Exonuclease-iii -propelled DNAzyme cascade for sensitive and reliable cervical cancer related miRNA analysis.

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences.

A simple and sensitive colorimetric approach for mecA gene analysis via exonuclease-III catalyzed signal cascade.

Analysis of mecA gene in Staphylococcus aureus (S. aureus) is essential for controlling infections in intensive care units (ICU) and preventing the use of ineffectual empirical treatments. However, quantitative determination of the mecA gene remains difficult. Herein, we propose a simple and sensitive colorimetric approach by integrating exonuclease-III (Exo-III) assisted signal cascade and G-quadruplex/hemin DNAzymes (G4 DNAzymes) catalyzed 2,2'-azino-bis (3-ethylben-zothiazoline-6-sulfonic acid) (ABTS) based color reaction. In this method, signal amplification does not necessitate the use of complex experimental components, such as multiple enzymes and primer design, while still maintaining a high signal amplifying efficiency. Therefore, the method has a broad mecA gene detection range from 10 fM to 1 nM and a low limit of detection down to 3.4 fM level. Taking the merit of simplicity and high sensitivity, the approach is promising in analyzing mecA gene in S. aureus and diagnosing infections.

A highly sensitive fluorescence biosensor for aflatoxins B1 detection based on polydiacetylene liposomes combined with exonuclease III-assisted recycling amplification.

A fluorescence biosensor for determination of aflatoxin B1 (AFB1) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB1 aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB1 by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1-100 ng/mL with a determination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheat samples. The stability, accuracy, and specificity of the method demonstrated the applicability for real sample analysis.

Exonuclease III-propelled DNAzyme walker: an electrochemical strategy for microRNA diagnostics.

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100 pM and achieves a detection limit of 5 fM (3σ criterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics.

An Exo III-powered closed-loop DNA circuit architecture for biosensing/imaging.

With their regulated Boolean logic operations in vitro and in vivo, DNA logic circuits have shown great promise for target recognition and disease diagnosis. However, significant obstacles must be overcome to improve their operational efficiency and broaden their range of applications. In this study, we propose an Exo III-powered closed-loop DNA circuit (ECDC) architecture that integrates four highly efficient AND logic gates. The ECDC utilizes Exo III as the sole enzyme-activated actuator, simplifying the circuit design and ensuring optimal performance. Moreover, the use of Exo III enables a self-feedback (autocatalytic) mechanism in the dynamic switching between AND logic gates within this circulating logic circuit. After validating the signal flow and examining the impact of each AND logic gate on the regulation of the circuit, we demonstrate the intelligent determination of miR-21 using the carefully designed ECDC architecture in vitro. The proposed ECDC exhibits a linear detection range for miR-21 from 0 to 300 nM, with a limit of detection (LOD) of approximately 0.01 nM, surpassing most reported methods. It also shows excellent selectivity for miR-21 detection and holds potential for identifying and imaging live cancer cells. This study presents a practical and efficient strategy for monitoring various nucleic acid-based biomarkers in vitro and in vivo through specific sequence modifications, offering significant potential for early cancer diagnosis, bioanalysis, and prognostic clinical applications.

Highly sensitive and stable fluorescent aptasensor based on an exonuclease III-assisted amplification strategy for ATP detection.

Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L-1 to µmol L-1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.

Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity.

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3'-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks. To assay the activity of Exo III, we applied the previous reports in that the peroxidase-mimicking activity of nanoceria is inhibited due to the aggregation induced by the electrostatic attraction between DNA and nanoceria. In the presence of Exo III, the substrate DNA (subDNA), which inhibits nanoceria's activity, is degraded, thereby restoring the peroxidase-mimicking activity of nanoceria. Consequently, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate is oxidized, leading to a color change from colorless to blue, along with an increase in the absorbance intensity. This approach enabled us to reliably detect Exo III at a limit of detection (LOD) of 0.263 units/mL across a broad dynamic range from 3.1 to 400 units/mL, respectively, with an outstanding specificity. Since this approach does not require radiolabels, complex DNA design, or sophisticated experimental techniques, it provides a simpler and more feasible alternative to standard methods.

A novel ratiometric electrochemical aptasensor for highly sensitive detection of carcinoembryonic antigen.

A novel ratiometric electrochemical aptasensor was proposed for carcinoembryonic antigen (CEA) detection based on exonuclease III (Exo III)-assisted recycling and rolling circle amplification (RCA) strategies. A thiolated ferrocene-labeled hairpin probe 2 (Fc-HP2) was fixed on the gold nanoparticles (AuNPs)-modified gold electrode (AuE) surface through Au-S bonds. The presence of CEA led to the release of trigger, which hybridized with the 3'-protruding of hairpin probe 1 (HP1) and triggered the Exo III cleavage reaction, accompanied by the releasing of trigger and generation of new DNA fragment which was used for the successive hybridization with Fc-HP2. After the Exo III cleavage process, the remaining Fc-HP2 fragments hybridized as primers with the RCA template to initiate the RCA process, and long single-stranded polynucleotides were produced for methylene blue (MB) binding. Such changes resulted in the signal of Fc (IFc) decreased and that of MB (IMB) increased, achieving a linear relationship between IMB/IFc and logarithm of CEA concentrations ranging from 1.0 pg mL-1 to 100.0 ng mL-1 with a detection limit of 0.59 pg mL-1. Additionally, the developed aptasensor had been successfully applied to detect CEA in human serum samples. Therefore, the proposed strategy might provide a new platform for clinical detections of CEA.

Target-mediated competitive hybridization of hairpin probes for kanamycin detection based on exonuclease III cleavage and DNAzyme catalysis.

Based on aptamer recognition and target-mediated competitive hybridization of hairpin probes, we developed a fluorescence sensor for kanamycin (KAN) detection. The aptamer and KAN binding will open hairpin H1 to release the trigger DNA fragment, which can initiate the competitive hybridization between hairpins H2 and H3. Then, exonuclease III (Exo III) can cleave H2 and H3 to produce numerous DNA3 and DNA4. Through the synergetic hybridization among DNA1, DNA2, DNA3, and DNA4, an active Mg2+-DNAzyme can be formed. The cleavage reaction toward FAM-BHQ-modified DNA2 will produce a high fluorescence signal for KAN assay. Through Exo III-guided cleavage and Mg2+-DNAzyme-based catalysis, the sensor exhibits high sensitivity, with a detection limit of 3.1 fM. This method is robust and has been applied to the detection of KAN in milk and water samples with good accuracy and reliability. Our developed fluorescence sensor exhibits the advantages of simple operation, high sensitivity, and good robustness, which are beneficial for KAN detection in food samples.

A low-noise ratiometric fluorescence biosensor for detection of Pb2+ based on DNAzyme and exonuclease III-assisted cascade signal amplification.

As a common heavy metal ion with strong toxicity and wide distribution, lead ions (Pb2+) had great harm to the human body. In this work, a low-noise ratiometric fluorescence biosensor was developed based on Pb2+-dependent DNAzyme and exonuclease III (Exo III)-assisted cascade signal amplification. Firstly, the substrate chain of DNAzyme (S-DNA) was modified on the surface of magnetic beads (MBs) through the combination of biotin and streptavidin, and then the enzyme chain of DNAzyme (E-DNA) was connected to the MBs by forming a double-stranded DNA (dsDNA) with S-DNA. A hairpin DNA (HP) labelled with Cy3 and Cy5 respectively at both ends was used as a fluorescence probe. The emission peaks of Cy3 and Cy5 can appear at 562 nm and 665 nm respectively, and their fluorescence intensity ratio (F562/F665) was chosen as the acquisition signal. The ratiometric sensor can reduce the interference of detection environment and avoid false positive reactivity. Due to the cleavage of DNAzyme and the release of single-stranded DNA (ssDNA) in the presence of Pb2+, the hairpin structure of HP was opened and the FRET between two fluorophores disappeared, resulting in the strengthened signal of Cy3 and the weakened signal of Cy5. Furthermore, the ratio [Formula: see text] signal increased gradually with the increase of Pb2+ concentration. When the concentration of Pb2+ was in the range of 0.1-1000 nM, [Formula: see text] had a good linear relationship with [Formula: see text], the correlation coefficient (R2) was 0.997, and the limit of detection (LOD) was 77 pM. The presented ratiometric fluorescence biosensor had lower LOD and wider detection range via comparing with other methods. At the same time, the sensor also obtained the satisfactory results for detection of Pb2+ in tap water, tea, and rice flour samples. The provided ratiometric biosensor has great potential in the monitoring of various targets. A low-noise ratiometric fluorescence biosensor based on the FRET between two fluorophores was developed, and the DNAzyme and exonuclease III-assisted cascade signal amplification was used to improve the sensitivity of the method. The biosensor had a detection limit as low as 77 pM.

Ultrasensitive colorimetric strategy for Hg2+ detection based on T-Hg2+-T configuration and target recycling amplification.

A novelty aptasensor for ultrasensitive detection of Hg2+ is developed, exploiting the combination of plasmonic properties of gold nanoparticles (AuNPs) and exonuclease III (Exo III)-assisted target recycling for signal amplification. In the presence of Hg2+, a DNA duplex can be formed due to the strong coordination of Hg2+ and T bases of single-stranded DNA (ssDNA) probe. Exo III digests the DNA duplex from the 3' to 5' direction, resulting in the releasing of Hg2+. Then, the released Hg2+ binds with another ssDNA probe through T-Hg2+-T coordination. After Exo III-assisted Hg2+ cycles, numerous ssDNA probes are exhausted, which promotes poly(diallyldimethylammonium chloride) (PDDA)-induced AuNP aggregation, leading to an obvious color change and aggregation-induced plasmon red shift of AuNPs (from 520 to 610 nm). Therefore, this biosensor is ultrasensitive, which is applicable to the detection of trace level of Hg2+ with a linear range from 5 pM to 0.6 nM and an ultralow detection limit of 0.2 pM. Furthermore, it enables visual detection of Hg2+ as low as 50 pM by the naked eye. More importantly, the assay can be applied to the reliable determination of spiked Hg2+ in sea water samples with good recovery.

An aptamer-exonuclease III (Exo III)-assisted amplification-based lateral flow assay for sensitive detection of Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7 (E. coli O157:H7), one of the most widespread foodborne pathogens, can cause a series of diseases and even lead to death. In this study, a highly sensitive method was developed by combining aptamer-exonuclease III (Exo III)-assisted amplification with lateral flow assay (LFA) based on gold nanoparticles (AuNP). The compound of single-stranded (ss) DNA-anti-E. coli O157:H7 aptamer (ssDNA-aptamer) was formed by hybridization between designed target ssDNA and aptamer. When E. coli O157:H7 was present, target bacteria were bound with the aptamer, and the free target ssDNA was hybridized with the probes of the designed hairpin (HP) structure. Exo III digests the 3' double-stranded blunt end of the complex and releases the enzyme product. Because the remaining sequence of the HP of the designed enzyme product was the same as the target ssDNA sequence, the target ssDNA could be amplified. Finally, the enhanced target ssDNA was combined with AuNP-LFA to achieve visual detection of E. coli O157:H7. The quantitative ability of this platform for E. coli O157:H7 was 7.6 × 101 cfu/mL in pure culture, and the detection limit in milk was 8.35 × 102 cfu/mL. This LFA was highly specific to E. coli O157:H7, and the time for detection of E. coli O157:H7 in milk was 4 h. Hence, this system has important application prospects in the detection of pathogenic bacteria in dairy products.

One-step and ultrasensitive ATP detection by using positively charged nano-gold@graphene oxide as a versatile nanocomposite.

A versatile nanocomposite was simply prepared based upon the electrostatic adsorption of positively charged gold nanoparticles with negatively charged graphene oxide (nano-gold@GO), and utilized as a novel fluorescence quenching platform for ultrasensitive detection of adenosine triphosphate (ATP). In the designed system, DNA-stabilized Ag nanoclusters (DNA/AgNCs) were used as fluorescent probes, DNA duplex was formed in the presence of ATP, and they can electrostatically adsorb onto the surface of nano-gold@GO to quench the fluorescence signal. Upon the addition of exonuclease III (Exo III), the DNA duplex would be hydrolyzed into DNA fragments and resulted in the recovery of the fluorescence signals due to the diffusion of AgNCs away from nano-gold@GO. Based on these, sensitive detection of ATP was realized with a detection range of 5.0 pM-20 nM. Notably, a good recovery in the range of 94-104% was obtained when detecting ATP in human serum samples, indicating a promising application value in early disease diagnosis. Graphical abstract A functional positively charged nano-gold@graphene oxide was fabricated and utilized as an enhanced fluorescence quenching platform for the detection of ATP, coupled with exonuclease III-assisted signal amplification.

A label-free electrochemical magnetic aptasensor based on exonuclease III-assisted signal amplification for determination of carcinoembryonic antigen.

A novel label-free and exonuclease III (Exo III)-assisted signal amplification electrochemical aptasensor was constructed for the determination of carcinoembryonic antigen (CEA) via magnetic field-induced self-assembly of magnetic biocomposites (Fe3O4@Au NPs-S1-S2-S3). The magnetic biocomposites were acquired by modifying double-stranded DNA (S1-S2-S3) on the surface of Fe3O4@Au nanoparticles (Fe3O4@Au NPs). Among them, Fe3O4@Au NPs were used as carriers for magnetic separation, thiolated single-stranded DNA (S1) provided signal sequence, CEA aptamer (S2) worked as a recognition element, and complementary strand (S3) was used to form double strands. In the presence of CEA, S2 bonded with CEA competitively; the exposed S1 could not be cleaved since Exo III was inactive against ssDNA. The G-quadruplex/hemin complexes finally formed with the existence of K+, and the high electrochemical signal of G-quadruplex/hemin complexes was recorded by differential pulse voltammetry (DPV) at - 0.6 V. Conversely, in the absence of CEA, dsDNA was cleaved from the 3' blunt end by Exo III; the disappearance of G-rich sequence blocked the generation of the signal. This method exhibited good selectivity and sensitivity for the determination of CEA; the linear range was from 0.1 to 200 ng mL-1 and the limit of detection was 0.4 pg mL-1. Graphical abstract.

A simple enzyme-assisted cascade amplification strategy for ultrasensitive and label-free detection of DNA.

A simple fluorescence biosensor is developed based on the enzyme-assisted cascade amplification strategy. The amplification system consists of a hairpin-structure DNA (H-DNA) and exonuclease III. The target DNA can hybridize with the H-DNA and initiate exonuclease III-assisted target recycling amplification to generate abundant G-rich DNA (G-DNA). One region of G-DNA is designed to possess the same sequence as target DNA. Thus, the G-DNA can also hybridize with H-DNA and initiate the digestion of H-DNA. The cascade strategy in this amplification system causes the concentration of G-DNA to grow exponentially. The fluorescence intensity of N-methylmesoporphyrin IX (NMM) is highly enhanced due to the formation of G-quadruplex configuration. Under optimal conditions, the cascade system could achieve an admirable sensitivity with a detection limit of 52 fM for HIV DNA, and guarantees a satisfactory specificity. Moreover, the cascade system could be implemented for other target DNA detections by substituting the recognition region of the H-DNA. In this way, a detection limit of 65 fM for HBV DNA could be achieved by the cascade system. The target DNA analysis in a real serum sample further indicates that this biosensor has potential for future application in clinical diagnosis. Graphical abstract A simple and label-free cascade amplification strategy is developed by exploiting hairpin DNA and EXO III for sensitive DNA detection.

Target-induced in-situ formation of fluorescent DNA-templated copper nanoparticles by a catalytic hairpin assembly: application to the determination of DNA and thrombin.

A fluorometric method is described for nucleic acid signal amplification through target-induced catalytic hairpin assembly with DNA-templated copper nanoparticles (Cu NPs). The toehold-mediated self-assembly of three metastable hairpins is triggered in presence of target DNA. This leads to the formation of a three-way junction structure with protruding mononucleotides at the 3' terminus. The target DNA is released from the formed branched structure and triggers another assembly cycle. As a result, plenty of branched DNA becomes available for the synthesis of Cu NPs which have fluorescence excitation/emission maxima at 340/590 nm. At the same time, the branched structure protects the Cu NPs from digestion by exonuclease III. The unreacted hairpins are digested by exonuclease III, and this warrants a lower background signal. The method can detect ssDNA (24 nt) at low concentration (44 pM) and is selective over single-nucleotide polymorphism. On addition of an aptamer, the strategy can also be applied to the quantitation of thrombin at levels as low as 0.9 nM. Graphical abstractSchematic representation of target-induced catalytic hairpin assembly to form branched DNA template for the in situ synthesis of fluorescent Cu nanoparticles.

A fluorometric aptamer-based assay for ochratoxin A by using exonuclease III-assisted recycling amplification.

A fluorometric assay is described for ochratoxin A (OTA) using an aptamer. The method is based on exonuclease-assisted recycling amplification. The OTA-binding aptamer partially hybridizes with complementary DNA (cDNA) that is released when the aptamer recognizes OTA. Then, cDNA hybridizes with a specifically designed hairpin DNA. Next, short ssDNA and cDNA are, respectively, released by exonuclease III catalyzed hydrolysis of the dsDNA. The cDNA induces the next ring opening and digestion. The short ssDNA captures the sDNA that is labeled with fluorescent FAM and is absorbed on graphene oxide (GO). The green fluorescence of the sDNA/GO system is quenched but is recovered if the sDNA is released from GO. This assay is high sensitive, works in the 5 nM to 200 nM OTA concentration range and has a 0.96 nM lower detection limit. It was applied to the quantitation of OTA in spiked wine and coffee samples. Graphical abstractSchematic of a fluorometric assay based on exonuclease-assisted recycling amplification for quantitative monitoring of OTA without the need of sample separation and multiple washing steps.

Graphene oxide and enzyme-assisted dual-cycling amplification method for sensitive fluorometric determination of DNA.

A fluorometric method is described for the determination of DNA. It involves the use of graphene oxide (GO), exonuclease III (Exo III), and two specially designed fluorophore-labeled hairpin probes (HP1 and HP2). Different from other GO-based DNA assays, the method takes advantage of the distinct binding abilities of GO with hairpin DNA probes and single nucleotides. GO serves as a strong quencher for fluorescent labels to ensure a very low background signal. Two reaction cycles mediated by Exo III are employed to enhance the signals. The combination of GO-induced quenching and Exo III-mediated dual regeneration of analytes leads to a detection limit as low as 1 pM for the model analyte human hemochromatosis protein (HFE) gene. The method is also applicable for the determination of HFE gene spiked into fetal bovine serum. Graphical abstract Schematic representation of a GO-based, Exo III-assisted method for dual-signal amplified detection of DNA, for which human haemochromatosis protein (HFE) gene is designed as the model target. The assay involves graphene oxide (GO), exonuclease (Exo III), and two specially designed, fluorophore-labelled hairpin probes (HP1 and HP2).

Fluorometric determination of mercury(II) by using thymine-thymine mismatches as recognition elements, toehold binding, and enzyme-assisted signal amplification.

A highly sensitive fluorometric method is described for the determination of mercury(II) ions. It is based on (a) the use of a DNA probe containing thymine-thymine mismatches that are employed as Hg(II) recognition elements, (b) subsequent toehold binding, and (c) endocuclease-assisted signal amplification. Target recycling is triggered by exonuclease III. This produces a large amount of ssDNA (defined as primer). Then, the generated primer-initiated strand displacement reaction with the help of polymerase and nicking endonuclease releases the free fluorophore-labelled probe. Under excitation at 532 nm, the fluorescent probe displays emission with a peak at 582 nm. The sensitivity of this method is improved by introduction of nicking endonuclease. The working range of the assay extends from 20 pM to 10 nM, and the detection limit is as low as 6 pM of Hg(II). Graphical abstract Schematic presentation of the fluorometric method for determination of mercury(II). By using a special structure of thymine-thymine mismatches, target-induced toehold binding and enzyme-assisted signal amplification strategy were employed. This method is selective and good performance in real sample application.

Fluorometric determination of mercury(II) using positively charged gold nanoparticles, DNA-templated silver nanoclusters, T-Hg(II)-T interaction and exonuclease assisted signal amplification.

The authors describe a method for detection of Hg2+ by using positively charged gold nanoparticles ((+)AuNPs) as a quencher of the fluorescence of DNA-capped silver nanoclusters (DNA-AgNCs) which are negatively charged. In the presence of Hg2+, a DNA duplex is formed through T-Hg2+-T coordination chemistry. The duplex can be digested by exonuclease III to form smaller DNA fragments. This leads to the release of the AgNCs and the recovery of fluorescence, best measured at excitation/emission wavelengths of 460/530 nm. The (+)AuNPs and Hg2+ are also released and can be reused for target recycling signal amplification. Based on these findings, a method is worked out for the determination of Hg2+ that works in the 5.0 pM to 10 nM concentration range and has a detection limit as low as 2.3 pM. It is highly selective because of the highly specific formation of T-Hg2+-T bonds. Graphical abstract By using ultrastable and positively charged gold nanoparticles as fluorescence quenchers and exonuclease assisted signal amplification, a method is developed for the sensitive and selective detection of Hg2+ in water samples.