Fluorescent and polarity-switchable photoelectrochemical dual-mode homogeneous sensing platform for ultrasensitive kanamycin detection based on EXO III-driven signal amplification.

A fluorescent and photoelectrochemical (PEC) dual-mode biosensor based on target biorecognition-triggered cyclic amplification was constructed for Kana detection. With the assistance of the catalyzed reaction of exonuclease III, a Kana-aptamer DNA duplex was designed for conducting the cyclic release of G-rich DNA sequence as well as output DNA S2. The released G-rich sequence triggers the fluorescence (FL) of thioflavin T (ThT), the intensity of which is positively correlated with the Kana concentration. The linear range is 0.2 to 30 nM, and the detection limit reaches 0.07 nM. Simultaneously, the released output DNA S2 was captured by Fe3O4@CdTe-probe ssDNA and then combined with methylene blue to realize the transduction of polarity-reversed PEC signal, leading to the sensitive detection of Kana with a linear range of 0.2 to 40 nM and a calculated detection limit of 0.2 nM. The outstanding performance endows the dual-mode biosensor a promising prospect for practical application.

AgI Precipitation Induced Polarity Reversal with Formation of Z-Type Heterojunction for Photoelectrochemical Sensing.

Regulating photocurrent polarity is highly attractive for fabricating photoelectrochemical (PEC) biosensors with improved sensitivity and accuracy in practical samples. Here, a new approach that adopts the in situ generated AgI precipitate and AgNCs to reversal Bi2WO6 polarity with formation of Z-type heterojunction was proposed for the first time, which coupled with a high-efficient target conversion strategy of exonuclease III (Exo III)-assisted triple recycling amplification for sensing miRNA-21. The target-related DNA nanospheres in situ generated on electrode with loading of plentiful AgI and AgNCs not only endowed the photocurrent of Bi2WO6 switching from the anodic to cathodic one due to the changes in the electron transfer pathway but also formed AgI/AgNCs/Au/Bi2WO6 Z-type heterojunction to improve the photoelectric conversion efficiency for acquiring extremely enhanced PEC signal, thereby significantly avoiding the problem of high background signal derived from traditional unidirectional increasing/decreasing response and false-positive/false-negative. Experimental data showed that the PEC biosensor had a low detection limit down to 0.085 fM, providing a new polarity-reversal mechanism and expected application in diverse fields, including biomedical research and clinical diagnosis.

DNA domino circuits based on a hairpin exonuclease assistance signal transmission architecture for temporal logic operations.

DNA circuits are important fundamental tools for performing temporal logic operations with complex structures, but they lack sequence orthogonality. Here, we developed a simple and orthogonal hairpin exonuclease assistance signal (H-EAST) architecture to construct DNA domino circuits with time-delay characteristics and temporal logic operations, which has potential applications in biomolecular computing.

Inverted Sandwich-Type e-LCR Aided by Lambda Exonuclease-RecJf Combination Enables Ultrasensitive Detection of Low-Frequency EGFR-L858R Mutation in NSCLC.

Highly sensitive detection of low-frequency EGFR-L858R mutation is particularly important in guiding targeted therapy of nonsmall-cell lung carcinoma (NSCLC). To this end, a ligase chain reaction (LCR)-based electrochemical biosensor (e-LCR) with an inverted sandwich-type architecture was provided by combining a cooperation of lambda exonuclease-RecJf exonuclease (λ-RecJf exo). In this work, by designing a knife-like DNA substrate (an overhang ssDNA part referred to the "knife arm") and introducing the λ-RecJf exo, the unreacted DNA probes in the LCR were specially degraded while only the ligated products were preserved, after which the ligated knife-like DNA products were hybridized with capture probes on the gold electrode surface through the "knife arms", forming the inverted sandwich-type DNA structure and bringing the methylene blue-label close to the electrode surface to engender the electrical signal. Finally, the sensitivity of the e-LCR could be improved by 3 orders of magnitude with the help of the λ-RecJf exo, and due to the mutation recognizing in the ligation site of the employed ligase, this method could detect EGFR-L858R mutation down to 0.01%, along with a linear range of 1 fM-10 pM and a limit detection of 0.8 fM. Further, the developed method could distinguish between L858R positive and negative mutations in cultured cell samples, tumor tissue samples, and plasma samples, whose accuracy was verified by the droplet digital PCR, holding a huge potential in liquid biopsy for precisely guiding individualized-treatment of NSCLC patients with advantages of high sensitivity, low cost, and adaptability to point-of-care testing.

A recognition-induced three-dimensional bipedal DNA walker for highly sensitive detection of APE1.

In contrast to the unipedal DNA walker, a bipedal DNA walker features a broader walking area and exhibits faster walking kinetics, leading to enhanced amplification efficiency. In this study, we designed a stochastic three-dimensional (3D) bipedal DNA walker, capable of navigating AuNP-based 3D tracks, driven by exonuclease III (Exo III). This detection system enables the linear detection of the non-invasive biomarker apurinic/apyrimidinic endonuclease 1 (APE1) activity across a range of 0 to 120 U per mL, with a detection limit of 0.03 U per mL. The platform not only offers a novel DNA walker for sensitive APE1 detection in cell lysate but also facilitates the precise assessment of NCA's inhibitory effect on APE1. This research holds promise for future screening of other potential APE1 inhibitors.

Label-free ratiometric fluorescence detection of Pb2+via structure-specific fluorescent dyes and dual signal amplification.

Lead ions (Pb2+) are a widely distributed and highly toxic heavy metal pollutant, which seriously threatens the environment, economy and human safety. Here, a label-free ratiometric fluorescent biosensor was constructed for Pb2+ detection using DNAzyme-driven target cycling and exonuclease III (Exo III)-mediated DNA cycling as a dual signal amplification strategy. The SYBR Green I (SGI) and N-methyl mesoporphyrin IX (NMM) used in this study are characterized by low cost, storage resistance, and short preparation time compared with conventional signaling probes labeled with fluorescent groups. Unlike the single-emission fluorescence strategy, monitoring the fluorescence intensity ratio of SGI and NMM can effectively reduce external interference to achieve accurate detection of Pb2+. DNAzyme structures on the surface of magnetic beads (MBs) can recognize Pb2+ and activate the target circulatory system to cleave single-stranded DNA (ssDNA). The ssDNA further initiated the Exo III-assisted DNA circulatory system to digest double-stranded DNA (dsDNA) and release guanine-rich G1. Finally, the fluorescence signals of SGI and NMM were weakened and enhanced, respectively. The sensing strategy achieved a wide linear range from 0.5 to 500 nM and a low limit of detection (LOD) of 26.4 pM. Furthermore, its anti-interference ability and potential applicability for Pb2+ detection in actual samples were verified. This work ingeniously combines the dual signal amplification strategy with the ratiometric sensing strategy constructed by structure-specific fluorescent dyes, which provides a promising method for constructing sensitive and accurate fluorescent biosensors.

Hairpin self-assembly powered by exonuclease III for highly sensitive and cross-validated miRNA-155 detection.

Cancer is one of the most important causes of human death and poses a serious threat to human health. As a cancer biomarker, microRNA-155 (miRNA-155) is highly expressed in various types of cancer tissues and is involved in the proliferation of tumor cells. Therefore, developing a miRNA-155 detection technology with high specificity and sensitivity is of great significance for the early detection, accurate treatment and prognostic evaluation of tumors. Here, we developed a fluorescence detection method using exonuclease III-assisted target cycling and catalytic hairpin assembly (CHA) as a signal amplification technique. This study developed a biosensor for the detection of miRNA-155, utilizing a DNA hairpin (Hp) for target recognition and generating double-stranded DNA (dual-Hp-T). The 3' flat end of the double-stranded DNA can be cleaved by exonuclease III to achieve the target cycle, and a large amount of single-stranded DNA (fuel) can trigger CHA to achieve signal amplification. Simultaneously, the fluorescence resonance energy transfer (FRET) of signal probes with different fluorescence labels on H1 and H2 ends occurs with the CHA reaction. The two fluorescence signals obtained can be used to cross-validate the experimental results. The biosensor exhibits excellent performance of high recovery, high sensitivity and high operability, which can achieve the specific detection of miRNA-155 with a detection limit as low as 8.3 pM. Additionally, the detection efficacy in a human serum environment is also highly satisfactory. This technology provides strong technical support for the development of nucleic acid probes and the diagnosis and treatment of cancer, demonstrating significant practical application value.

Construction of a DNA walker nanomachine aptasensor for simultaneous detection of dual-cancer biomarkers.

While it is recognized that early diagnosis of cancer-related biomarkers can become an effective avenue for timely treatment and successfully improve patient survival, it remains challenging to get accurate inspection results. Currently, most reported cancer biomarker sensing methods are focused on the quantitative detection of a single type of biomarker, which makes accurate medical diagnostics difficult. In this work, we constructed a DNA walker nanomachine aptasensor based on gold nanoparticles for the simultaneous sensing of dual cancer biomarkers. The aptamers, labelled with a fluorophore, hybridized with complementary strands on the gold nanoparticle surface, serve as a walking track. Target analytes bind to their specific aptamers, leading to the dissociation of the unstable double-strand spherical nucleic acid. Exonuclease I (Exo I) selectively digested the aptamers bound with the target analytes, then the released targets go back to the next apamers on the gold nanopareticles surface for walking. The use of spherical nucleic acid probes improved the sensitivity of analyte detection. Exo I provided a driving power for target recycling and considerably improved the sensitivity of the aptasensor as well. The DNA walker nanomachine aptasensor was successfully applied for the detection of carcinoembryonic antigen (CEA) in the range of 0.167 to 3.34 ng mL-1, and mucin-1 (MUC-1) in the same range. Moreover, we used the two aptamers to construct the DNA walker nanomachine and achieved the simultaneous detection of CEA and MUC-1, thus having great potential for biomolecular logic gate construction and early disease diagnosis.

Stochastic bipedal dual-DNA walkers for fast and sensitive detection of apurinic/apyrimidinic endonuclease1 and inhibitor screening.

DNA walkers have emerged as a powerful tool in various biosensors, enabling the detection of low-abundance analytes with their precise programmability and efficient signal amplification capacity. However, many existing approaches are hampered by limited reaction kinetics. Herein, we designed a stochastic bipedal dual-DNA walkers (SBDW) that can traverse at high speed on AuNP-based three-dimensional (3D) tracks powered by Exo III. The SBDW exhibited superior reaction kinetics and are up to least 2.25 times faster than traditional DNA walkers, reaching a plateau within 40 min. This advancement allows for rapid and highly sensitive fluorescence detection of a significant base excision repair enzyme of APE1 with a detection limit of 0.001 U/mL. In comparison to traditional DNA walkers, this platform enables highly sensitive and specific APE1 assays in cell lysate and facilitates rapid and accurate screening of APE1 inhibitors. Given its rapid, sensitive, specific, and reliable analysis features, the strategy shows great promise in drug discovery and clinical diagnosis.

A sensitive tobramycin electrochemical aptasensor based on multiple signal amplification cascades.

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

Exonuclease III/Cas12a Cascade Amplification Strategy and Smartphone-Based Portable Fluorescence Detector to Repurpose the Commercial AFP Strip for the POCT of Multiple RNAs.

Point of care testing (POCT) of nucleic acid (NA) contributes to the timely disease diagnosis, like bacteria and virus screening in households or resource-constrained areas, but its development has always been stagnant. Herein, we proposed an exonuclease III cascaded with CRISPR/Cas12a (Exo-III/Cas12a) amplification strategy and constructed a smartphone-based portable fluorescence detector (SPFD) to repurpose the commercial alpha-fetoprotein (AFP) strip for the ultrasensitive and hand-held detection of NA samples. In detail, the target-initiated-Exo-III/Cas12a strategy realizes the signal amplification and liberates AFP from magnetic beads through the trans-cleavages of activated Cas12a toward the AFP aptamer. After magnetic separation and migration, the fluorescence signals of the test (FT) and control (FC) lines on the AFP strip were digitally output by the SPFD, and the FT/FC was employed for the quantitative analysis to minimize external disturbances and improve accuracy. We experimentally assessed the universe applicability of the proposed NA-POCT platform toward miRNA-155, 16S rRNA of Staphylococcus aureus, and ORF1a/b RNA of Covid-19 pseudovirus, achieving favorable detection limits of 42 aM, 18 CFU/mL, and 87 copies/µL, respectively. Moreover, its simplicity, universality, and admirable detection performance demonstrate a great potential in the aspect of rapidly transforming the existing POCT devices for multiple new applications at the time of need.

An exonuclease III-driven dual-mode aptasensor based on Au-Pd@Fc nanozyme and magnetic separation pretreatment for aminoglycoside antibiotics detection.

A novel dual-mode aptasensor was constructed for aminoglycoside antibiotics (AAs) detection by using a broad-spectrum aptamer as a biorecognition element, and Au-Pd@Fc functionalized by signal DNA as nanoprobes. In electrochemical mode, the target-induced cyclic amplification reaction run under the action of exonuclease-III, which increased the number of nanoprobes on the electrode surface. AAs could be quantitatively detected with LOD of 0.0355 ± 0.00613 nM. In colorimetric mode, the Au-Pd@Fc nanozyme catalyzed the color reaction of 3,3',5,5'-tetramethylbenzidine. The blue-shifted absorbance will be observed with the change of AAs concentration, and the LOD was 0.0458 ± 0.00572 nM. Furthermore, a magnetic molecular-imprinted material capable of specific adsorption of AAs was prepared on milk sample pretreatment. The aptasensor was used to detect 10 kinds of AAs in milk and the recoveries were 97.19 ± 4.41% âˆ¼ 98.70 ± 4.45% and 96.38 ± 3.53%-97.54 ± 4.13% in electrochemical and colorimetric methods. This work provided a theoretical basis for the application of aptamers in simultaneous detection of antibiotics.

Visual detection of microRNAs using gold nanorod-based lateral flow nucleic acid biosensor and exonuclease III-assisted signal amplification.

An ultrasensitive method for the visual detection of microRNAs (miRNAs) in cell lysates using a gold nanorod-based lateral flow nucleic acid biosensor (GN-LFNAB) and exonuclease III (Exo III)-assisted signal amplification. The Exo III-catalyzed target recycling strategy is employed to generate a large number of single-strand DNA products, which can be detected by GN-LFNAB visually. With the implementation of a unique recycling strategy, we have demonstrated that the miRNA in the concentration as low as 0.5 pM can be detected without the need for instrumentation, providing a detection limit that surpasses previous reports. The new biosensor is ultrasensitive and can be applied to the reliable monitoring of miRNAs in cell lysates with high accuracy. The approach offers a simple and rapid tool for cancer diagnosis and clinical biomedicine, thanks to its flexibility, simplicity, cost-effectiveness, and convenience. This new method has the potential to significantly improve the detection and monitoring of cancer biomarkers, ultimately contributing to more effective cancer diagnosis and treatment.

The biochemical characterization of a TatD nuclease from Thermus thermophilus.

Nucleases play pivotal roles in DNA repair and apoptosis. Moreover, they have various applications in biotechnology and industry. Among nucleases, TatD has been characterized as an exonuclease with various biological functions in different organisms. Here, we biochemically characterized the potential TatD nuclease from Thermus thermophilus. The tatD gene from T. thermophilus was cloned, then the recombinant TatD nuclease was expressed and purified. Our results revealed that the TthTatD nuclease could degrade both single-stranded and double-stranded DNA, and its activity is dependent on the divalent metal ions Mg2+ and Mn2+. Remarkably, the activity of TthTatD nuclease is highest at 37 °C and decreases with increasing temperature. TthTatD is not a thermostable enzyme, even though it is from a thermophilic bacterium. Based on the sequence similarity and molecular docking of the DNA substrate into the modeled TthTatD structure, several key conserved residues were identified and their roles were confirmed by analyzing the enzymatic activities of the site-directed mutants. The residues E86 and H149 play key roles in binding metal ions, residues R124/K126 and K211/R212 had a critical role in binding DNA substrate. Our results confirm the enzymatic properties of TthTatD and provide a primary basis for its possible application in biotechnology.

Simultaneous Hg2+ and Pb2+ detection in water samples using an electrochemical aptasensor with dual signal amplification by exonuclease III and metal-organic frameworks.

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.

Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

Exo â ¢-assisted amplification signal strategy synergized with Au@Pt NFs/CoSe2 for sensitive detection of enrofloxacin.

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.

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

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

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.

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.