TALANTA
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2025
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IF 6.1
Tao Meng,Qi Kang,Jiashuo Xu,Shuang Zhao,Tianqi Liu,Dianming Zhou,Xiaoqun Gong,Jianyu Zhang
The CRISPR/Cas12a system has become a powerful tool in biosensing because of its specific target recognition ability and highly efficient trans -cleavage activity. However, a problem faced by the CRISPR/Cas12a system when directly used for trace detection is the linear amplification efficiency of single-cycle digestion. Here, we present a novel hairpin reporter-driven CRISPR/Cas12a (HR-CRISPR) amplification system that establishes a positive feedback loop within the CRISPR/Cas12a platform to finish an exponential and sensitive signal amplification in a one-step reaction. As proof of concept, we applied this strategy to the terminal deoxynucleotidyl transferase (TdT) activity assay without pre-amplification procedure. The polyT strand extended by TdT hybridizes with crRNA, activating Cas12a, which then cleaves the FQ-hairpin reporter. The cleavage products are further elongated by reverse transcriptase using crRNA as a template, reactivating Cas12a and producing exponentially amplified fluorescence signals. This assay offers a simple yet highly sensitive approach for quantifying TdT activity, achieving a low detection limit of 4.55 × 10 −6 U. Moreover, it is applicable for inhibitor screening and monitoring TdT activity in human serum samples.
研究领域:
分子生物学
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生物技术
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医学检测
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
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2017
·Vol. 409
·期 28
·6677-6688
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IF 3.431
Hu Yufang,Zhang Qingqing,Xu Lihua,Wang Jiao,Rao Jiajia,Guo Zhiyong,Wang Sui
Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient “signal-on” electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3’-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H 2 O 2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3′-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery.
Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.
研究领域:
生物化学
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分子生物学
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分析化学
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2024
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IF 8
Wenjun Dong,Caizhao Cheng,Jiayun Chen,Xingyu Zhao,Zhiyong Guo,Yufang Hu,Zhihe Qing
Terminal deoxynucleotidyl transferase (TdT) plays a pivotal role in lymphocyte development, antigen receptor diversity, and leukemia progression. In this study, a smartphone-assisted dual-mode detection system is introduced for the precise and visual quantification of TdT activity in cells. The system leverages the Ag(I)-o-phenylenediamine (OPD) reaction, where fluorescence intensity, UV–vis absorption and colorimetric changes generate multimodal signals. The inhibition of Ag(I)-catalyzed OPD oxidation results from the formation of stable C-Ag-C complexes, driven by strong interactions between Ag(I) and cytosine-rich DNA, which sequester free Ag(I) and shield its catalytic sites. Unlike other ions, Ag(I) specifically catalyzes OPD oxidation due to its elevated redox potential and noble metal properties, as evidenced by significant signal reduction and a distinct color transition. Furthermore, the integration of smartphone technology enables real-time RGB value analysis, creating a portable and cost-effective platform suitable for point-of-care diagnostics. The system demonstrates exceptional sensitivity, with detection limits of 0.00034 U/mL (fluorescence) and 0.0049 U/mL (UV-Vis), making it be conducive to the early detection of acute lymphoblastic leukemia. Owing to the intrinsic specificity of TdT elongation activity, the method exhibits high selectivity, accurately quantifying TdT activity in Molt-4 cells and distinguishing leukemia cells from normal cells. Additionally, the platform extends to inhibitor screening through the use of positive and negative small-molecule controls, demonstrating its practical utility in biochemical research. Compared to traditional TdT assay kits, the system offers superior sensitivity, enhanced diagnostic accuracy, and broader accessibility, representing a significant advancement in clinical diagnostics and personalized medicine.
研究领域:
生物化学
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医学诊断
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分子生物学
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产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2024
·
IF 8
Shan He,Yiyu Chen,Huiting Lian,Xuegong Cao,Bin Liu,Xiaofeng Wei
Terminal deoxynucleotidyl transferase (TdT) is a crucial biomarker for diseases such as lymphoblastic leukemia . Herein, a pho tocatalytic co lorimetry and f luorescence dual-signal biosensor (PhoCoF-sensor) for simple, low-cost and sensitive detection of TdT was developed, which integrated with the TdT-triggered polymerization reaction and the fluorescence-photocatalytic dual performance of double-strand DNA/SYBR Green-I (dsDNA/SG-I) complex. The target TdT catalyzed the elongation of single-stranded DNA in the dTTP substrate pool, and then hybridized with complementary DNA to form dsDNA/SG-I complex that exhibited obvious fluorescent and excellent photocatalytic activity . Reactive oxygen species were generated from photosensitization of the dsDNA/SG-I complex acted as the visual colorimetric readout. The PhoCoF-sensor was successfully used to detect TdT activity with a series of color and fluorescence changes in the concentration range from 0.5 U/mL to 20.0 U/mL. The PhoCoF-sensor further improves the assay simplicity and accuracy and reduces the time and cost, which overcomes the limitations of traditional detection TdT methods and single-signal sensors. Additionally, this PhoCoF-sensor can readily be integrated with centrifugal microfluidic device for one-step test and achieve quantitative determination without sophisticated instruments. Moreover, the PhoCoF-sensor has good sensitivity and good practical reliability and is suitable for instant determination in early disease diagnosis and drug discovery .
研究领域:
生物化学
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医学诊断
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分子生物学
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产品关联
DOI
高影响因子
JOURNAL OF ELECTROANALYTICAL CHEMISTRY
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2022
·
IF 4.598
Qiong Liu,Kaiyue Hu,Yuqi She,Yufang Hu
Cancer cells require higher ROS levels to maintain their growth state of rapid division and spread·H 2 O 2 , as a kind of usual ROS molecule, is monitored by designing a new in-situ growth G4-nanowire-driven electrochemical biosensor, which is further used for probing H 2 O 2 released by rat astroglioma cell. In this process, the electrocatalytic performance to H 2 O 2 of G4-nanowire (containing G-quadruplex/hemin nanostructures) is employed as signal output, which is constructed by terminal deoxynucleotidyl transferase (TdT)-mediated polymerization in the dNTPs pool of dTTP and dGTP (4: 6). Then, the method is applied for H 2 O 2 monitoring in the range of 0.0003 ∼ 6 mM and the detection limit is estimated to be 0.075 μM (S/N = 3). More importantly, the content of H 2 O 2 generated in rat astroglioma cell is also calculated roughly, displaying an excellent feasibility for the related physiological and pathological studies. Later, TdT activity and its inhibitors are also probed orderly, and a wide linear range is presented with a detection limit of 0.81 mU/mL. It is hoped that the designed platform will provide a reference for exploring new ways to continuous dynamic surveillance of diseases in diagnosis and treatment stage.
研究领域:
分析化学
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生物化学
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癌症研究
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分子生物学
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DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2021
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IF 10.618
Keqin Deng,Jing Xiao,Zhang Liu,Chunxiang Li,Jinglun Wang,Qingfeng Yi,Haowen Huang,Hu Zhou
ABSTRCT Photocathode-based assay is anti-interference for real sample detection . Photocathode produces low photocurrent signal and gives rise to poor sensitivity. Herein, a novel cathode photoelectrochemical (CPEC) sensing platform based on Ag-AgI-CNTs as photocathode material and K 3 [Fe(CN) 6 ] as photoelectron acceptor was established. Since [Fe(CN) 6 ] 3- effectively accepted photoelectrons from Ag-AgI-CNTs, it greatly enhanced the CPEC response. Combining a surface multisite strand displacement amplification (SMSDA) strategy, the CPEC platform was applied for the activity assay of terminal deoxynucleotidyl transferase (TdT). In this proposal, oligo dT primer tethered on CPEC platform was in-situ extended to generate a polyA tail. Then the polyA tail formed a stable multi-point hybrid structure with the adjacent oligo dT. After launching the SMSDA, the CPEC platform was covered by more elongated polynucleotide chains and network, which acutely hampered the photoelectron transfer (eT) between photocathode and electron acceptor and caused a reduced photocurrent. The CPEC sensor possessed a satisfactory linear response from 6 × 10 -5 -0.1 U and a low detection limit of 1.1 × 10 -5 U. The strategy offered a more specific and sensitive method for TdT activity assay . It was feasible in the field of TdT-based biochemical research, drug screening, and disease diagnosis.
研究领域:
生物化学
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分析化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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