Exonuclease III-assisted Target Recycling Research Articles

Label-Free and Ultrasensitive Electrochemical Detection of Nucleic Acids Based on Exonuclease III-Assisted Target Recycling Amplification Strategy at Heated Gold Disk Electrode

Label-Free and Ultrasensitive Electrochemical Detection of Nucleic Acids Based on Exonuclease III-Assisted Target Recycling Amplification Strategy at Heated Gold Disk Electrode

A Simple Ratiometric Electrochemical Aptasensor Based on Exonuclease III-Assisted Target Recycling for Ultrasensitive Detection of Prostate Specific Antigen

Prostate specific antigen (PSA) is considered as an important biomarker in prostate cancer diagnosis. Herein, an ultrasensitive ratiometric electrochemical aptasensor was constructed for analyzing PSA. The ferrocene (Fc)-labeled thiolated hairpin probe 2 (Fc-HP2) was conjugated to the gold nanoparticles (AuNPs)-modified gold electrode (AuE) surface by Au-S bonds. With the introduction of PSA, the aptamer region of hairpin probe 1 (HP1) preferred to bind with PSA, and the rest of HP1 could hybridize with Fc-HP2 to form Fc-HP2/HP1/PSA complex with a blunt 3′ terminus, which triggered the exonuclease III (Exo III) cleavage process accompanied by Fc leaving from the electrode and recycling of HP1/PSA. The remaining Fc-HP2 fragments left on the electrode surface were then hybridized with methylene blue-labeled DNA (MB-DNA). This resulted in an enhancement of MB signal (IMB) and a decrement of Fc signal (IFc). Under the optimal conditions, the proposed aptasensor showed good analytical performance for sensitive detection of PSA with a linear range from 100 fg·mL−1 to 10 ng·mL−1 and a detection limit of 34.7 fg·mL−1. The fabricated aptasensor had been applied to detect PSA in diluted human serum samples and obtained satisfied results, suggesting it had great potential in clinical diagnosis.

Assembling G-wire-induced pyrene excimer switching for microRNA amplified detection based on exonuclease III-assisted target recycling and Fe3O4@PDA nanoparticles

We reported a novel assembled G-wire-induced pyrene excimer switching probe for microRNA (miRNA) amplified detection on the basis of exonuclease III (Exo III)-assisted target recycling and Fe3O4@PDA nanoparticles. The multifunctional hairpin (HP) probe recognized and effectively bound to target miRNA, which formed a 3′-blunt terminal for Exo-III digestion and miRNA regeneration to initiate another Exo III-mediated cycling cleavage. After digestion, numerous HP-caged c-myc sequences were released, which were positively related to target miRNA and folded to the parallel G-quadruplex with K+. Additionally, Fe3O4 polydopamine nanoparticles (Fe3O4@PDA NPs)-based magnet separation was conducted to reduce background fluorescence signal, and parallel G-quadruplexes self-asembled into filamentous G-wires with Mg2+. G-wires generated from 5′-terminal pyrene-labeled c-myc sequences made close distance between two labeled pyrene molecules, and induced the “turn-on” fluorescence signal of pyrene excimer switching. Moreover, gamma-cyclodextrin (γ-CD) was added, which promoted pyrene moiety, causing signal amplification in pyrene excimer fluorescence. Therefore, miRNA was efficiently measured within the homogeneous solution. This method detected miRNA within the dynamic range of 1 pM to 5 nM, with the miR-141 limit of detection (LOD) being 99 fM. It distinguished perfect-matched and mismatched target miRNAs, and showed an accurate detection of miRNA levels in different cell lines and favorable reproducibility when analyzing miRNAs within spiked serum samples. We propose a promising method for G-wire-based biosensing in disease-related biomarkers detection.

A fluorometric graphene oxide-based assay for determination of agrB gene transcription in methicillin-resistant Staphylococcus aureus by coupling exonuclease III-assisted target recycling and hybridization chain reaction

Schematic representation of the graphene oxide-based fluorometric bioassay for determination of agrB gene transcription in MRSA by coupling exonuclease III-assisted target recycling and hybridization chain reaction.

A graphene-oxide-based aptasensor for fluorometric determination of chloramphenicol in milk and honey samples utilizing exonuclease III-assisted target recycling and Nb.BbvCI-powered DNA walker cascade amplification

Herein, a graphene oxide (GO)-based fluorescence aptasensor was developed for the sensitive and selective detection of chloramphenicol (CAP), based on exonuclease III (Exo III)-assisted target recycling and Nb.BbvCI-driven DNA walker cascade amplification. Interactions between CAP, hairpin1(HP1), hairpin2 (HP2), and 3′-amino modified hairpin3 (HP3) labeled with carboxyfluorescein (FAM) and covalently coupled to GO enabled efficient CAP detection. CAP was quantitatively assayed by measuring fluorescence at excitation/emission wavelengths of 480/514 nm, resulting from the accumulation of released FAM. A good linear range of 1 fM to 1 nM and a limit of detection (LOD) of 0.875 fM (signal-to-noise (S/N)= 3) were achieved. This aptasensor can distinguish the CAP from interference antibiotics with good specificity and selectivity, even if the concentration of the interfering substance is ten-fold higher than the target concentration. Moreover, the developed fluorescence aptasensor was successfully applied for the detection of CAP in spiked milk and honey samples. Thus, this method is potentially applicable for assaying CAP in foods and provides a promising strategy for the development of fluorescence aptasensors for environmental sample analysis.

An ultrasensitive biosensor for virulence ompA gene of Cronobacter sakazakii based on boron doped carbon quantum dots-AuNPs nanozyme and exonuclease III-assisted target-recycling strategy

This work presented an electrochemical biosensor for the detection of virulence outer membrane protein A (ompA) gene of Cronobacter sakazakii (C. sakazakii), which was based on the mimic peroxidase activity of boron doped quantum dots-Au nanoparticles (BQDs-AuNPs) and a signal amplification strategy of exonuclease III (Exo III)-assisted target-recycling (EATR). The electrochemical signal was come from the electrochemical reduction of H2O2 by BQDs-AuNPs nanozyme. Due to the enhanced peroxidase-mimic electrocatalytic efficiency of BQDs-AuNPs and the EATR strategy, the biosensor showed a broad linear range (1.0 × 10-15 – 1.0 × 10-8 mol L-1) and a low limit of detection (LOD, 4.0 × 10-17 mol L-1). The constructed biosensor could also be applied in direct detection of extracted DNA from C. sakazakii. A good linear relationship (7.8 – 7.8 × 106 CFU mL−1) between the logarithm concentration of C. sakazakii and electrochemical signal was obtained with a LOD of 2.6 CFU mL−1. The biosensor was applied in the detection of impA gene segments in contaminated infant formula with recoveries ranged in 83.4 – 108.2%.

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.

Target biorecognition-triggered assembly of a G-quadruplex DNAzyme-decorated nanotree for the convenient and ultrasensitive detection of antibiotic residues

The abuse of kanamycin (Kana) in many fields has led to increasing antibiotic pollution problems and serious threats to public health. Therefore, determining how to develop methods to realize the convenient detection of antibiotics in complicated environmental matrices is highly desirable. In this study, we utilized a target biorecognition-triggered hybridization chain reaction (HCR) assembly of a G-quadruplex DNAzyme (G-DNAzyme)-decorated nanotree to develop a novel homogeneous colorimetric biosensing method for the convenient and ultrasensitive detection of Kana antibiotic residues in real samples. Through the designed aptamer-recognition reaction, an Mg2+-dependent DNAzyme (MNAzyme) strand can be liberated. Thus, its catalyzed cleavage of the hairpin substrates anchored at a DNA nanowire will cause the assembled formation of an HCR-initiator; this process can be greatly amplified by the exonuclease III-assisted target recycling and the MNAzyme-catalyzed release of another MNAzyme strand. Based on the DNA-nanowire-accelerated HCR assembly of many G-DNAzyme-decorated DNA duplexes on the two sides of the nanowire, a DNA nanotree decorated by numerous G-DNAzymes will form to realize the ultrasensitive colorimetric signal output. Under the optimal conditions, this method exhibited a wide five-order-of-magnitude linear range and a very low detection limit of 28 fg mL−1. In addition, excellent selectivity, repeatability, and reliability were also demonstrated for this homogeneous bioassay method. These unique features along with its automatic manipulation and low assay cost show promise for practical applications.

Ultrasensitive PEC aptasensor based on one dimensional hierarchical SnS2|oxygen vacancy-WO3 co-sensitized by formation of a cascade structure for signal amplification

Herein, we developed a photoelectrochemical (PEC) aptasensing platform that relies on a dual-signal amplification strategy. The proposed PEC system consists of a one dimensional (1D) hierarchical SnS2|OV (oxygen vacancy)-WO3 nanorods (NRs) sensitized by CdS quantum dots (QDs)/TCPP (TCPP: meso-tetra(4-carboxyphenyl)-porphine), and exonuclease III-assisted target recycling. As a photoactive heterojunction, SnS2|OV-WO3 nanocomposite exhibits a PEC signal ~11 times higher than pure WO3 NRs due to the enhanced separation efficiency of photo-generated carriers. Moreover, it was also used to immobilize a hairpin DNA3 probe labeled with CdS QDs/TCPP, which formed a co-sensitization cascade structure with SnS2|OV-WO3 NRs on the sensor surface due to the well-matched energy levels. This special cascade structure effectively shortened the electron-transfer path and inhibited charge recombination, thereby improving the PEC performance. The addition of the target vascular endothelial growth factor 165 (VEGF165) triggered the exonuclease III-assisted target recycling to generate plentiful DNA sequences (S1). S1 was specifically hybridized with HP3 to unfold its hairpin structure. As a result, CdS QDs/TCPP detached from the sensor surface and SnS2|OV-WO3 NRs, which destructed the co-sensitization cascade structure and minimized the PEC signals. The proposed PEC aptasensor exhibited a dynamic determination range of 0.5 fM-10 nM, and a detection limit of 0.34 fM for VEGF165.

A novel DNA sensor of homogeneous electrochemical signal amplification strategy

A simple homogeneous electrochemical biosensing platform was developed for efficient and ultrasensitive detection of K-rat sarcoma (K-ras) gene fragment via exonuclease III-assisted target recycling and π–π stacking between graphene and nucleotide bases. The presence of fully complementary target DNA, triggers the cycle of target DNA with the aid of exonuclease III (Exo III), resulting in short single strand DNA probe formation. Furthermore, a novel biferrocene was newly synthesized and used for probe labeling; the two ferrocene units provide a large electrochemical signal compared with single. The cyclic release biferrocene is adsorbed on graphene electrode due to DNA conformational change. Moreover, the method has a simple operation process, and is cost effective. Taking advantage of the unique surface property of graphene and high efficiency of DNA Exo III, our sensor shows good ability toward K-ras detection. The detection limit of 20.4 fM could be achieved possesses great potential in detect K-ras normal gene.

A highly sensitive and selective fluorescence biosensor for hepatitis C virus DNA detection based on δ-FeOOH and exonuclease III-assisted signal amplification

Developing the high selectivity and sensitivity strategy for nucleic acid detection is crucial for early diagnosis and therapy of diseases. In this work, a novel low back-ground fluorescent sensor platform for the detection of nucleic acid has been developed based on δ-FeOOH nanosheets integrating with exonuclease III-assisted target-recycling signal amplification. Because of the strong binding ability between the single-strand DNA (ssDNA) and the δ-FeOOH nanosheets, the dye-labeled ssDNA probe would be quenched by δ-FeOOH nanosheets through fluorescence resonance energy transfer (FRET). By using magnetic separate properties of δ-FeOOH, the background signal was separated from the sensor system, and the low background sensor system was obtained. After adding the target DNA, a double-strand DNA complex (dsDNA) would be formed between the target DNA and dye-labeled ssDNA probe. Then, the dye-labeled ssDNA probe in the dsDNA complex would be stepwise hydrolyzed into short fragments from 3′-terminus by Exonuclease III, and the fluorescence signal was recovered due to the weak bind affinity between the short fragments and δ-FeOOH nanosheets. By using the fluorescence quenching ability of δ-FeOOH nanosheets and enzyme-assisted target-recycling signal amplification, this strategy could show an excellent selectivity toward hepatitis C virus DNA with a low detection limit of 10 pM. By simply changing the dye-labeled ssDNA probe sequence, this sensing platform can be developed as a universal approach for the simple, sensitive, and selective detection of different target DNA.

Signal-on electrochemical detection of DNA methylation based on the target-induced conformational change of a DNA probe and exonuclease III-assisted target recycling

A promising electrochemical system was explored for DNA methylation detection according to the construction of a signal-on biosensor. Based on the ingenious design of probe DNA and auxiliary DNA, methylated target DNA triggered the exonuclease III (Exo III) digestion of auxiliary DNA from 3′-terminus, resulting in the conformational change of probe DNA with an electroactive methylene blue (MB) tag at 5′-terminus. Consequently, the MB tag in the probe DNA was close to the electrode surface for electron transfer, generating an increased current signal. Because of the target recycling of methylated DNA, significant signal amplification was obtained. Moreover, bisulfite conversion conferred an efficient approach for the universal analysis of any CpG sites without the restriction of specific DNA sequence. As a result, the target DNA with different methylation statuses were clearly recognized, and the fully methylated DNA was quantified in a wide range from 10 fM to 100 pM, with a detection limit of 4 fM. The present work realized the assay of methylated target DNA in serum samples with satisfactory results, illustrating the application performance of the system in complex sample matrix.

Fluorometric determination of HIV DNA using molybdenum disulfide nanosheets and exonuclease III-assisted amplification.

A convenient and ultrasensitive fluorometric method is described for the determination of HIV DNA. It exploits the strong difference in the affinities of MoS2 nanosheets for long ssDNA versus short oligonucleotide fragments. In addition, efficient signal amplification is accomplished by exonuclease III-assisted target recycling. When absorbed on the MoS2 nanosheets, the fluorescenceof the FAM-labeled ssDNA probe (FP) is quenched. However, in the presence of HIV DNA, the FP hybridizes with target to form a duplex. As a result, the FP in the duplex will be stepwise hydrolyzed into short fragments by Exo III, and the fluorescence signal thus isretained because short fragments have low affinity for the MoS2 nanosheets. By using the Exo III-assisted target recycling amplification, the detection sensitivity is strongly improved. The sensor can detect DNAin a concentration as low as 5.3 pM (at an S/N ratio of 3), and the analytical range extends from 0.01nM to 10nM. The assay is simple, sensitive and specific, and conceivably represents a valuable tool in clinical studies related to the HIV. Graphical abstract Schematic presentation of fluorometric determination of HIV DNA based on molybdenum disulfide nanosheets and Exo III. When the fluorescence-tagged ssDNA probe hybridized with target to form a duplex, the Exo III-assisted target recycling amplification is generated. The method can detect as low as 5.3 pM HIV DNA.

A ratiometric electrochemical biosensor for ultrasensitive and highly selective detection of the K-ras gene via exonuclease III-assisted target recycling and rolling circle amplification strategies

A ratiometric electrochemical biosensor for ultrasensitive and highly selective detection of the K-ras gene via Exo III-assisted target recycling and RCA strategies.

Highly sensitive detection of nucleic acids using a cascade amplification strategy based on exonuclease III-assisted target recycling and conjugated polyelectrolytes.

In this paper, a novel ratiometric and cascade amplification strategy was developed by combining the unique signal amplification and effective fluorescence resonance energy transfer (FRET) property of conjugated polymers with the Exo III-assisted target recycling method. The target DNA (ssDNAc) could be hybridized with the duplex-stranded probe to trigger the cyclic digestion of the probe strands and lead to the continuous release of fluorescein from the probe. The proposed strategy thus shows enhanced sensitivity toward target DNA with a detection limit of 0.38 nM, which is more sensitive than the previously reported comparable biosensors based on conjugated polyelectrolytes. Furthermore, this method exhibited an improved performance to discriminate single mismatched targets through an efficient FRET-based ratiometric detection method using a conjugated polymer as a donor and an optical transducer. More importantly, this cascade amplification approach offers the advantages of simplicity, which avoids multiple utilization of probes and complex assay steps required in traditional amplification methods.

Colorimetric detection of genetically modified organisms based on exonuclease III-assisted target recycling and hemin/G-quadruplex DNAzyme amplification.

An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2- causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420nm) at concentrations as low as 0.23nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs. Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.

Fe3+ doped ZnO-Ag photocatalyst for photoelectrochemical sensing platform of ultrasensitive Hg2+ detection using exonuclease III-assisted target recycling and DNAzyme-catalyzed amplification

Abstract Herein we designed a novel photoelectrochemical sensing platform based on Fe3+ doped ZnO-Ag photocatalyst (Fe3+/ZnO-Ag) for ultrasensitive Hg2+ detection by using exonuclease III-assisted target recycling and DNAzyme-catalyzed amplification. Firstly, Fe3+/ZnO-Ag was synthesized to provide photocurrent under visible light irradiation. Secondly, target Hg2+ was determined by hairpin DNA probes on the Fe3+/ZnO-Ag modified ITO based on T-Hg2+-T, and then Hg2+ was released by the digestion of exonuclease III to double-stranded DNA. Finally, the photocurrent markedly decreased due to catalysis of hemin/G-quadruplex toward 4-chloro-1-naphthol. Under optimal conditions, the photocurrents were linearly related to Hg2+ concentrations form 0. 5 nM to 100 nM with a detection limit of 0. 1 nM at the 3Sblank criterion. More importantly, the developed biosensor exhibited good selectivity, repeatability, and stability, meanwhile, this analysis platform held great potential for testing real water samples in future.

Smart DNA Machine for Carcinoembryonic Antigen Detection by Exonuclease III-Assisted Target Recycling and DNA Walker Cascade Amplification.

A synthetic DNA machine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA walker biosensor for label-free detection of carcinoembryonic antigen (CEA) is developed for the first time by a novel cascade amplification strategy of exonuclease (Exo) III-assisted target recycling amplification (ERA) and DNA walker. ERA as the first stage of amplification generates the walker DNA, while the autonomous traveling of the walker DNA on the substrate-modified silica microspheres as the second stage of amplification produces an ultrasensitive fluorescent signal with the help of N-methylmesoporphyrin IX (NMM). The DNA machine as a biosensor could be applied for transducing and quantifying signals from isothermal molecular amplifications, avoiding the complicated reporter elements and thermal cycling. The present biosensor achieves a detection limit of 1.2 pg·mL-1 within a linear range of 10 pg·mL-1 to 100 ng·mL-1 for CEA, along with a favorable specificity. The practical applicability of the biosensor is demonstrated by the detection of CEA in human serum with satisfactory results; thus, it shows great potential in clinical diagnosis.

Cascade Dual-Signal Enhancement by Integrating Exonuclease III-Assisted Target-Recycling and Rolling Circle Amplification for Ultrasensitive Electrochemical Detection of DNA

Based on a cascade dual-signal enhancement strategy by integrating exonuclease III (Exo III)-assisted target-recycling (EATR) with rolling circle amplification (RCA), a highly sensitive electrochemical biosensor was developed for the detection of target DNA (tDNA), a kind of Alzheimer's disease biomarkers. When tDNA was hybridized with hairpin DNA (hDNA) previously modified in the electrode surface by Au-SH bond, the subsequent introduction of Exo III triggered EATR through cleaving hDNA, releasing tDNA and further hybridizing with other hDNA. After repeated recycling, the remained DNA (rDNA) fragments of hDNA as initiators of RCA were extended, generating numerous copies of G-rich base sequence. The presence of hemin resulted in the in situ assembly of multiple hemin/G-quarduplex structures in the resultant electrode surface, which were acted as both label-free redox probes and mimicking peroxidase catalysts for the electrochemical signal readout and amplification, producing significantly enhanced electrochemical response dependent on tDNA. Thus, the developed biosensor exhibited ultrahigh sensitivity with a limit of detection of 3.3 aM. By combining EATR with subsequent RCA, the cascade dual-signal amplification strategy would be promising and potential in biological analysis and clinical diagnosis.

A facile and green strategy for preparing newly-designed 3D graphene/gold film and its application in highly efficient electrochemical mercury assay

In this work, we report a facile and green strategy for in situ and one step preparation of a novel 3D graphene/gold (G/Au) film. Triggering with unique driving force from hydrothermal growth, a 3D interlaced graphene framework with hierarchically porous structures was directly attached on a gold substrate pretreated with a self-assembled monolayer. Simultaneously, highly dispersive Au nanoparticles with tunable morphologies were anchored on the framework utilizing generated graphene as an endogenous reductant. Newly-designed 3D G/Au film possessed excellent properties of significantly large specific area, good electrical conductivity, high structure stability and substrate binding strength, etc. As a paradigm, an electrochemical Hg2+ biosensor was constructed on 3D G/Au film, in which an exonuclease III-assisted target recycling was introduced. Impressively, an ultralow detection limit of 50 aM (S/N=3), a wide linear range from 0.1 fM to 0.1μM, a high selectivity and a good reliability for Hg2+ assay in real water and serum samples were realized using prepared biosensor. It is highly envisioned that this work opens the door towards simply fabricating varying types of 3D graphene based hybrid films, and such G/Au film will have widespread applications in electroanalysis and electrocatalysis.