MICROCHEMICAL JOURNAL
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2025
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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
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分子诊断学
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基因编辑技术
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DOI
高影响因子
JOURNAL OF THE ELECTROCHEMICAL SOCIETY
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2019
·Vol. 166
·期 12
·B1028
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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
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分析化学
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医学诊断
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DOI
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
BIOSENSORS & BIOELECTRONICS
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2016
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IF 7.476
Kai Shi,Baoting Dou,Jianmei Yang,Ruo Yuan,Yun Xiang
Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3′-OH termini on the sensor electrode . Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion. The sensor shows a linear range of 0.5 pM–10.0 nM and a detection limit of 0.12 pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.
研究领域:
生物医学
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生物分析学
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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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
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肿瘤诊断
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分子生物学
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2025
·
IF 5.1
Jian Yang,Jiahua Li,Xuan Yi,Shiyuan Feng,Wei Kang,Gang Ren,Jianli Liu,Chen Mai,Jinfeng Miao,Yuanyuan Xu
Energy levels in organisms are closely linked to metabolic diseases, with ATP—the universal energy currency—playing a central role in this relationship. This paper presents a novel electrochemical biosensor for ATP, integrating homogeneous bio-recognition reactions with terminal deoxynucleotidyl transferase (TdT)-mediated polymerization to enable precise ATP detection in metabolic research. Initially, a DNA oligonucleotide composed of a camouflaged aptamer and exposed hairpin DNA was designed to react homogeneously with ATP in the presence of exonuclease I (Exo I). This reaction generates blunt-ended hairpin DNA, which can activate the assembly of an ordered DNA-rich nanostructure layer on the gold electrode. Subsequently, the DNA strand is elongated through TdT-induced polymerization in the presence of deoxynucleotide triphosphates (dNTPs). A significant amount of Ru(NH 3 ) 6 3+ can be adsorbed onto the negatively charged DNA phosphate backbone via strong electrostatic interactions, effectively amplifying the detection signal and enhancing the sensor's performance. Under optimal conditions, the system exhibits excellent linearity over the target concentration range of 10 −2 to 10 3 nM, with a detection limit as low as 8.37 pM, enabling the detection of trace amounts of ATP. The biosensor demonstrates high stability and reliability, with recovery rates ranging from 97.8 % to 110 %. This method serves as a diagnostic tool to assess organismal metabolic status by monitoring ATP levels, enabling the tracking of metabolic disease progression. By integrating molecular monitoring with clinical diagnosis, it offers a potential approach for the preventing and intervention of metabolic diseases.
研究领域:
生物电化学
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生物传感器
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分子诊断
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核酸扩增
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代谢研究
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临床诊断
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DOI
高影响因子
COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS
·
2024
·
IF 4.9
Hang Wu,Lei Xie,Liang Zhu,Tengbo Yu,Yingze Zhang
As a common malignant bone tumor in adolescents, osteosarcoma (OS) possesses a very poor prognosis and lacks effective treatments. In this study, we developed a multifunctional nanotherapeutic agent (WSe 2 /AIPH/GOx) that can combine photothermal therapy (PTT) /thermodynamic therapy (TDT) /starvation therapy (ST) against OS. The prepared WSe 2 /AIPH/GOx exhibited a high photothermal conversion efficiency of 37.3% under 808 nm near-infrared laser irradiation, which could effectively eliminate tumor cells. In addition, the local high temperature caused by photothermal heat induces the rapid release and decomposition of AIPH, generating oxygen-independent alkyl radicals that promote tumor cell death in the hypoxic microenvironment. More importantly, the introduction of GOx depletes glucose to starve tumor cells on the one hand, and on the other hand can indirectly increase the thermal sensitivity of cells. In vivo experiments have demonstrated that WSe 2 /AIPH/GOx can effectively inhibit tumor growth in mice due to the combined effects of multiple therapies. Overall, this work provides an effective strategy for potential osteosarcoma treatment based on combination therapies.
研究领域:
肿瘤学
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纳米医学
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生物材料学
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DOI
高影响因子
TALANTA
·
2025
·
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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DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 11.8
Chuang Wang,Yue Peng,Yuanyuan Miao,Xinyuan Sun,Qianlei Zhang,Zhaokun Zheng,Wenting Cheng,Jinlong Li
Aberrant DNA methylation has emerged as a critical epigenetic biomarker closely associated with bacterial pathogenicity and human tumorigenesis. In this study, a highly sensitive and label-free electrochemical biosensor has been developed for the detection of Dam MTase activity. 3′-aminated hairpin DNA probes (HP1) containing Dam MTase-specific recognition sites are immobilized onto the surface of a gold electrode. In the presence of Dam MTase, HP1 is methylated and subsequently cleaved by Dpn I, thereby exposing free 3′-hydroxyl groups. These hydroxyl groups serve as primers and are extended by terminal deoxynucleotidyl transferase (TdT) to generate long poly-thymine (polyT) tails. As an electrochemical redox indicator with numerous positive charges, ruthenium hexaammine trichloride (RuHex) can be captured by negatively charged DNA extension products. Accordingly, a remarkably enhanced electrochemical signal is produced, and the signal response is proportional to methyltransferase activity. This biosensor exhibits a good linear relationship in the range of 0.1 U/mL to 20 U/mL, and the limit of detection (LOD) is as low as 0.57 U/mL. Furthermore, its application value has been verified through the successful screening of existing MTase inhibitors (5-fluorouracil and gentamicin), indicating its potential application prospects in clinical diagnosis and antibacterial drug screening.
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DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Minqiao Feng,Chengyan Yin,Hailiang Qin,Wei Liu,Yunqi Wang,Ruth Antwi-Baah,Yao Rao,Shuxia Xu
Terminal deoxynucleotidyl transferase (TdT) is actively expressed in leukemia patients and plays an important role in maintaining the genetic diversity of an organism. Therefore, developing simple and sensitive analytical methods for the accurate detection of TdT in serum is of significant importance. Herein, an electrochemical biosensor based on a toluidine blue (TB)-loading mesoporous silica nanoparticles (MSN) was established for the detection of TdT in serum. First, MSN with amino group was synthesized and subsequently was carbonylated to obtain carboxyl-functionalized MSN (MSN-COOH), which was used to load TB. Thereafter, MSN-COOH was sealed with the substrate strand S(rA)-NH 2 of the Mg 2+ -dependent DNAzyme to form S(rA)-NH 2 /TB/MSN-COOH. In the presence of TdT, it can add deoxyadenosine triphosphate (dATP) to the 3’-OH end of Enc-5 A, activating its cleavage activity on the substrate chain S(rA)-NH 2 , thus releasing TB encapsulated in MSN-COOH. By measuring the change in the electrochemical signal of TB, the TdT concentration can be determined. The linear range is from 5 to 400 U/mL and the limit of detection (LOD) is 0.369 U/mL (3 σ/K). Since DNAzyme can be extended by TdT without the need of a template, the proposed biosensor demonstrates excellent selectivity towards TdT in the presence of other DNAzyme, such as T4 DNA ligase, phi29 DNA polymerase, DreamTaq DNA polymerase, and Klenow fragment ( exo -). Furthermore, the proposed biosensor was successfully applied to the detection of TdT in serum with the recovery rates ranged from 91.0 % to 94.7 %, indicating its potential for early detection of leukemia and other related diseases.
研究领域:
生物分析化学
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生物传感器
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分子诊断
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纳米生物技术
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肿瘤学
·
临床化学
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产品关联
DOI
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2026
·
IF 12.5
Shuang Guo,Jiashuo Xu,Huiquan Wang,Wen Li,Tao Meng,Zhe Zhao,Zhiguang Niu,Xiaoqun Gong
We reported a TdT combined with CRISPR/Cas12a driven cascade amplification platform for the sensitive detection of both heavy metal ions and viral targets. • Integrated with reverse fluorescence-enhanced lateral flow strip for visual readout, this platform achieves LODs of 2.5 × 10 −15 M for Pb 2+ and 3.6 × 10 −16 M for DENV-RNA. • The modular design allows flexible replacement of recognition modules, offering a universal solution for on-site rapid monitoring of diverse pollutants in complex environmental matrices. Currently, constructing flexible detection platforms capable of identifying diverse hazardous substances remains a significant challenge. Conventional detection methods are limited by target specificity and lack cross-class universality. Herein, we developed a general platform for sensitive detection of lead ions (Pb 2+ ) and dengue virus (DENV) as two distinct model targets. This platform specifically converted different types of targets into detectable nucleic acid molecules, which would be further recognized and signal amplified by the cascade cycling isothermal amplification system composed of terminal deoxynucleotidyl transferase (TdT) and CRISPR/Cas12a system. TdT has the property of extending the 3′ end of DNA molecule in a template-independent manner, providing long poly-T nucleic acid sequence for binding lots of crRNA, so as to activate the trans-cleavage activity of Cas12a to cleave the hairpin DNA reporters with exposing numerous novel 3′ ends for the same cycle. By combining this strategy with the reverse fluorescence-enhanced lateral flow test strip, the platform achieves a limit of detection (LOD) of 2.5 × 10 −15 M and 3.6 × 10 −16 M for Pb 2+ and DENV-RNA, respectively. Moreover, the detection platform for Pb 2+ in environmental water samples showed excellent agreement with standard methods. It also demonstrated high sensitivity for DENV-RNA detection in complex samples, confirming its reliability and broad applicability potential in practical scenarios. Download: Download high-res image (103KB) Download: Download full-size image
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DOI
高影响因子
FOOD CHEMISTRY
·
2019
·
IF 5.399
Bin Wang,Jiushang Zheng,Ailing Ding,Liqun Xu,Jiuncun Chen,Chang Ming Li
A novel aptasensor, based on a perylene probe (PAPDI; N,N′-bis(propylenetrimethylammonium)-3,4,9,10-perylenediimide), was developed for the detection of aflatoxin B1 (AFB1) in maize samples. AuNPs/DNA composites were synthesized and integrated with aptamers-modified magnetic nanoparticles (MNPs) via DNA hybridization. For AFB1 determination, AuNPs/DNA composites were released from MNPs through specific binding of AFB1 with the aptamer and used for assembly of the PAPDI probe. To enhance the method sensitivity, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA polymerization was performed to elongate DNA on AuNPs/DNA composites. As a result, more PAPDI probes were assembled on the AuNPs/DNA composites. Through a multiple signal amplification strategy, the proposed method exhibited high sensitivity towards AFB1, with a detection limit of 0.01 nM (3.1 pg/mL). In summary, the proposed method has great potential to be a universal strategy for monitoring AFB1 in food samples.
研究领域:
食品科学
·
分子生物学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）