MICROCHEMICAL JOURNAL
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2022
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IF 5.304
Wanling Cui,Xiaoyang Fan,Congcong Shen,Qingshuai Chen,Xiumei Zhang,Enguang Lv,Chonghui Li,Qiang Li,Rui Wang,Shicai Xu,Jihua Wang
T4 polynucleotide kinase (T4 PNK) and T4 DNA ligase are two important repair enzymes that play a significant role in maintaining the stability and integrity of genome. Here, we proposed a multiple primers-mediated exponential rolling circle amplification strategy for label-free and highly sensitive fluorescent analysis of T4 PNK and T4 DNA ligase activity. Firstly, we designed a target strand that contained two C-rich sequences, a complementary sequence of the primer, and three cleavage sites of Nt.BbvCI scattered among the three sequences. The 5′-hydroxyl terminus of target strand was catalyzed by T4 PNK to form a phosphorylated target strand. After the phosphorylated target strand was combined with the primer, T4 DNA ligase catalyzed the formation of circular DNA. Then, the primer triggered an exponential rolling circle amplification reaction along the circular DNA template, exponentially generating G-rich sequences. N-methyl mesoporphyrin IX as signal molecules specifically bound to the G-quadruplexs folded by G-rich sequences, generating a detectable fluorescent signal. Benefiting from exponential signal growth, this strategy showed extremely high detection sensitivity with detection limits of 8.11 × 10 −5 U mL −1 of T4 PNK and 3.07 × 10 −4 U mL −1 of T4 DNA ligase, respectively. In addition, the proposed strategy exhibited excellent inhibitor evaluation effects for two repair enzymes, and could detect T4 PNK in HeLa cell lysate. The obtained results showed that the proposed strategy had excellent application prospects in disease diagnosis and treatment evaluation.
研究领域:
生物化学
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分子生物学
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医学诊断
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DOI
高影响因子
Biosensors-Basel
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2023
·Vol. 13
·期 9
·875
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IF 5.4
Ziang Wu,Roujuan Kou,Kun Ni,Rui Song,Yuxuan Li,Tianhu Li,Hao Zhang
DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of phosphodiester bonds between juxtaposed 5′ phosphate and 3′ hydroxyl termini in double-stranded DNA. In addition to their critical roles in maintaining genomic integrity, DNA ligases have been recently identified as diagnostic biomarkers for several types of cancers and recognized as potential drug targets for the treatment of various diseases. Although DNA ligases are significant in basic research and medical applications, developing strategies for efficiently detecting and precisely quantifying these crucial enzymes is still challenging. Here, we report our design and fabrication of a highly sensitive and specific biosensor in which a stable DNA hairpin is utilized to stimulate the generation of fluorescence signals. This probe is verified to be stable under a wide range of experimental conditions and exhibits promising performance in detecting DNA ligases. We anticipate that this hairpin-based biosensor will significantly benefit the development of new targeting strategies and diagnostic tools for certain diseases.Keywords:DNA ligases;extraordinarily stable hairpins;biomarker detection;fluorescence probes;molecular sensing
研究领域:
分子生物学
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医学诊断
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生物化学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2023
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IF 8.4
Qing Han,Xinlan Bian,Yue Chen,Bingzhi Li
Flap endonuclease I (FEN1) is a structure-specific nuclease that participates in DNA repair, and its overexpression is related to an increased risk of cancer. Currently, few FEN1 assays can meet the requirements for diagnostic purposes due to their high complexity, low specificity, and limited sensitivity. Herein, we developed a biosensor adopting synthetic biology strategy, called CLAIM (cleavage-ligation-activation-involved monitoring) for FEN1 quantification. CLAIMintegrates ligation-based T7 promoter repair, RNA generation, and CRISPR/Cas13a. FEN1 cleavages the DNA probe with a 5′-flap to generate a nick site that can be ligated to form a T7 promoter. Subsequent transcription produces abundant RNA that can activate Cas13a, generating a fluorescence signal due to the trans -cleavage of Cas13a to a fluorogenic reporter. CLAIM was able to detect FEN1 with low femtomolar sensitivity, and its accuracy was validated using samples of cancer and non-tumorigenic cell lines. Interestingly, we found that the utilization of thermostable T4 DNA ligase is critical to make the cascade of FEN1 cleavage, ligation, and transcription take place in one-pot. CLAIM was also repurposed for validating FEN1 inhibitors, indicating its potential in drug screening. This work provides a diagnostic tool for FEN1 analysis and opens up opportunities for FEN1 biosensing and related drug discovery.
研究领域:
分子生物学
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合成生物学
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癌症诊断
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生物技术
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2021
·
IF 4.821
Qian Zhao,Guangyin Zhang,Dai Lu,Kejun Feng,Xingbo Shi
Herein, an ultra-sensitive aptasensor based on dual-enzyme mediated cascade- signal amplification (dubbed Demcsa aptasensor) for ampicillin (AMP) detection is proposed. The sensor’s activity involves two major steps, i.e., DNA ligase-assisted Exonuclease III (Exo III) catalyzed degradation reaction in a homogeneous solution, signal generation/amplification through the biotin-streptavidin linkage and hybridization chain reaction (HCR) on a 96-well polystyrene plate. Firstly, a detection probe was formed via the hybridization reaction between biotinylated AMP aptamer (bio-AP), HCR initiator strand (H strand) and complementary DNA (cDNA). As a result of the dual-enzyme mediated degradation, a long APH strand was formed via the DNA ligase-catalyzed ligation of bio-AP and the H strand, which served as integrated initiator to trigger the HCR reaction, thereby producing a long double-stranded DNA polymer. Subsequently, the SYBR Green I, as fluorescent dye for DNA, was added and a strong fluorescence signal was generated. In the presence of AMP, the function of DNA ligase was effectively suppressed due to the higher affinity of target-AP strand complex. As a result, the HCR reaction could not be triggered without generating a fluorescence signal, because the bio-AP cannot ligate to the H strand even when captured on the plate. The cascade- signal amplification of biotin-streptavidin linkage and the HCR reaction endowed the Demcsa aptasensor with a limit of detection (LOD) of aM, which was comparable or superior to those of the previously reported sensors. The Demcsa aptasensor has been used for the screening of AMP in food samples with favorable recoveries. The results of this work pave a new, universal way for developing efficient sensors for the detection of other trace food hazards.
研究领域:
分析化学
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生物传感器
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食品科学
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分子生物学
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2014
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IF 6.451
Lu Lu,Jing Cao Si,Zhong Feng Gao,Yu Zhang,Jing Lei Lei,Hong Qun Luo,Nian Bing Li
A dual strategy that combines the adenosine triphosphate (ATP)-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection is employed to construct the biosensor. In this design, the methylene blue-labeled hairpin-structured DNA was self-assembled onto a gold electrode surface to prepare the modified electrode through the interaction of Au–S bond. In the procedure of ATP-dependent ligation reaction, when the specific cofactor ATP was added, the two split oligonucleotide fragments of 8-17 DNAzyme were linked by T4 DNA ligase and then released to hybridize with the labeled hairpin-structured DNA substrate. The linked 8-17 DNAzyme catalyzes the cleavage of the hairpin-structured substrate by the addition of Zn 2+ , causing the methylene blue which contains high electrochemical activity to leave the surface of the gold electrode, therefore generating a dramatic decrease of electrochemical signal. The decrease of peak current was readily measured by square wave voltammetry and a relatively low detection limit (0.05 nM) was obtained with a linear response range from 0.1 to 1000 nM. By taking advantage of the highly specific cofactor dependence of the DNA ligation reaction, the proposed ligation-induced DNAzyme cascades demonstrate ultrahigh selectivity toward the target cofactor ATP. A catalytic and molecular beacons strategy is further adopted to amplify the electrochemical signal detection achieved by cycling and regenerating the 8-17 DNAzyme to realize enzymatic multiple turnover, thus one DNAzyme can catalyze the cleavage of several hairpin-structured substrates, which improves the sensitivity of the newly designed electrochemical sensing system.
研究领域:
生物化学
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电化学
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生物传感器
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纳米技术
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DOI
PubMed
高影响因子
NEW JOURNAL OF CHEMISTRY
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2019
·Vol. 43
·期 36
·14327-14335
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IF 3.069
Wancun Zhang,Fang Hu,Xianwei Zhang,Wei Meng,Yaodong Zhang,Yinsen Song,Huanmin Wang,Peng Wang,Yueqing Gu
In this study, a simple, robust, label-free electrochemical biosensor based on the ligase chain reaction (LCR) was developed for the highly sensitive and selective detection of single nucleotide polymorphisms (SNPs) using Fe(CN)63−/Fe(CN)64− as a redox indicator. High sensitivity and specificity were achieved by using the LCR, which employs a thermostable and single-base discerning Hifi Taq DNA ligase to repeatedly ligate adjacently hybridized LCR-specific probes during thermal cycles, resulting in the highly specific exponential amplification of DNA targets. The electrochemical signal of the biosensor was further decreased using stem-loop probe DNA (SL-DNA), which specifically hybridized with LCR products on the electrodes. The linear dynamic range and high sensitivity of the mutant DNA (MutDNA) and wild-type DNA (WtDNA) LCR-based electrochemical biosensors for the detection of MutDNA and WtDNA were studied in vitro, with a broad linear dynamic range of 10 aM to 10 pM and detection limits of 1.18 aM and 0.35 aM, respectively. In addition, the MutDNA and WtDNA LCR-based electrochemical biosensors were able to accurately detect allele frequency changes as low as 0.1%. In summary, the developed LCR-based electrochemical biosensor provides a useful platform to monitor SNPs in a highly sensitive and specific manner.
研究领域:
生物传感
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分子生物学
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电化学
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遗传分析
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
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2020
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IF 5.977
Fang Hu,Wancun Zhang,Wei Meng,Yuxiang Ma,Xianwei Zhang,Ying Xu,Peng Wang,Yueqing Gu
Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A′, and B′, where B′ is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A′B′ in 2 n . Subsequently, immobilization of the Fc-tagged duplex AB-A′B′ on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A′B′, such strategy realized directly analysis of LCR products without the need for purification . By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM–10 pM), and the recovery rates (95.3%–107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.
研究领域:
生物化学
·
生物技术
·
分析化学
·
分子生物学
·
医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）