Exonuclease-assisted Signal Amplification Research Articles

Fluorescent aptasensors for sensitive detection of lead ions based on structure-switching DNA beacon probe and exonuclease I-mediated signal amplification

Herein, two simple fluorescent signal-on sensing strategies for detecting lead ions (Pb2+) were established based on structure-switching aptamer probes and exonuclease-assisted signal amplification strategies. Two hairpin-structure fluorescent probes with blunt-ended stem arms were designed by extending the base sequence of Pb2+ aptamer (PS2.M) and labelling the probes with FAM (in probe 1) and 2-aminopurine (2-AP) (in probe 2), respectively. In method 1, graphene oxide (GO) was added to adsorb probe 1 and quench the fluorescence emission of FAM to achieve low fluorescent background. In method 2, fluorescent 2-AP molecule inserted into the double-stranded DNA of probe 2 was quenched as a result of base stacking interactions, leading to a simplified, quencher-free approach. The addition of Pb2+ can induce the probes to transform into G-quadruplex structures, exposing single DNA strands at the 3′ end (the extended sequences). This exposure enables the activation of exonuclease I (Exo I) on the probes, leading to the cleavage effect and subsequent release of free bases and fluorophores, thereby resulting in amplified fluorescence signals. The two proposed methods exhibit good specificity and sensitivity, with detection limits of 0.327 nM and 0.049 nM Pb2+ for method 1 and method 2, respectively, and have been successfully applied to detect Pb2+ in river water and fish samples. Both detection methods employ the structure-switching aptamer probes and can be completed in two or three steps without the need for complex analytical instruments. Therefore, they have a broad prospect in the sensitive and simple detection of lead ion contamination in food and environmental samples.

Simultaneous detection of acute myocardial infarction -related miR-199a and miR-499 based on a dual-emission CdTe fluorescent probe and T7 exonuclease-assisted signal amplification

The early diagnosis of acute myocardial infarction (AMI) was a tough nut in both clinical medicine and forensic science since the occurrence of gross changes needed at least 12 h. Compared to other diagnostic methods, microRNA was reported as an earlier cardiac biomarker with higher sensitive. In this work, we designed a novel fluorescent analytical strategy using a dual-emission probe based on T7 Exo-assisted target recycling amplification for simultaneous detection of AMI-related miR-199a and miR-499, which could indicate myocardial ischemia or damage in AMI. The dual-emission probe was conducted by yellow-emission and red-emission CdTe quantum dots capped with beta-cyclodextrin. The florescence of probe was firstly quenched by graphene oxide (GO) and then recovered in the presence of target miR-199a and miR-499. Under the optimized conditions, the limit of detection as low as 0.1 pM was obtained and the overall test could be performed within 50 min. Besides, the simultaneous detections of microRNAs in spiked serum samples were achieved with the recovery of 81.3–109.6%, which suggested the potential method for diagnosis in practical. The advantages of this method include easy operation, high accuracy and low labor cost, which makes point-of-care test possible both in hospital and forensic application.

Sensitive Fluorescent Determination of Chloramphenicol Based upon Graphdiyne and RecJf Exonuclease-Assisted Signal Amplification

A sensitive and selective method for determining chloramphenicol (CAP) based on RecJf exonuclease-assisted signal amplification and graphdiyne (GDY) is reported. GDY possesses a strong absorptivity for single-stranded DNA and is proposed for the first time as a new specific and eminently sensitive type of fluorescence sensing platform for chloramphenicol. Under the optimized conditions, the assay operates from 10 to 80 µg/L chloramphenicol with a 43.6 ng/L detection limit. The procedure was employed to determine chloramphenicol in food samples, with recoveries from 93.60% to 107.90%. The proposed technique offers a novel development in food safety using sensitive aptasensors for detecting small molecules.

RecJf exonuclease-assisted signal amplification for the sensitive detection of di(2-ethylhexyl)phthalate by a Pd@Au NB labeled electrochemical aptasensor based on Au@Ni-CoHNB/PEI-g-C3N4

Electrochemical aptasensor based on nanocomposite and RecJf exonuclease-assistant signal amplification strategy for di(2-ethylhexyl)phthalate (DEHP) detection.

Sensitive electrochemical aptasensor for determination of sulfaquinoxaline based on AuPd NPs@UiO-66-NH2/CoSe2 and RecJf exonuclease-assisted signal amplification

The authors designed a sensitive label-free electrochemical aptasensor for the detection of sulfaquinoxaline (SQX), including the AuPd NPs@UiO-66-NH2/CoSe2 nanocomposites and RecJf exonuclease-assisted target recycle signal amplification strategy. AuPd NPs@UiO-66-NH2/CoSe2 nanocomposite with excellent conductivity and numerous active sites was successfully synthesized to provide a favorable sensing platform and load more double-strand DNA (dsDNA) on the electrode surface. The negatively charged phosphate group of the oligonucleotide and [Fe (CN)6] 3-/4- repel each other electrostatically, resulting in very low electrical signals. In the presence of SQX, its corresponding aptamer will be released from the double-stranded structure and then digested by RecJf exonuclease, which resulted in the SQX being released to initiate the next recycling to help amplify the DPV signal. Under the optimal conditions, the peak current has a linear relationship with the logarithmic of SQX concentration in the range of 1 pg/mL∼100 ng/mL and the obtained detection limit was 0.547 pg/mL. Furthermore, the contrasted aptasensor possess reliable specificity, reproducibility and stability toward SQX, and has been applied to detect SQX in pork samples with a satisfied recovery varied from 94.40% to 95.98%.

An electrochemical aptasensor based on PEI-C3N4/AuNWs for determination of chloramphenicol via exonuclease-assisted signal amplification.

Anelectrochemical aptasensor, including the polyethyleneimine-graphite-like carbon nitride/Au nanowire nanocomposite (PEI-C3N4/AuNWs) and exonuclease-assisted signal amplification strategy was constructedfor the determination of chloramphenicol (CAP). Initially, a nanocomposite with substantial electrocatalytic property was synthesized by PEI-C3N4/AuNWs. This improves the conductivity and specific surface area of the PEI-C3N4/AuNW-modified gold electrode. Next, a DNA with a complementary sequence to a CAP aptamer (cDNA) was immobilized on the PEI-C3N4/AuNW-modified electrode, followed by the CAP aptamer hybridized with cDNA. The lower signal at this time is due to the negatively charged phosphate group of the oligonucleotide and [Fe (CN)6]3-/4- electrostatically repelling each other. The presence of the CAP would cause aptamer on the electrode surface to fall off and be digested by Recjf exonuclease, which resulted in target recycling, and a significant increase in DPV signal can be observed at a potential of 0.176V (vs. Ag/AgCl). Under optimal conditions, there is a linear relationship between the peak current and the logarithm of CAP concentration in the range 100 fM-1μM, and the detection limit of this aptasensor is 2.96 fM (S/N = 3). Furthermore, the resultant aptasensor has excellent specificity, reproducibility, and long-term stability, and has been applied to the detection of CAP in milk samples. Graphical abstract The detection principle of the electrochemical aptasensor for CAP detection was based on PEI-C3N4/AuNWs and exonuclease-assistant signal amplification. It is based on the fact that PEI-C3N4/AuNWs nanocomposites on the surface of the electrode can effectively improve the performance of the aptasensor, and Recjf exonuclease initiates the target recycling process, causes signal amplification.

A signal-on fluorescence-based strategy for detection of microRNA-21 based on graphene oxide and λ exonuclease-based signal amplification.

MicroRNA (miRNA) expression is perturbed in various diseases. Herein, we have aimed to develop a novel and rapid fluorescence-based assay for detecting microRNA-21 (miR-21) activity based on FAM molecular signal amplification and graphene oxide (GO) quenching. In this system, a single stranded DNA (ssDNA) with a phosphate group at the 5'-end is labeled with a FAM molecular label at the 3'-end. In the presence of miR-21, this ssDNA forms a DNA/RNA duplex, which is cleaved by λ exonuclease (λ-exo), releasing FAM and resulting in fluorescence signal amplification at 530 nm. However, the DNA/RNA duplex is not generated in the absence of miR-21, which impedes λ-exo cleavage; subsequently, GO quenches the fluorescence intensity. The results show a detection limit of 0.02 nM and a wide linear range of 0.02-5 nM. The high sensitivity and easy operability of this assay can be applied for detecting miR-21 during clinical diagnosis of certain diseases and in biological research.

Amplified Fluorescent Aptasensor for Ochratoxin A Assay Based on Graphene Oxide and RecJf Exonuclease.

In this study, we developed an aptamer-based fluorescent sensing platform for the detection of ochratoxin A (OTA) based on RecJf exonuclease-assisted signal amplification and interaction between graphene oxide (GO) and the OTA aptamer (OTA-apt). After optimizing the experimental conditions, the present aptamer-based sensing system can exhibit excellent fluorescent response in the OTA assay, with a limit of detection of 0.07 ng/mL. In addition to signal amplification, this strategy is also highly specific for other interfering toxins. Furthermore, this aptasensor can be reliably used for assessing red wine samples spiked with different OTA concentrations (2.4, 6 and 20 ng/mL). The proposed assay plays an important role in the field of food safety and can be transformed for detecting other toxins by replacing the sequence that recognizes the aptamer.

Improving the sensitivity and selectivity of a DNA probe using graphene oxide-protected and T7 exonuclease-assisted signal amplification.

The accurate analysis of single-nucleotide polymorphisms is of great significance for clinical detection and diagnosis. Based on the hybridization hindrance caused by graphene oxide (GO) and hairpin probe, we report a T7 Exo-assisted cyclic amplification technique to distinguish single-base mismatch for highly sensitive and selective detection of mutant-type DNA. When the mutant-type target is completely complementary to the probe, the T7 Exo hydrolyzes the probe and releases the fluorescent molecule from the GO surface, resulting in a fluorescence signal. Conversely, when the wild-type mismatch target is present, the weak hybridization prevents the release of FAM-labeled probe from the GO surface. Therefore, the FAM-labeled probe cannot be degraded efficiently by T7 Exo, and the fluorescence is still quenched by GO. The detection limit of the proposed method can be as low as 34 fM due to the cyclic signal amplification. The experimental results showed that the established method could be used to detect single-nucleotide polymorphisms accurately and sensitively at low cost.

Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection

The expression level of miRNA-21 is closely related to the occurrence and development of cancer, especially in gastrointestinal cancer. Monitoring miRNA-21 has clinical application in the diagnosis and evaluation of gastrointestinal cancer. A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease-mediated cyclic enzymatic amplification method was developed for miRNA-21 determination by using carbon dots (CDs) and FAM-labeled ssDNA as the signal source. CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy. In the absence of miRNA-21, FAM-labeled ssDNA would be adsorbed and quenched by CDs. The addition of miRNA-21 induced cycle hydrolysis from the 5′ end by the T7 exonuclease and then released the short-cleaved FAM-labeled oligonucleotides. Then, the increased FAM signal (FFAM) and the stable CD signal (FCDs) would be tested through a ratiometric routine for the quantification of miRNA-21. The FFAM/FCDs value showed a good linear relationship with the concentration of miRNA-21 in the range of 0.05–10 nM, and the detection limit for miRNA-21 was 1 pM with excellent selectivity and reproducibility. Furthermore, this sensor successfully evaluated the expression level of miRNA-21 in clinical blood samples from healthy individuals and gastrointestinal cancer patients, and the results were highly consistent with those of qRT-PCR, suggesting the great clinical application value in the diagnosis of cancer associated with miRNA-21 expression levels.

An electrochemical aptasensor based on DNA-AuNPs-HRP nanoprobes and exonuclease-assisted signal amplification for detection of aflatoxin B1

It is very essential and meaningful for detection and quantitation of aflatoxin in food and agricultural commodities to establish a rapid and accurate detection method. Herein, a novel electrochemical aptasensor for sensitive and selective detection of aflatoxin B1 (AFB1) was successfully constructed. The thiolated complementary strand (cDNA) of the AFB1 aptamer (Apt) was immobilized on the surface of the glassy carbon electrode (GCE) modified with gold nanoparticles (AuNPs) through Au–S bond. Then Apt was attached on the surface of GCE via by specific base pairing. In the presence of AFB1, the Apt was detached from the electrode surface by specific recognition between the AFB1 and Apt, forming Apt-AFB1 conjugates in solution. The conjugates were digested by exonuclease I (Exo I) to trigger AFB1 recycling. Finally, DNA-AuNPs-horseradish peroxidase (DNA-AuNPs-HRP) nanoprobes was bound with cDNA on the surface of electrode by specific base pairing. HRP could catalyze the oxidation of hydroquinone (HQ) to benzoquinone (BQ) by hydrogen peroxide (H2O2) for producing a strong electrochemical signal. Under the optimized conditions, the electrochemical signals increased with increasing concentrations of AFB1 in a range from 10−3 ng mL−1 to 200 ng mL−1 with a low detection limit of 3.3 × 10−4 ng mL−1. Moreover, the aptasensor has been successfully applied for the determination of AFB1 in peanuts and corn samples and the recoveries were 88.5 %–110.2%, which would provide a novel method for the rapid detection of AFB1 in food samples.

Fluorometric determination of microRNA by using target-triggered cascade signal amplification and DNA-templated silver nanoclusters.

A highly sensitive fluorometric method is described for the determination of let-7a microRNA. It is based on the use of target-triggered cascade signal amplification that involves the use of (a) catalytic hairpin assembly (CHA), (b) exonuclease-assisted signal amplification (EASA), and (c) DNA-templatedfluorescent silver nanoclusters (DNA-AgNCs) having excitation/emission maxima at 535/616nm. The CHA reaction is initiated by the hybridization of the microRNA with hairpin probe HP1. The opening of HP1 results in the assembly of HP1 and another hairpin probe (HP2) to form a duplex. This releases the microRNA that which initiates another CHA reaction. The HP1-HP2 duplex binds to hairpin probe HP3 to form a stable Y-shaped junction structure HP2-HP1-HP3. The Y-shaped junction structure is cleaved by λ exonuclease to recycle the HP1-HP2 duplex to initiate the EASA reaction. This generates a large number of single-stranded reporter sequences. These act as scaffolds for the synthesis of fluorescent AgNCs by reduction of Ag (I) ions. A remarkably amplified fluorescent signal is observed whose intensity increases linearly in the 1 fM to 10nM let-7a microRNA concentration range. The detection limit is 0.89 fM. The method can well discriminate let-7a microRNA from other microRNAs of the same family. Graphical Abstract Schematic representation of a fluorometric method based on target-triggered cascade signal amplification and DNA-templated silver nanoclusters (DNA-AgNCs) for sensitive detection of microRNA (miRNA).

A microchip electrophoresis-based assay for ratiometric detection of kanamycin by R-shape probe and exonuclease-assisted signal amplification

Excessive intake of kanamycin (KANA) can cause some serious drug-resistant diseases, so it is urgent to develop some accurate and rapid analytical methods for monitor KANA residues in foodstuffs with complex matrix. Recently, many ratiometric assays were reported to be capable of overcoming matrix interference. Herein, a ratiometric and homogeneous assay for KANA detection based on microchip electrophoresis (MCE) was developed. First, by one single strand DNA (S-DNA) and one hairpin DNA (H-DNA), a novel R shape DNA probe (R-DNA) was prepared. After the probe was incubated with KANA, the S-DNA-KANA complex was formed, and H-DNA was released. Moreover, in the presence of exonuclease I (Exo-I), S-DNA-KANA complex would be digested to release the captured KANA for triggering target recycling and signal amplification. With the reaction going on, the fluorescence intensity of H-DNA (IH) increased and that of R-DNA (IR) decreased. They can be separated at different voltage intensities and converted to fluorescent signals for signal readout by MCE. The signal ratio of IH/IR was found to be linear toward target from 0.5 pg mL−1 to 10 ng mL−1, and the limit of detection was 150 fg mL−1. Moreover, it was successfully employed for KANA detection in milk and fish samples with consistent results of enzyme linked immune sorbent assay (ELISA). The R-DNA probe can quantitatively convert the amount of target to the intensity of DNA without label by MCE, and achieved exonuclease assisted signal amplification in homogenous solution. It was valuable to detect antibiotics residues in foodstuff with complex matrix. This approach broadened the application field of MCE to detect antibiotics without derivatization, which provided a promising platform for rapid screening of antibiotic residues in food.

Application Progress of Exonuclease-Assisted Signal Amplification Strategies in Biochemical Analysis

Abstract As an important member of the tool enzymes, exonuclease is a kind of hydrolytic enzymes without strict base sequence dependence. In recent years, by taking advantage of different hydrolysis ways of exonuclease and nanotechnology, cycle effect of enzyme digestion, aptamer, non Watson-Crick base pairing system by metal ions, fluorescent nucleic acid probes, electrochemical methods etc., a series of exonuclease-assisted signal amplification strategies have been developed, which have played a key role in improving the sensitivity of detection method. Therefore, exonucleases have been widely used in highly sensitive detection of nucleic acids, proteins, ions, small molecules and so on. To understand it better and apply it well in the future, the application progresses of exonuclease-assisted signal amplification strategies in biochemical analysis have been summarized in this review.

Sensitive and Selective DNA Detection Based on Lambda Exonuclease Assisted Signal Amplification

Abstract A new versatile chemiluminescence biosensing platform with G-Quadruplexes/Hemin DNAzyme and lambda exonuclease (λexo) assisted signal amplification was designed for sensitive detection of DNA. The system involved target DNA, phosphate-DNA, auxiliary DNA and hairpin DNA. The selective digestion of λexo to 5′-phosphorylated strand of phosphate-DNA-auxiliary DNA-target DNA duplex sandwich structure resulted in the release of target DNA and auxiliary DNA. The released target DNA hybridized with another phosphate-DNA-auxiliary DNA duplex to trigger the target DNA recycling, while the released auxiliary DNA hybridized with hairpin DNA to activate DNAzyme segments. The activated DNAzyme segments interacted with hemin to form stable G-Quadruplexes/ Hemin DNAzymes that could catalyze H 2 O 2 -mediated oxidation luminol to produce chemiluminescence signals. In the presence of 10 units λexo, 1.0 × 10 −3 M luminol, 3.0 × 10 −2 M H 2 O 2 and pH 9.0 buffer solution, the relative chemiluminescence intensity of luminol was linearly related with the concentrations of target DNA in the range of 2.0 × 10 −12 M to 8.0 × 10 −9 M. This unique analysis strategy provided a detection limit down to 7.0 × 10 −14 M. The present method was successfully applied to the determination of target DNA in serum samples, which was also capable of discriminating mismatched DNA from perfectly matched target DNA with a high selectivity.

A sensitive strategy for the fluorescence detection of DNA methyltransferase activity based on the graphene oxide platform and T7 exonuclease-assisted cyclic signal amplification.

In this work, a simple fluorescence strategy based on the graphene oxide (GO) platform and T7 exonuclease (T7 Exo)-assisted cyclic signal amplification is developed for the fast and sensitive detection of DNA methyltransferase (MTase) activity and inhibition. In the sensing design, Dam MTase was used as a model analyte. In the presence of Dam MTase, a hairpin probe (HP) was methylated, and then specially recognized and cleaved by Dpn I endonuclease, releasing a ssDNA fragment. The released ssDNA subsequently hybridized with a FAM-labeled signal probe (DP) to form a duplex with a blunt 5'-terminal of DP and a 4-mer overhang at the 5'-end of the released ssDNA. This would trigger the T7 Exo-assisted cyclic signal amplification by repeating the hybridization and digestion of DP, liberating the fluorophore. The liberated fluorophore could not be adsorbed on the GO surface due to low affinity and the fluorescence signal was retained. In contrast, no enzymatic degradation of the DP occurred in the absence of Dam MTase. Thus the intact DP was then adsorbed on the GO surface, resulting in fluorescence quenching. By combining the efficient digestion ability of T7 Exo and the super fluorescence quenching efficiency of GO, the present strategy exhibits a high signal-to-background ratio, providing a satisfying sensitivity for the Dam MTase activity assay. In addition, this method does not require a specific recognition sequence for enzymatic cyclic amplification and dual labels with fluorophore/quencher pairs, making the design easy and low cost. Furthermore, the proposed method was also applied to assay the inhibition of Dam MTase activity. This approach may offer potential applications in clinical diagnostics, drug screening and some other related biomedical research.

A highly sensitive homogeneous electrochemical assay for alkaline phosphatase activity based on single molecular beacon-initiated T7 exonuclease-mediated signal amplification.

Alkaline phosphatase (ALP), a class of enzymes that catalyzes the dephosphorylation of a variety of substrates, is one of the most commonly assayed enzymes in routine clinical practice, and an important biomarker related to many human diseases. Herein, a facile and highly sensitive homogeneous electrochemical biosensing strategy was proposed for the ALP activity detection based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. One 3'-phosphorylated and 5'-methylene blue (MB) labeled hairpin probe (HP) is ingeniously designed. In the presence of ALP, the dephosphorylation of HP, the subsequent Klenow fragment (KF) polymerase-catalyzed elongation and T7 exonuclease-catalyzed digestion of the duplex stem of HP take place, releasing MB-labeled mononucleotides and the trigger DNA (tDNA). tDNA then hybridizes with another HP and initiates the subsequent cycling cleavage process. As a result, a large amount of MB-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the ALP activity assay. A directly measured detection limit as low as 0.1 U L(-1) is obtained, which is comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy previously reported. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits the advantages of simplicity and convenience, because the assay is carried out in the homogeneous solution phase and sophisticated electrode modification processes are avoided. Therefore, the homogeneous electrochemical method we proposed here is an ideal candidate for ALP activity detection in biochemical research and clinical practices.