Heavy metal pollution in the environment has become a significant global concern due to its detrimental effects on human health and the environment. In this study, we report an electrochemical aptasensor for the simultaneous detection of Hg2+ and Pb2+. Gold nanoflower/polyethyleneimine-reduced graphene oxide (AuNFs/PEI-rGO) was introduced on the surface of a gold electrode to improve sensing performance. The aptasensor is based on the formation of a T-Hg2+-T mismatch structure and specific cleavage of the Pb2+-dependent DNAzyme, resulting in a dual signal generated by the Exo III specific digestion of methylene blue (MB) labeled at the 3\' end of probe DNA-1 and the reduction of the substrate ascorbic acid (AA) catalyzed by the signal label. The decrease of MB signal and the increase of AA oxidation peak was used to indicate the content of Hg2+ and Pb2+, respectively, with detection limits of 0.11 pM (Hg2+) and 0.093 pM (Pb2+). The aptasensor was also used for detecting Hg2+ and Pb2+ in water samples with good recoveries. Overall, this electrochemical aptasensor shows promising potential for sensitive and selective detection of heavy metals in environmental samples.

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.

Overuse of enrofloxacin (ENR) has posed a potential threat to ecosystems and public health, so it is critical to sensitive and accurate determination of ENR residues. In this work, a novel ultra-sensitive and specific electrochemical aptasensor was fabricated based on the cobalt diselenide loaded gold and platinum nanoflowers (Au@Pt NFs/ CoSe2) and Exonuclease III (Exo III)-assisted cycle amplification strategy for the detection of ENR. Au@Pt NFs/ CoSe2 nanosheets as the substrate material, with large surface area, accelerate electron transfer and attach more DNA probes on the electrode substrate, have effectively enhanced the electrochemical performance of the electrode. With the existence of Enrofloxacin (ENR), the aptamer recognizes and binds to ENR, thus the signal probe cDNA was released and immobilized onto the electrode surface to hybridized with methylene blue (MB) labelled DNA (MB-DNA), thereby triggering the Exo III-assisted cycle for further signal amplification. As expected, the prepared aptasensor demonstrated excellent sensitivity and selectivity, with a wide linear range from 5.0 × 10-6 ng/mL to 1.0 × 10-2 ng/mL for ENR, a low detection limit of 1.59 × 10-6 ng/mL. Consequently, this strategy provided a promising avenue for ultrasensitive and accurate detection of ENR in milk samples.

BACKGROUND: Sensitive and selective analysis of low content nucleic acid sequences plays an important role in pathogen analysis, disease diagnosis and biomedicine. The electrochemical biosensor based on toehold-mediated strand displacement reaction (TMSD) is highly attractive in nucleic acid detection due to their improved sensitivity and rapid response. But the traditional TMSD carried out on the electrode always with low displacement efficiency and complicated electrode operation, resulting in compromised sensing performance. There is a great need to construct a novel TMSD based electrochemical detection strategy to overcome such challenges in nucleic acid detecting.
RESULTS: Herein, a triple signal amplification electrochemical aptasensor was developed for ultrasensitive detection of CYFRA21-1 DNA. The dual-output toehold mediated strand displacement reaction (dTMSD) can convert one input to two strands output within one strand displacement cycle. So that it possesses a higher efficiency for improving the sensitivity in comparison with the single-output TMSD. And the fuel strand was configured with a tail to realize successive DNA circuits through self-propelling as a DNA walker. All the above processes were carried out on magnetic beads, which is conducive to achieving effective sample purification and minimizing the background signals. Besides, Exonuclease III was further amplified signal. As a result, through the cascade use of above three technologies, the proposed biosensing strategy realized sensitive detection of target DNA with a low detection limit of 0.35 fM (S/N = 3) and wide linear range (0.5 fM-500 pM).
CONCLUSIONS: The proposed novel dTMSD combining multiple signal amplification strategies for electrochemical detection of CYFRA21-1 DNA with easy operation not only possesses excellent sensitivity and selectivity, but also has potential application value for monitoring DNA in serum. Meanwhile, the development of highly sensitive and specific CYFRA21-1 DNA detection methods is very important for the prevention and treatment of lung cancer.

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.

Dual-mode sensing has attracted more attentions which provide more accurate and reliable approach of cancer-related biomarkers. Herein, we developed a novel SERS/electrochemical dual-mode biosensor for miRNA 21 detection based on Exo III-assisted signal transformation. Firstly, the Au NPs were deposited on electrode as SERS substrate and Mn3O4/S4(DNA signal strand) was modified on Au NPs/S5 by the DNA strands S5-S4 pairing principle as hydrogen peroxide catalyst, leading to an obviously high DPV electrical signal without Raman signal. Subsequently, the presence of miRNA 21 will activate the Mn3O4/S4 to be decomposed under exonuclease III-assisted process, then the S3\' chains modified with Raman molecular Cy3(Cy3-S3\') is continuously connected to the Au NPs/S5 by DNA stands S5-S3\' pairing principle, leading to the Raman signal response and DPV signal reduction. The biosensor shows good linear calibration curves of both SERS and electrochemical sensing modes with the detection limit of 3.98 × 10-3 nM and 6.89 × 10-5 nM, respectively. This work finds an ingenious mode for dual detection of microRNA on a same interface, which opens a new strategy for SERS and electrochemical analysis.

This study presents a technique for detecting 3\'-5\' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3\'-5\' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5\'-end of a hairpin probe (HP), forming a blunt end. When the 3\'-end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3\'-end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

BACKGROUND: Tobramycin (TOB), an essential aminoglycoside antibiotic in human life, poses potential threats due to its residues in the environment. The primary concern is the adverse impact of excessive TOB on human kidneys, hearing, and other organs, significantly affecting human health. Constructing a sensitive electrochemical platform for simple and rapid trace detection is crucial. Herein, to enhance the sensitivity of TOB detection in the environment and mitigate the risks associated with residual antibiotics, an ultrasensitive electrochemical aptasensor was developed.
RESULTS: The sensor employs a dual-cycle amplification strategy involving catalytic hairpin assembly (CHA) and exonuclease III (Exo III) for efficient signal amplification. Simultaneously, the electrode performance was optimized by incorporating gold nanowires (AuNWs) onto the surface of reduced graphene oxide (PDA-rGO). Specifically, in the presence of TOB, which binds to the aptamer (Apt), dsDNA dissociates, releasing cDNA to open hairpin 1 (HP1) and initiate the CHA cycle with the participation of hairpin 2 (HP2). Exo III shears HP1 in the HP1/HP2 complex, freeing HP2 to participate in the CHA cycle again. Ultimately, a significant amount of signal label is retained on the electrode by hybridizing with sheared HP1, generating a robust electrical signal.
CONCLUSIONS: Through the signal amplification strategy, the aptasensor design provides a broad linear range of 0.005-500 nM, with a low detection limit of 0.112 pM for TOB. It is worth mentioning that the aptasensor displayed favorable stability, specificity, and reproducibility, and has been successfully applied to practical samples, demonstrating its utility in practical applications.

At present, a large number of studies have demonstrated that miRNAs can be used as biological indicators for the diagnosis and treatment of diseases such as tumours and cancer, so it is important to develop a new miRNA detection platform. In this work, miRNA-122 is used as the basis for targeting detection agents. We have designed an unlabelled DNA1 that undergoes partial hybridisation and has a 20 T base long strand. The fluorescent signal in this experiment is derived from copper nanoclusters (CuNCs) generated on the circular T-long strand of DNA1. At the same time, DNA1 is able to react with miRNA-122 and achieve hydrolysis of the part bound to miRNA-122 via the action of nucleic acid exonuclease III (Exo III), leaving a part of the DNA, called DNA3, while releasing miRNA-122 to participate in the next reaction, thus achieving circular amplification. DNA3 is able to react with DNA2, which is bound to streptavidin magnetic beads (SIBs) and separated from the reaction solution via the application of a magnetic field. Overall, this is a fluorescence signal reduction experiment, and the strength of the fluorescence signal from the copper nanoclusters can determine whether the target miRNA-122 is present or not. The degree of fluorescence reduction indicates how much DNA1, and thus the amount of target miRNA-122, has been hydrolysed. By evaluating the variations in the fluorescence signal under optimised conditions, we discovered that this method has good sensitivity, with a detection limit as low as 0.46 nM, better than many other previous works on fluorescence signal-based biosensors for miRNA detection. This technique offers high discrimination and selectivity and can serve as a persuasive reference for early diagnosis.

The development of a contamination-free and on-site nucleic acid detection platform with high sensitivity and specificity but low-cost for the detection of pathogenic nucleic acids is critical for infectious disease diagnosis and surveillance. In this study, we combined the recombinase-aided amplification (RAA) with the exonuclease III (Exo III)-assisted signal amplification into a platform for sensitive and specific detection of nucleic acids of African swine fever virus (ASFV). We found that this platform enabled a naked eye visual detection of ASFV at a detection limit as low as 2 copies/μL in 30 min. As expected, no cross-reactivity was observed with other porcine viruses. In addition, to avoid aerosol contamination, a one-tube RAA-Exo III colorimetric assay was also established for the accurate detection of ASFV in clinical samples. Taken together, we developed a rapid, instrument-free, and low-cost Exo III-assisted RAA colorimetric-assay-based nucleic acid detection platform.