JOURNAL OF THE ELECTROCHEMICAL SOCIETY
·
2019
·Vol. 166
·期 12
·B1028
·
IF 3.12
Dandan Hu,Yufang Hu,Qingqing Zhang,Pingjian Ran,Xinda Liu,Yudi Zheng,Zhiyong Guo,Sui Wang,Di Wu
Monitoring DNA-related enzymes activity precisely and reliably is absolutely vital to better grasp their roles in the initiation and progression of cancer, thus further exploring their functions in cancer diagnosis and treat. Here, we establish a label-free electrochemical biosensor for investigating both terminal deoxynucleotidyl transferase (TdT) and exonuclease I (Exo I) activity by silver-based square wave stripping voltammetry (SWV). The strategy is achieved by two attractive points in design: a TdT-derived enzymatic synthesis of randomly arrayed C-rich DNA sequence and an Exo I-guided enzymatic degradation. This C-rich DNA-templated silver nanoclusters (AgNCs) serve as the electrochemical signal probe, and a signal on-off modulated process is displayed for the existence of TdT and Exo I, respectively. On account of AgNCs-based square wave stripping voltammetry, testing TdT has a linear concentration range of 0.001 to 5 U/mL with a detection limit of 0.00044 U/mL (S/N = 3). Additionally, Exo I with a detection limit of 0.027 U/mL (S/N = 3) can be analyzed with a linear range of 0.1 to 500 U/mL. The real application of the proposed method in their inhibitors screening is also discussed well. The biosensor provides a powerful idea for the ultrasensitive bio-analysis and shows broad clinical application prospect.
研究领域:
生物化学
·
分析化学
·
医学诊断
✓ 已验证
产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Shihua Luo,Lifeng Zhang,Xiaohe Zhang,Jiezhen Pan,Mei Wang,Ye Zhang,Aiping Qin,Wencheng Chen
The integrity of sperm DNA is essential for proper fertilization and embryonic development. Various methods exist to evaluate sperm DNA fragmentation. However, they are frequently hindered by high costs, complex procedures, and low precision. Herein, a simple and sensitive fluorescence biosensor was developed using the terminal deoxynucleotidyl transferase (TdT) and CRISPR/Cas12a system for analyzing sperm DNA fragmentation. This biosensor efficiently measures the number of 3′-OH, which correspond to the mean number of sperm DNA breakpoints (MDB). In the presence of TdT, the dATP nucleotide polymerized at the 3′-OH end of the primer, generating numerous polyA-tails, which were detected by CRISPR-guided RNA (crRNA) at the same time. Ultimately, the trans -cleavage activity of CRISPR/Cas12a was activated, which then cleaved the spherical nucleic acids (SNA) reporter, generating amplified fluorescence signal. The proposed method exhibited a broad linear detection for the quantity of 3′-OH ends in single-stranded DNA, from 1 fM to 10 pM, with a limit of detection of 2.5 fM. Moreover, the platform was successfully validated with clinical sperm samples and demonstrated a stronger correlation with sperm motility compared to traditional DNA fragmentation index (DFI) test. Therefore, the developed method offers advantages such as ease of use, elevated sensitivity, and mimimal instrumentation requirements, making it suitable for clinical application.
研究领域:
生殖生物学
·
分子诊断学
·
基因编辑技术
✓ 已验证
产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Chao Li,Haoyu Wang,Rong Wei,Jingzhi Ren,Mengyang Zhou,Chao Yan,Lin Huang
Exosome-based liquid biopsy has shown great potential in the diagnosis and prognosis of tumors, thus developing a convenient and reliable detection method is crucial for its clinical application. Herein, we construct a separable visual aptasensor for the sensitive detection of exosomes by using double spherical nucleic acids (DSNAs) and terminal deoxynucleotidyl transferase (TdT). In this system, CD63 aptamer-modified magnetic beads (MNPs-Apt CD63 , the first spherical nucleic acids) could efficiently capture exosomes, and avoid the aggregation of gold nanoparticles (GNPs) induced by complex components in the reaction system through magnetic separation. As the second spherical nucleic acids, abundant nucleolin aptamer-modified GNPs (GNPs-Apt nucleolin ) are used to specially identify exosomes. Then, those nucleolin aptamers can further produce long polyT sequences by the extension of TdT and induce polyA release from the GNPs-polyA (polyA-attached gold nanoparticles) by hybridization, thus resulting in the salt-induced aggregation of GNPs to produce color change for visual analysis of exosomes. Based on the above ingenious designed strategies, this method achieved a limit of detection (LOD) as low as 45 particles/μL, and showed accurate visual analysis for the clinical samples. We expect this method to provide a new opportunity for the exosomes-based point-of-care diagnosis of tumors.
研究领域:
生物医学工程
·
肿瘤诊断
·
分子生物学
✓ 已验证
产品关联
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Jing Zhu,Liutao Chen
Fluorescent dye DITO-1 has almost no fluorescence in the absence of nucleic acid . G bases in single strand DNA can induce maximum fluorescent enhancement followed by the A bases when it binds the DITO-1. However, the incorporation efficiency of the dATP was higher than dGTP in terminal transferase (TdT) polymerization. As a consequence, ploy (A) n , rather than ploy (G) n via TdT polymerization had the superior photoluminance when it binded DITO-1 fluorescent dye. Here, we developed a high selective and sensitive sensing strategy for assaying TdT and T4 polynucleotide kinase activity (T4 PNK) based on the ploy (A) n -DITO-1 fluorescent probe. An increasing amounts of TdT enzyme could promote the distinct incorporation of dATP on the DNA primer and form poly (A) n ssDNA with a difference in length. A good linear relationship between the ΔF and the concentrations of TdT in a range of 0.2–50 U/mL was obtained and the detection limit was 0.05 U/mL. Based on the experimental results for TdT, we further expanded the application of this method for detection of a series of concentrations of T4 PNK. The ΔF and the logarithm concentrations of T4 PNK in the range of 0.1–10 U/mL showed a good linear response and the detection limit of 0.02 U/mL was obtained. In addition, the detection of T4 PNK in Hela cell lysate was achieved, demonstrating that the proposed method had the potential application in complex system. The ploy (A) n -DITO-1 fluorescent probe had the excellent properties of one-step readout, robustness for target detection in complex system, and easiness operation, and showed the great potential in clinical diagnostics, inhibitor screening, and drug discovery.
研究领域:
分子生物学
·
生物化学
·
分析化学
·
生物医学工程
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）