Journal of Analysis and Testing
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2024
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IF 5.5
Xiao Qi,Liu Shuai,Wang Yali,Li Huihao,Yang Mingli,Fang Yi,Chen Sijing,Huang Shan
Human T-cell lymphophilic virus type 1 (HTLV-1), the known retrovirus causing cancer in humans, is closely associated with adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. Due to its ability to evade the host's defense mechanisms, early tracking of HTLV-1 becomes crucial. In this study, we integrate λ-Exonuclease (λ-Exo)-assisted target recycling with a terminal deoxynucleotidyl transferase (TdT)-mediated template-free DNA extension process to develop an electrochemical analysis platform for the specific and sensitive detection of HTLV-1 DNA. During the λ-Exo-assisted target recycling, HTLV-1 DNA is recognized by hairpin DNA (Hp-DNA), forming double-stranded DNA (dsDNA) through DNA hybridization. The dsDNA, featuring blunt 5' terminal phosphorylation, is cleaved by λ-Exo, generating abundant short output sequence (sDNA). HTLV-1 DNA is released, initiating a cyclic hybridization-cleavage process. Subsequently, thiol-labelled capture DNA (CP-DNA) assembled on gold electrode surface captures a substantial amount of the generated sDNA, forming CP-DNA-sDNA nanostructures. When TdT and dNTPs are present on the electrode surface, the 3'-OH terminal of sDNA extends to generate long single-stranded DNA (ssDNA) structure. Methylene blue (MB) is selected as the electrochemical signal molecule. MB not only binds with ssDNA but also interacts specifically with dsDNA, resulting in a significantly enhanced electrochemical signal on modified electrode surface. The detection limit of HTLV-1 DNA is as low as 19 amol/L ( S / N = 3) when the two signal amplification strategies are combined. The analysis platform exhibits excellent analytical performance and holds promise as a novel tool for the early tracing and diagnosis of HTLV-1 DNA.
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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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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DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2024
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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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DOI
高影响因子
MICROCHIMICA ACTA
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2018
·Vol. 185
·期 8
·1-8
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IF 5.705
Wen Dongxiao,He Minhui,Ma Kefeng,Cui Ying,Kong Jinming,Yang Huaixia,Liu Qingyun
The article describes an on-chip amplification scheme initiated by a terminal deoxynucleotidyl transferase (TdT) for highly sensitive fluorometric determination of protein. Two thrombin-binding aptamers were designed to capture thrombin as they can form a sandwich structure for improved specificity. An amino-modified aptamer (TBA29) was first immobilized on a silicon chip. After capture of thrombin, a second aptamer (TBA15) was conjugated to the second binding site of thrombin. The 3’-terminal of aptamer TBA15 is exposed on the chip surface, and then fluorescein-labeled 12-dATP associates to the 3’-terminal with the help of TdT. This results in signal amplification, and eventually leads to highly sensitive detection. Under optimal conditions, fluorescence intensity is linearly related to the logarithm of thrombin concentration in the range of 100 fM - 0.1 μM, and the detection limit is as low as 2.0 fM. The assay is sensitive and selective even over potentially interfering proteins and in the presence of human serum.
Schematic strategy for thrombin detection. Two thrombin-binding aptamers were designed to capture thrombin to form a sandwich structure for improved specificity. The protein detection is based on TdT initiated on-chip fluorescent amplification.
研究领域:
分析化学
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生物分子工程
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分子诊断
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2026
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IF 5.2
Chunlei Yu,Keyun Ren,Xuezhen Xu,Jinmiao Ma,Falan Li,Yanyan Zhang,Qingqing Yang
Most applications of CRISPR-Cas12a technology for mycotoxin detection rely on aptamers as recognition elements. However, although antibodies exhibit high affinity and specificity, they require converting immune signals into nucleic acid signals and cannot directly activate the CRISPR-Cas12a system, thereby limiting their utility for highly sensitive small-molecule target detection. In this work, we conjugated CRISPR-Cas12a activator DNA (aDNA) to antibodies via strain-promoted azide-alkyne cycloaddition (SPAAC) to achieve dual functionality: transducing small-molecule target signals into nucleic acid signals and partially immobilizing the Cas12a protein. Magnetic spherical nucleic acids (MSNAs) served as trans-cleavage substrates for the CRISPR-Cas12a system. Terminal deoxynucleotidyl transferase (TdT) was used to extend polyA sequences by adding dATP to the 3’-OH ends of numerous cleaved DNA fragments. This process, combined with high-concentration NaCl-induced aggregation and color change of gold nanoparticles (AuNPs), established a cascade signal amplification system. We developed a novel ultrasensitive immunosensor, termed Cas12a-TdT Cascade Amplification for Antigen-Toxin Immunoassay (CTCA-ATI), for mycotoxin quantification. The limits of detection for aflatoxin B1 and deoxynivalenol (DON) were 2.63 × 10 −5 ng/mL and 6.23 × 10 −4 ng/mL, respectively, with linear ranges of 1 × 10 −4 –10 ng/mL for aflatoxin B1 and 1 × 10 −3 –10 ng/mL for DON. This study provides new insights into the development of cascade-amplified biosensors using CRISPR-Cas12a and demonstrates substantial potential for integrating antibodies with CRISPR-Cas technology to detect other critical targets.
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高影响因子
ANALYTICA CHIMICA ACTA
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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
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生物化学
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分析化学
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生物医学工程
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DOI
PubMed
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
TALANTA
·
2018
·
IF 4.244
Guirong Liu,Wenli He,Chenghui Liu
As an important base excision repair (BER) enzyme, uracil-DNA glycosylase (UDG) can repair the uracil-induced DNA lesion and maintain the genomic integrity. Herein, we have proposed a sensitive UDG assay based on the terminal deoxynucleotidyl transferase (TdT)-assisted formation of fluorescent copper nanoclusters (CuNCs). In this study, a uracil-containing stem-loop DNA substrate is rationally designed with its 3′-end blocked with 2′, 3′-dideoxycytosine (ddC). UDG can remove the uracil in the DNA substrate to generate an apurinic/apyrimidinic (AP) site, which can then be specifically cleaved by endonuclease IV (Endo IV) to expose a 3′-OH terminus. TdT will initiate the template-free DNA extension along the exposed 3′-OH terminus to produce a quite long poly(T) tail, which will perfectly template the production of fluorescent CuNCs. By recording the fluorescence of the CuNCs, the UDG activity can be faithfully detected. In contrast, if UDG is absent, the 3′-ddC terminus of the DNA substrate cannot be recognized by TdT and thus no TdT-based extension and formation of CuNCs will occur. The use of ddC as a 3′-end blocker can greatly decrease the nonspecific DNA extension and improve the signal-to-noise ratio. Furthermore, TdT is a template-free and sequence-independent DNA polymerase, which can effectively catalyze the tailing process up to a maximum of thousands of thymines , and each tail can form many fluorescent CuNCs. Therefore, an ultrahigh sensitivity is achieved and as low as 0.00005 U/mL of UDG can be clearly detected. Moreover, with an additional AP site-contained poly(A) oligonucleotide during TdT-mediated extension, a branched amplification mechanism is also preliminarily devised, which can further push the detection limit of UDG to an extremely low level of 0.000002 U/mL.
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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2021
·
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
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分子生物学
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PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2022
·
IF 5.304
Xuemei Ma,Liu Shi,Haojie Xie,Deqi Kuang,Yetao Xu,Jiehua Ma,Jie Yang,Lizhou Sun
Fat mass and obesity-associated protein (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) motif in ssDNA or RNA, which plays a crucial role in maintaining the relative stability of m6A modification. Meanwhile, dysregulation of FTO expression can lead to cancers and cardiovascular diseases. So quantitative analysis of FTO is of great significance for the research related with its biological functions and application in disease diagnosis. Herein, we propose a highly sensitive biosensor for FTO detection based on terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification. To be specific, FTO can specifically demethylate the substrate probe, and the emerging double strands can be cleaved by DpnII which is a methylation-sensitive restriction endonuclease. The polymerase TdT in turn recognizes the free 3′OH and initiates polymerization to produce a mass of G-quadruplexes. Thioflavin T (ThT) can then be embedded into G-quadruplexes, producing a strong fluorescence. Experimental results manifest that the fluorescence intensity is positively correlated with the abundance of FTO. Moreover, this biosensor also has good specificity and anti-interference, and the limit of detection (LOD) is down to 30.9 fM. Therefore, our biosensor can provide an efficient strategy for FTO analysis without labeling and separation, showing great potential in clinical application.
研究领域:
生物化学
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分子生物学
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生物医学工程
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诊断学
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DOI
高影响因子
MICROCHIMICA ACTA
·
2019
·Vol. 186
·期 3
·1-7
·
IF 5.479
Tang Yafang,Liu Mingxiu,Zhao Zilin,Li Qing,Liang Xuehua,Tian Jianniao,Zhao Shulin
The authors describe a method for the determination of microRNA-122 by using terminal deoxynucleotidyl transferase (TdT). It is based on the use of polythymine and exonuclease III-aided cycling amplification. A 3′-phosphorylated hairpin probe 1 (H 1 ) and a hairpin probe 2 (H 2 ) were designed. In the presence of the microRNA, hybridization and enzymatic cleavage will occur and produce lots of 3′-hydroxylated ssDNA which can be tailed by TdT and converted into long polythymine (polyT) sequences. These can be used to synthesize copper nanoparticles (CuNPs) with fluorescence excitation/emission maxima at 350 nm/630 nm. This method shows good selectivity and high sensitivity with a linear response in the 1.00 × 10 2 fM and 1.00 × 10 6 fM microRNA concentration range and a 44 fM limit of detection. It was successfully applied to determination of microRNA in spiked serum samples.
A label-free and highly sensitive fluorometric method is described for the assay of microRNA on the basis of target-triggered two-cycle amplification and combining with terminal TdT. It produces a series superlong polyT that can be used for synthesis of copper nanoclusters (CuNCs) displaying red fluorecence.
研究领域:
分析化学
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分子生物学
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纳米技术
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生物医学工程
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DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
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2024
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IF 13.3
Haoyu Wang,Shiting Chen,Jiqing Liu,Wanyue Liu,Wei Chen,Lin Huang,Chao Yan
In recent years, RPA-CRISPR/Cas12a-based electrochemical biosensors have emerged as highly effective tools for nucleic acid detection. However, the random trans -cleavage characteristics of Cas12a on single-stranded nucleic acid sequences can result in the presence of uncertain nucleic acid residues on the electrode surface post-reaction. This situation complicates subsequent sensitization strategies. In this study, we devised a universal sensitization protocol mediated by terminal deoxynucleotidyl transferase (TdT) , a template-independent DNA polymerase and G-rich spherical nucleic acids (G-rich SNAs) with superior performance. Initially, Cas12a was activated by the specific RPA amplification product and cleaved the closed single stranded DNA (C-ssDNA) on the electrode surface. Then, the TdT enzyme extended residual single stranded DNA (R-ssDNA) on the electrode surface, forming G-Quadruplex structures. Subsequently, G-rich SNAs were immobilized on the electrode surface via π-π stacking interactions with the G-quadruplexes. Consequently, a substantial accumulation of hemin occurred within the G-quadruplex cluster structures, generating significant electrochemical signals. We applied this method to the trace detection of Staphylococcus aureus ( S. aureus ) and achieved a good linear calibration curve with concentrations ranging from 0 CFU/mL to 10 4 CFU/mL. Notably, the limit of detection (LOD) was as low as 1.29 CFU/mL, surpassing that of most similar sensors. These results demonstrate that our proposed universal sensitization method effectively addresses the limitations associated with the random trans -cleavage characteristics of Cas12a.
研究领域:
生物传感
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纳米技术
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分子生物学
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电化学
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DOI
高影响因子
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