ANALYST
·
2014
·Vol. 139
·期 24
·6387-6392
·
IF 3.906
Chunyan Gao,Henan Li,Yuanjian Liu,Wei Wei,Yuanjian Zhang,Songqin Liu
Strategies to detect the methylation of site specific DNA and assay of M.SssI methyltransferase (M.SssI MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaII and exonuclease III (Exo III). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaII and degradation reaction of Exo III, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.SssI MTase, the action of HpaII and Exo III was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The fluorescence intensity has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL−1 with a detection limit of 0.16 U mL−1 in terms of 3 times deviation of the blank sample. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.
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MICROCHIMICA ACTA
·
2019
·Vol. 186
·期 8
·1-7
·
IF 5.479
Li Mei,Xu Xiong,Cai QingYou,Luo XuJian,Zhou ZhongGao,Xu GuoHai,Xie YongRong
A graphene oxide-based method has been developed for ultrasensitive and selective determination of microRNA-141 by means of rolling circle amplification (RCA) and exonuclease III (Exo III)-assisted recycling amplification. The method uses (a) a padlock probe with a hybrid sequence that is complementary to the target microRNA-141 at both the 5'- and the 3'-end, and (b) a long binding region of a signalling reporter strand. On addition of microRNA-141, it acts as the primer for triggering the RCA reaction following ligation. This results in the formation of a repeatedly concatenated sequence of the padlock probe. Subsequently, the RCA product hybridizes with the fluorescein-labelled signal strand to form the duplex DNA containing the blunt 3'-termini of signal strand. Addition of Exo III causes signal strand digestion and leads to RCA product recycling and liberation of fluorescein. Added graphene oxide does not absorb the fluorescein liberated because of the poor mutual interaction. Therefore, microRNA-141 can be quantified by measurement of the green fluorescence under excitation/emission wavelengths of 470/520 nm. The method has a 100 aM detection limit towards microRNA-141 and works in the wide range from 1 fM to 1 nM. It can discriminate even single-mismatched microRNA and shows good selectivity and sensitivity when applied to spiked human serum.
Schematic representation of a graphene oxide (GO)-based method for fluorometric determination of microRNA by using rolling circle amplification and exonuclease III (Exo III)-aided recycling amplification. With microRNA, the fluorescein-labelled signal strand becomes digested, and this genererates a fluorescent signal.
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JOURNAL OF ELECTROANALYTICAL CHEMISTRY
·
2022
·
IF 4.598
Zhixian Liang,Meng Chen,Xing Huang,Yanli Tong,Qiyou Wang,Zuanguang Chen
Protein p53 induces cell growth arrest, differentiation, apoptosis and DNA repair. More than 50% of malignant tumors produce mutations in the p53 gene. Hence, p53 sequence-specific analysis helps in early cancer diagnosis, increasing the treatment success rate. The single nucleic acid amplification method limits sensitivity, so we innovatively integrated exonuclease III (ExoIII)-assisted target recycling amplification with multi-site enzyme polymerization labeling. The integration dramatically improved the biosensor’s sensitivity. Given the nonspecific reaction of ExoIII, we optimized the ExoIII amplification system to reduce false positives and background current. The single-strand DNA produced in the ExoIII system served as the bridge between the electrode and signal DNA tetrahedron (sTDN). Moreover, sTDN provided accurate enzyme loading and well-organized spatial arrangement for multi-site enzymatic labeling. Compared with single-stranded signal probes, sTDN modified by terminal transferase provided higher catalytic efficiency. Under the optimal experimental conditions, the cathodic current exhibited a logarithmic relation over 1.0–5.0 × 10 6 fM p53, with a detection limit of 0.47 fM. Applying this biosensor has successfully detected p53 in serum samples, providing a potential tool for cancer diagnosis and treatment.
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ANALYST
·
2022
·Vol. 147
·期 22
·4991-4999
·
IF 5.227
Jinyue Shi,Yu Lin,Weiling Qin,Mingxiang Li,Yuyi Zhou,Yeyu Wu,Hu Luo,Ke-Jing Huang,Xuecai Tan
Graphdiyne (GDY) is an sp and sp2 co-hydrocarbon allotrope whose particular structure endows it with many fascinating properties, including abundant chemical bonds, high conjugation, natural pores, high carrier mobility, high conductivity and stability, etc. In this work, two-dimensional graphdiyne is prepared as an electrode substrate material coupling with an exonuclease III-assisted amplification strategy to construct a superior-performance self-powered biosensor based on enzymatic biofuel cells for highly sensitive detection of the tumour marker miRNA-21. Glucose oxidase (GOD) is first immobilized on the GDY/AuNP composite to prepare a bioconjugate. GDY/AuNP modified carbon cloth is used as an enzyme biofuel cell electrode, which is then modified with bilirubin oxidase as a biocathode. The bioconjugate binds to GOD through specific binding to the bioanode. When miRNA-21 is present, specific recognition by exonuclease III in the system results in cleavage of the capture probe, and miRNA-21 is recovered and involved in the cycle. The target miRNA-21 then causes corresponding changes in the open-circuit voltage of the self-powered system. Based on this, a sensitive detection method was constructed, within the scope from 0.1 fM to 0.1 nM with a shallow detection limit of 55.2 aM (S/N = 3). The new approach triumphantly has been used to detect miRNA-21 in serum, which provides a compelling new way for early diagnosis of related cancers.
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高影响因子
ACS Applied Materials & Interfaces
·
2021
·
IF 9.229
Senyao Wang,Baoshan He,Ying Liang,Huali Jin,Min Wei,Wenjie Ren,Zhiguang Suo,Jinshui Wang
Herein, a hierarchically porous Zr-MOF-labeled electrochemical aptasensor based on the composite of PtPd@Ni-Co hollow nanoboxes (PtPd@Ni-Co HNBs) and poly (diallyldimethylammonium chloride)-functionalized graphene (PDDA-Gr) was developed for ultrasensitive detection of chloramphenicol (CAP). PtPd@Ni-Co HNBs have excellent conductivity and provide binding sites for aptamers; the functionalized PDDA-Gr improves its dispersibility and conductivity as a substrate material, which can be successfully used to increase the electrode surface area and support more PtPd@Ni-CoHNBs. Besides, hierarchically porous Zr-MOFs (HP-UiO-66) were utilized as signal probes and showed a stronger load capacity for signal molecules than conventional UiO-66. In the presence of CAP, two ingeniously designed Exo III-assisted cyclic amplification strategies further improved the sensitivity of the aptasensor: CAP causes cycle I to release a large amount of trigger DNA (Tr DNA), and then, Tr DNA initiated cycle II, which causes the exposed capture DNA to further bind the signal probes. With these advantages, the constructed aptasensors performed with satisfactory sensitivity in a wide linear range (10 fM–10 nM) and a detection limit of 0.985 fM. Several signal amplification strategies adopted in this study have effectively improved the performance of the sensor, providing a new avenue for the development of ultrasensitive sensors in the food analysis field.
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Journal of Chemistry
·
2023
·
IF 3
Ping Wang,Yaoyao Xie,Zhimin Guan,Huikai Ma,Shoumin Xi
Prostate specific antigen (PSA) is considered as an important biomarker in prostate cancer diagnosis. Herein, an ultrasensitive ratiometric electrochemical aptasensor was constructed for analyzing PSA. The ferrocene (Fc)-labeled thiolated hairpin probe 2 (Fc-HP2) was conjugated to the gold nanoparticles (AuNPs)-modified gold electrode (AuE) surface by Au-S bonds. With the introduction of PSA, the aptamer region of hairpin probe 1 (HP1) preferred to bind with PSA, and the rest of HP1 could hybridize with Fc-HP2 to form Fc-HP2/HP1/PSA complex with a blunt 3′ terminus, which triggered the exonuclease III (Exo III) cleavage process accompanied by Fc leaving from the electrode and recycling of HP1/PSA. The remaining Fc-HP2 fragments left on the electrode surface were then hybridized with methylene blue-labeled DNA (MB-DNA). This resulted in an enhancement of MB signal (IMB) and a decrement of Fc signal (IFc). Under the optimal conditions, the proposed aptasensor showed good analytical performance for sensitive detection of PSA with a linear range from 100 fg·mL−1 to 10 ng·mL−1 and a detection limit of 34.7 fg·mL−1. The fabricated aptasensor had been applied to detect PSA in diluted human serum samples and obtained satisfied results, suggesting it had great potential in clinical diagnosis.
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LUMINESCENCE
·
2024
·Vol. 39
·期 1
·e4675
·
IF 3.2
Gerile Oudeng,Junguo Ni,Hao Wu,Honglian Wu,Mo Yang,Chunyi Wen,Yuanwei Wang,Hui Tan
The coronavirus disease-2019 pandemic reflects the underdevelopment of point-of-care diagnostic technology. Nuclei acid (NA) detection is the “gold standard” method for the early diagnosis of the B.1.1.529 (Omicron) variant of severe acute respiratory syndrome-coronavirus disease-2. Polymerase chain reaction is the main method for NA detection but requires considerable manpower and sample processing taking ≥ 3 h. To simplify the operation processes and reduce the detection time, exonuclease III (Exo III)-aided MoS 2 /AIE nanoprobes were developed for rapid and sensitive detection of the oligonucleotides of Omicron. Molybdenum disulfide (MoS 2 ) nanosheets with excellent optical absorbance and distinguishable affinity to single-strand and duplex DNAs were applied as quenchers, and aggregation-induced emission (AIE) molecules with high luminous efficiency were designed as donor in fluorescence resonance energy transfer-based nanoprobes. Exo III with catalytic capability was used for signal amplification to increase the sensitivity of detection. The composite nanoprobes detected the mutated nucleocapsid (N)-gene and spike (S)-gene oligonucleotides of Omicron within 40 min with a limit of detection of 4.7 pM, and showed great potential for application in community medicine.
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ANALYTICA CHIMICA ACTA
·
2021
·
IF 6.558
Xinyan Yang,Lihan Wang,Lidong Pang,Shiqian Fu,Xue Qin,Qing Chen,Chaoxin Man,Yujun Jiang
A novel fluorescent platform of DNA-stabilized silver nanoclusters (DNA-AgNCs) has been developed based on exonuclease III (Exo III) amplification-assisted for simple and sensitive detection of Salmonella Typhimurium ( S. Typhimurium). The platform was designed by using magnetic beads, aptamer, its complementary DNA , hairpin probe (HP), Exo III, AgNO 3 , and NaBH 4 . The functionalized HP contained a cytosine-rich oligonucleotide loop (C-rich loop), which served as an effective template for the chemical reduction of Ag + with NaBH 4 to synthesize DNA-AgNCs. In the presence of S. Typhimurium, the C-rich loop was converted into an open form of ssDNA by the recycle digestion of Exo III, leading to a corresponding decrease in fluorescence intensity . Based on the fluorescence changes of the formed DNA-AgNCs, the sensitive detection of S. Typhimurium was achieved. Under the optimal conditions, a wide linear relationship was observed in the concentration of S. Typhimurium ranging from 4.6 × 10 2 to 4.6 × 10 7 cfu mL −1 with the limit of detection (LOD) being 82 cfu mL −1 . The method showed good selectivity for detecting S. Typhimurium. In addition, the platform could be used for the detection of S. Typhimurium in milk samples. The LOD reached 6.6 × 10 2 cfu mL −1 with a good linear range, indicating that the method had excellent practicability in complex food samples.
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BIOSENSORS & BIOELECTRONICS
·
2016
·
IF 7.476
Wei Wang,Ting Bao,Xi Zeng,Huayu Xiong,Wei Wen,Xiuhua Zhang,Shengfu Wang
In this work, a novel and ultrasensitive electrochemical biosensor was constructed for DNA detection based on functionalized gold clusters/graphene nanohybrids (AuNCs/GR nanobybrids) and exonuclease III (Exo III)-aided cascade target recycling. By utilizing the capacity of GR as universal template, different metal nanoclusters including AuNCs/GR nanobybrids and PtNCs/GR nanohybrids were synthesized through convenient ultrasonic method. Exo III-aided cascade recycling was initiated by target DNA, generating the final cleavage product (S2), which acted as a linkage between capture probe and the functionalized metal nanoclusters/GR conjugates in the construction of the biosensor. The AuNCs/GR-DNA-enzyme conjugates acted as interfaces of enzyme-catalyzed silver deposition reaction, achieving DNA detection ranging from 0.02 fM to 20 pM with a detection limit of 0.057 fM. In addition, PtNCs/GR-DNA conjugates presented peroxidase-like activity and the functionalized PtNCs/GR nanohybrids-based electrochemical biosensor also realized DNA detection by catalyzing the 3,3′,5,5′-tetramethylbenzidine-hydrogen peroxide (TMB-H 2 O 2 ) system to produce electrochemical signal. This metal clusters/GR-based multiple-amplified electrochemical biosensor provided an universal method for DNA detection.
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Analytical Methods
·
2022
·Vol. 14
·期 7
·734-740
·
IF 3.532
Mei Liu,Shasha Liu,Yue Ma,Baoxin Li
Specific and sensitive detection of hazardous mycotoxins in agricultural crops is one of the most important goals of food safety. A fluorescence biosensor for sensitive detection of ochratoxin A (OTA) was constructed via magnetic beads and the exonuclease III (Exo III)-assisted trigger DNA circle amplification approach. Exo III-assisted trigger DNA circle amplification can be utilized as an effective strategy for the sensitive detection of OTA. The employment of streptavidin labeled magnetic beads offers a manner for the accumulation and separation of the hairpin signal probe sDNA-FAM in solution. After target specific recognition, the aptamers combined with OTA were released and the remaining block DNA (bDNA) probes captured the signal probes on magnetic bead modified fluorophores. Subsequently, the enzyme digestion reaction leads to the fluorophore free solution. Exo III-assisted DNA circle amplification contributed to the high sensitivity of the presented OTA fluorescence aptasensor. The experimental results demonstrate that the aptasensor is sensitive with the limit of detection as low as 0.28 ng mL−1 for OTA, which was lower than that of the proposed aptasensors reported by the other literature on fluorescence methods. Additionally, the developed aptasensor with the diverse aptamer sequence shows promising potential applications in food monitoring.
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CHEMOMETRICS AND INTELLIGENT LABORATORY SYSTEMS
·
2018
·
IF 2.701
Cai-Xia Shi,Qing Liu,Zeng-Ping Chen,Ru-Qin Yu
Exonuclease III assisted target recycling amplification strategy can be used to enhance the sensitivity of mass spectrometry for the detection of short DNA sequences. However, the distribution pattern of DNA fragments produced by exonuclease III assisted target recycling amplification is generally different for samples with different concentrations of the target DNA sequence, which hinders the extraction of both qualitative and quantitative information of the target DNA from mass spectral measurements using traditional univariate or multivariate models. In this contribution, an advanced model was derived based on a reasonable assumption for the qualitative and quantitative analysis of the mass spectral measurements of DNA fragments produced by exonuclease III-assisted target recycling amplification. Experimental results demonstrated that the integration of exonuclease III assisted target recycling amplification, mass spectrometry and the advanced model could achieve sensitive and accurate quantitative results for a target short DNA sequence in complex biological medium with a detection limit of 50 pM and a mean recovery rate within the range of 89.5%–106.7%. More interestingly, the proposed model could unambiguously identify single nucleotide polymorphisms based on the distribution patterns of residual DNA fragments. Therefore, with the aid of the proposed model, mass spectrometry based on exonuclease III assisted recycling amplification has great potential for the reliable, sensitive, selective, and relatively low-cost detection and quantification of short DNA sequences in clinical diagnosis and biomedical research.
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MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 7
·1-12
·
IF 5.3
Tao Chunxu,Wang Junyan,Zhu Ying,Ding Chao,Shen Zhuoyue,Sun Danni,Cao Shanshan,Jiang Xinrong,Li Yaqi,Liu Chang,Zhang Qi,Li Shijie,Zhang Xinyan,Shi Qiaoqiao,Kong Dezhao
A fluorescence biosensor for determination of aflatoxin B 1 (AFB 1 ) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB 1 aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB 1 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. Graphical abstract
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危险品化学品经营许可证（带存储）