementary chain (cDNA). In the absence of LPS, the CuNPs were synthesized on DNA double-strands, and a strong readout corresponding to the CuNPs was achieved at 0.10 V (vs. SCE). In the presence of LPS, the fabricated aptamer could detach from the DNA double-strand to form a complex with LPS, disrupting the template for the synthesis of CuNPs on the electrode. Meanwhile, RecJf exonuclease could hydrolyze the cDNA together with this single-stranded aptamer, releasing the LPS for the next round of aptamer binding, thereby enabling target recycling amplification. As a result, the electrochemical signal decreased and could be used to indicate the LPS content. The fabricated electrochemical aptasensor exhibited an extensive dynamic working range of 0.01 pg mL-1 to 100 ng mL-1, and its detection limit was 6.8 fg mL-1. The aptasensor also exhibited high selectivity and excellent reproducibility. Moreover, the proposed aptasensor could be used in practical applications for the detection of LPS in human serum samples.

MOF-based nanocomposites coupled with RecJf exonuclease-assisted target recycling amplification: Dual signal amplification for ultrasensitive detection of vanillin

A “signal off” electrochemical aptasensor based on metal-organic framework (MOF) - based nanocomposites and RecJf exonuclease-assisted target recycling was constructed for ultrasensitive detection of vanillin (VAN). In this work, benefited from the high catalytic activity of the synthesized gold nanoparticle/iron-based MOF/polyetherimide-reduced graphene oxide (AuNPs/NH2-MOF-235(Fe)/PEI-rGO) and the AuNPs/Ce-MOF nanocomposites toward thionine (Thi), the electrochemical signal was amplified. And the target recycling assisted by RecJf exonuclease were triggered with the presence of VAN, which induced the strongly reduced electrochemical signal. On account of dual amplification strategies, the performance of the aptasensor was significantly improved to achieve ultrasensitive detection of VAN. Under optimal conditions, the linear relationship was fitted for VAN with a linear range of 2 pM - 0.2 mM and the detection limit of 1.14 pM (S/N = 3). Furthermore, this assay for VAN has excellent specificity, long-term stability, and satisfactory results of the detection in the real samples. In summary, the aptasensor could be further used for the specific quantitative determination of other targets in foods.

Bi-directionally amplified ratiometric electrochemical aptasensor for the ultrasensitive detection of alpha-fetoprotein

Alpha-fetoprotein (AFP) is always employed as a biomarker for liver cancer. The detection of AFP with high sensitivity and good anti-interference property is extremely imperative for clinical cancer diagnosis and effective treatment. In this work, a novel bi-directionally amplified ratiometric electrochemical aptasensor based on exonuclease-assisted target recycling and bioconjugate probe was developed for the ultrasensitive detection of AFP. The aptasensor was fabricated by attaching ferrocene-labeled capture probe (Fc-CP) onto electrode surface through the hybridization with thiolated DNA1. The recognition of AFP caused the disassociation of Fc-CP from the DNA1/Fc-CP duplex, and further triggered the cleavage process of RecJf exonuclease and recycling of AFP. As a result, numerous Fc labels were released, accompanied by plenty exposed DNA1 on the electrode surface for the hybridization with bioconjugate probe containing vast methylene blue (MB) molecules. This strategy significantly amplified the decrease of Fc signal and the increase of MB signal, finally achieving a wide detection range of seven orders of magnitude and an ultralow AFP detection limit down to 269.4 ag/mL. The anti-interference property of the aptasensor was verified by analyzing clinical human serum samples. This aptasensor is promising for the development of reliable electrochemical devices for point-of-care diagnosis.

An aptamer based fluorometric assay for amyloid-β oligomers using a metal-organic framework of typeRu@MIL-101(Al) and enzyme-assisted recycling.

Amyloid-beta (Aβ) oligomers causing neuron damage are regarded as potential therapeutic targets and diagnostic markers for Alzheimer's disease (AD). A homogeneous turn-on fluorometric aptasensor is described for Aβ oligomers. It is highly selective and non-invasive and based on (a) the use of a luminescent metal-organic framework carrying aptamer-modifiedAuNPs (L-MOF/Apt-Au) as tracking agent, and (b) enzyme-assisted target recycling signal amplification. The tracking agent doesnot emit fluoresce by fluorescence resonance energy transfer (FRET) between theluminescent MOF as donor and Apt-Au as the acceptor under the excitation wavelength of 466nm. When Aβ oligomers are added to the tracking agent solution, the Apt-Au on tracking agent can preferentially bind with Aβ oligomers and then be released. This turns the "off" signal of theluminescent MOF tracer to the"on" state. The enzyme (Rec Jf exonuclease) added into the supernatant further improves sensitivity due to enzyme-assisted target-recycling signal amplification. The assay has anexcellent linear response to Aβ oligomers from 1.0pM to 10nM, with a detection limit of 0.3pM. This homogeneous turn-on fluorometric method is expected to have potential and applications in clinical diagnosis. Graphical abstractSchematic representation of fluorometric assay for amyloid-β oligomers based on luminescence metal-organic framework nanocomposites as tracking agent with exonuclease-assisted target recycling.

Plasmon Coupling-Enhanced Raman Sensing Platform Integrated with Exonuclease-Assisted Target Recycling Amplification for Ultrasensitive and Selective Detection of microRNA-21.

A "signal-off" surface-enhanced Raman scattering (SERS) platform has been constructed for ultrasensitive detection of miRNA-21 by integrating exonuclease-assisted target recycling amplification with a plasmon coupling enhancement effect. On this platform, Raman-labeled Au nanostar (AuNS) probes can be covalently linked with the thiolated aptamer (Apt) on the Au-decorated silicon nanowire arrays (SiNWAs/Au) substrate, creating a coupled electromagnetic field between the substrate and the probes to enhance Raman signal. In the presence of miRNA-21, T7 exonuclease specifically hydrolyzed Apt on Apt/miRNA duplex to release miRNA-21. The regenerated element could then initiate another cycle of Apt/miRNA duplex formation and Apt cleavage. Correspondingly, the capture ability of substrate toward probes and the plasmon coupling effect between them were both diminished, giving a prominent attenuation of Raman intensity that can work as the detection signal. Due to the cascading integration between the target cycle process and the plasmon coupling effect, the present platform displayed a very low detection limit (0.34 fM, 3σ) for miRNA-21 detection. Furthermore, it was proven to be effective for analyzing miRNA-21 in biological samples and distinguishing the expression levels of miRNA-21 in MCF-7 cells and NIH3T3 cells, which became a promising tool to monitor miRNA-21 in cancer auxiliary diagnosis and drug screening.

Label-free and ultrasensitive electrochemical biosensor for the detection of EBV-related DNA based on AgDNCs@DNA/AgNCs nanocomposites and lambda exonuclease-assisted target recycling

A label-free and efficient electrochemical biosensor was developed for the ultrasensitive detection of EBV-related DNA by combing AgDNCs@DNA/AgNCs nanocomposites with noncanonical lambda exonuclease (λ exo)-assisted target recycling (LNTR). The conjugates of AgDNCs, DNA/AgNCs and probe DNA (pDNA-AgDNCs@DNA/AgNCs conjugates) worked as not only ideal nanocarriers but also efficient electrochemical tags. LNTR didn't require phosphorylated substrates and could be triggered specifically by target DNA, leading to the recycling use of target DNA and the liberation of plentiful linker probes (LP). Subsequently, the LP hybridized with the capture probes on the electrode and then bond to pDNA-AgDNCs@DNA/AgNCs conjugates, generating a sensitive electric signal directly. What's more, the signal amplification effects of DNA/AgNCs and LNTR were investigated. Under the optimal conditions, the proposed method exhibited a wide linear range of 1 fM to 1 nM and the detection limit down to 0.38 fM. In addition, the developed biosensing method exhibited excellent specificity and was successfully applied to detect target DNA in complex biological matrix. The proposed biosensor without extra bio-labels may provide a promising platform in bioanalysis and biochemical research.

Amplified oxygen reduction signal at a Pt-Sn-modified TiO2 nanocomposite on an electrochemical aptasensor

In this work, a metallic composite with strong electrocatalytic property was designed by uniformly decorating Pt and Sn nanoparticles on the surface of TiO2 nanorods (Pt–Sn@TiO2). A detection scheme was then developed based on a dual signal amplification strategy involving the Pt–Sn@TiO2 composite and exonuclease assisted target recycling. The Pt–Sn@TiO2 composite exhibited an enhanced oxygen reduction current owing to the synergistic effect between Pt and Sn, as well as high exposure of Pt (111) crystal face. Initially, a Pt–Sn@TiO2 modified glassy carbon electrode produced an amplified electrochemical signal for the reduction of dissolved oxygen in the analyte solution. Next, a DNA with a complementary sequence to a streptomycin aptamer (cDNA) was immobilised on the Pt–Sn@TiO2 modified electrode, followed by the streptomycin aptamer that hybridised with cDNA. The corresponding oxygen reduction current was diminished by 51% attributable to the hindrance from the biomolecules. After a mixture of streptomycin and RecJf exonuclease was introduced, both the streptomycin-aptamer complex and the cDNA were cleaved from the electrode, making the Pt–Sn and Pt (111) surface available for oxygen reduction. RecJf would also release streptomycin from the streptomycin-aptamer complex, allowing it to complex again with aptamers on the electrode. This has then promoted a cyclic amplification of the oxygen reduction current by 85%, which is quantitatively related to streptomycin. Under optimal conditions, the aptasensor exhibited a linear range of 0.05–1500 nM and a limit of detection of 0.02±0.0045 nM streptomycin. The sensor was then used in the real-life sample detection of streptomycin to demonstrate its potential applications to bioanalysis.

Building a Fluorescent Aptasensor Based on Exonuclease-Assisted Target Recycling Strategy for One-Step Detection of T-2 Toxin

In this work, a rapid and accurate assay was successfully developed for T-2 toxin detection based on exonuclease-catalyzed target recycling strategy. Upconversion nanoparticles (UCNPs) were conjugated with T-2 aptamer and used as signal probes, while magnetic nanoparticles (MNPs) were conjugated with the complementary DNA of T-2 aptamer (cDNA) and used as capture probes. The results reveled that good linear correlation (R2 = 0.9988) was achieved for T-2 toxin detection over the concentration range of 0.1–100 ng/mL with a detection limit as low as 0.035 ng/mL (S/N = 3). In addition, the reliability of the proposed method was also applied to the determination of T-2 toxin contents in real food samples and the average recoveries ranged from 95.97 to 104.00%. The sensing platform developed in our study demonstrated great potential for simple and sensitive detection of T-2 toxin contents in food samples.

An amperometric aptasensor for ultrasensitive detection of sulfadimethoxine based on exonuclease-assisted target recycling and new signal tracer for amplification

The risks caused by veterinary drug residues in animal foodstuffs are of great concern to the public. Accordingly, this work reported an amperometric aptasensor for highly sensitive detection of sulfadimethoxine (SDM). Functionalised fullerene (C60)-doped graphene (C60-rGO) nanohybrid was designed and prepared to load electroactive toluidine blue (Tb) through the π-π stacking, forming a C60-rGO-Tb nanocomposite. Furthermore, the as-prepared nanocomposite was decorated with gold nanoparticles and used for the immobilization of signal probes to form a new signal tracer, which was coupled with exonuclease-catalyzed target recycling for amplification. To construct the aptasensor, a thiolated double-stranded DNA (dsDNA) of aptamer-capture probe complex was immobilised on a gold electrode surface through strong Au-S bond. In the presence of SDM, the aptamer preferred to form an aptamer-SDM complex, which led to the dissociation of dsDNA. Then aptamer could be selectively digested by RecJf exonuclease, resulting in liberated SDM molecules to participate in the next reaction cycling and achieve signal amplification. Then, capture probes released from the cyclic processes were hybridized with the signal tracer, which could further enhance electrochemical signal responses. On the basis of cascade signal amplification strategies, the proposed aptasensor exhibited a wide linear range from 10 fg/mL to 10 ng/mL for SDM with high sensitivity, good selectivity and satisfactory stability.

A POCT colorimetric aptasensor for streptomycin detection using porous silica beads- enzyme linked polymer aptamer probes and exonuclease-assisted target recycling for signal amplification

A point of care testing (POCT) colorimetric aptasensor was developed to detect streptomycin (STR) using porous SiO2 micro beads (pore size of 200nm) −enzyme linked polymer (PowerVision™, PV) probes and exonuclease-assisted target recycling for signal amplification. The probe was fabricated by single- stranded DNA binding protein labelled SiO2 micro particles (P-SiO2-SSB) as capture probes and Au NPs & aptamer (Apt) co-immobilized on PV (Apt-Au-PV) as nanotracer. In the presence of STR and exonuclease I (Exo I), the nanotracer would combine with STR to form the complex (STR/Apt-Au-PV). Hereafter, Exo I further digest the aptamer on STR/Apt-Au-PV, then STR was released again to participate new cycling, the approach lead to generate more nanotracers into supernatant to improve the sensitivity. Moreover, the dendrimer PV which contains large quantities of HRPs can efficiently catalyzed H2O2-TMB (3,3′,5,5′- tetramethylbenzidine) system for color development and the results can observed by the naked eye. Due to the excellent multiple signal amplification effects for porous SiO2 micro particles to label more nanotracer, PV and exonuclease-assisted target recycling, the aptasensor exhibit a low detection limit with 1pgmL−1 (S/N=3). In addition, the developed method might provide a prospect assay for food tests at point of care.

A triple-amplification SPR electrochemiluminescence assay for chloramphenicol based on polymer enzyme-linked nanotracers and exonuclease-assisted target recycling

The present study aimed to explore a novel triple-amplification electrochemiluminescence (ECL) assay for detecting of chloramphenicol (CAP). This strategy was based on single-stranded DNA-binding protein (SSB) and horseradish peroxidase (HRP) enzyme-linked polymer (EnVision reagent, EV) labeled on Au nanoparticles (EV–Au–SSB) as nanotracer and exonuclease-assisted target recycling. The composite probes were prepared via immunoreactions between the CdS nanocrystal (CdS NC)-functionalized partial complementary DNA and aptamer (CdSNCs/Apt-ssDNA1) as capture probes, and EV–Au–SSB as nanotracer. When the composite probe solution co-existed with CAP and Exo I, the aptamer on the capture probes preferentially combined with CAP, and then CAP–Apt and nanotracer complex were released into the solution. Subsequently, Exo I in the solution could further digest the CAP–Apt from the 3′-end of the aptamer and release CAP, which could participate in further reaction with the probes. It was worth mentioning that EV contained a large number of HRPs on its dendritic chain. In the EV–Au–SSB, Au could enhance ECL intensity of CdS NCs by surface plasmon resonance. What's more, HRPs on EV could catalyze the reaction of H2O2, which could obviously enhance ECL intensity of CdS NCs. This study demonstrated excellent performance of the triple-amplification ECL assay, which makes this aptasensor system suitable and promising for the practical application of CAP residues in fish samples. Moreover, the assay might provide a promising avenue to develop efficient aptasensors to determine small-molecule harmful substances in environmental monitoring and food safety.

A sensitive electrochemical aptasensor for multiplex antibiotics detection based on high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted cascade target recycling

A multiplex electrochemical aptasensor was developed for simultaneous detection of two antibiotics such as chloramphenicol (CAP) and oxytetracycline (OTC), and high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted target recycling was used to improve sensitivity. The cascade amplification process consists of the exonuclease-assisted target recycling amplification and metal ions encoded magnetic hollow porous nanoparticles (MHPs) to produce voltammetry signals. Upon the specific recognition of aptamers to targets (CAP and OTC), exonuclease I (Exo I) selectively digested the aptamers which were bound with CAP and OTC, then the released CAP and OTC participated new cycling to produce more single DNA, which can act as trigger strands to hybrid with nanotracers to generate further signal amplification. MHPs were used as carriers to load more amounts of metal ions and coupling with Exo I assisted cascade target recycling can amplify the signal for about 12 folds compared with silica based nanotracers. Owing to the dual signal amplification, the linear range between signals and the concentrations of CAP and OTC were obtained in the range of 0.0005–50ngmL−1. The detection limits of CAP and OTC were 0.15 and 0.10ngmL−1 (S/N=3) which is more than 2 orders lower than commercial enzyme-linked immunosorbent immunoassay (ELISA) method, respectively. The proposed method was successfully applied to simultaneously detection of CAP and OTC in milk samples. Besides, this aptasensor can be applied to other antibiotics detection by changing the corresponding aptamer. The whole scheme is facile, selective and sensitive enough for antibiotics screening in food safety.

An ultrasensitive fluorescence aptasensor for chloramphenicol based on FRET between quantum dots as donor and the magnetic SiO2@Au NPs probe as acceptor with exonuclease-assisted target recycling

In this work, a fluorescence aptasensor has been developed to detect trace chloramphenicol based on FRET and exonuclease-assisted target recycling. Firstly, the composite probe for CAP was prepared through the immunoreactions between the capture probe based on dsDNA antibody labeled on Fe3O4@Au nanoparticles and the nanotracer using double strand aptamer (aptamer hybrid with its complementary DNA) labeled on core-shell SiO2@Au. When the composite probe solution was mixed with CAP, the aptamer on the nanotracer preferentially bounded with CAP, and then released cDNA-SiO2@Au and CAP-Apt complex to the supernatant. This is because anti-DNA on capture probes can’t recognize the ssDNA. The exonuclease I (Exo I) added into the supernatant can further digest the aptamer-CAP from the 3′-end of aptamer and release CAP again, which can further participate in new cycle to react with the probes. The supernatant containing numerous SiO2@Au could efficiently quench the fluorescence response of CdSe QDs by FRET. Experimental results showed the CAP detection owning a linearity range of 0.001–10ngmL−1 and detection of limit (LOD) of 0.0002ngmL−1. Besides, the results of our method for CAP detection in the fish samples agreed well with those from ELISA, verifying its accuracy and reliability.

An exonuclease-assisted amplification electrochemical aptasensor for Hg(2+) detection based on hybridization chain reaction.

In this work, a novel electrochemical aptasensor was developed for Hg(2+) detection based on exonuclease-assisted target recycling and hybridization chain reaction (HCR) dual signal amplification strategy. The presence of Hg(2+) induced the T-rich DNA partly folded into duplex-like structure via the Hg(2+) mediated T-Hg(2+)-T base pairs, which triggered the activity of exonuclease III (Exo III). Exo III selectively digested the double-strand DNA containing multiple T-Hg(2+)-T base pairs from its 3'-end, the released Hg(2+) participated analyte recycle. With each digestion cycle, a digestion product named as help DNA was obtained, which acted as a linkage between the capture DNA and auxiliary DNA. The presence of help DNA and two auxiliary DNA collectively facilitated successful HCR process and formed long double-stranded DNA. [Ru(NH3)6](3+) was used as redox indicator, which electrostatically bound to the double strands and produced an electrochemical signal. Exo III-assisted target recycling and HCR dual amplification significantly improved the sensitivity for Hg(2+) with a detection limit of 0.12 pM (S/N=3). Furthermore, the proposed aptasensor had a promising potential for the application of Hg(2+) detection in real aquatic sample analysis.

Mediator-free triple-enzyme cascade electrocatalytic aptasensor with exonuclease-assisted target recycling and hybridization chain reaction amplification

The amplified sensitive detection of protein is essential to biomedical research as well as clinical diagnosis. Here, we developed an ultrasensitive mediator-free triple-enzyme cascade electrocatalytic aptasensor for thrombin detection on the basis of exonuclease-assisted target recycling and hybridization chain reaction (HCR) amplification strategy. The double strands constructed by the hybridization of thrombin binding aptamer (S1) with its complementary strand (S2) were firstly assembled on the electrode. Upon addition of target to the system, the S1 recognized thrombin and left off electrode to make space for assembly of hybrid-primer probe (H0). Then, the H0 triggered the HCR to form the multi-functional hemin/G-quadruplex DNAzyme nanowires. In the mediator-free triple-enzyme cascade electrocatalytic amplification system, the hemin/G-quadruplex DNAzyme nanowires here simultaneously played three roles: the redox probe, NADH oxidase and HRP-mimicking DNAzyme, respectively, which effectively avoided the fussy redox probe and enzyme labeling process, serving a useful alternative or supplement to conventional assays that typically suffer from complexity and poor sensitivity. Additionally, in order to improve the assembly amount of hemin/G-quadruplex DNAzyme nanowire, the exonuclease-assisted target recycling amplification was used for the continuous removal of S1. As a result, the proposed method can detect thrombin specifically with a detection limit as low as 20fM.