MICROCHEMICAL JOURNAL
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2022
·
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
CURRENT MICROBIOLOGY
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2025
·Vol. 82
·期 6
·1-10
·
IF 2.6
Zhang Pei,Yang Huanggen,Ahmad Malik Taj,Zheng Qi,Nie Guochao,Ahmad Ayesha,Raza Muslim,Raza Saleem
Biofilm formation presents significant challenges in healthcare, food processing, and water treatment, contributing to antibiotic resistance and persistent infections. Effective strategies to combat biofilm-associated infections are urgently needed. This study introduces a novel approach to biofilm removal by bio-functionalizing T4 bacteriophage with modified Fe 3 O 4 nanoparticles (NPs) and Nisin, an antibacterial peptide, to form the Fe 3 O 4 -Phage-T4 + Nisin complex. The aim is to enhance antimicrobial efficacy and biofilm eradication. The Fe 3 O 4 -Phage-T4 + Nisin complex was synthesized by conjugating T4 bacteriophage with modified Fe 3 O 4 NPs and Nisin. The antimicrobial and antibiofilm activity of the complex was evaluated against multidrug-resistant Pseudomonas aeruginosa strains ( PA01 and PA14 ) using biofilm inhibition and eradication assays. Stability and efficacy were further tested across a pH range of 5 to 8. The Fe₃O₄-Phage-T4 + Nisin complex exhibited superior biofilm removal compared to its individual components. The integration of Nisin broadened the antibacterial spectrum, targeting both Gram-positive and Gram-negative bacteria , while the modified Fe₃O₄ NPs enhanced phage penetration and bacterial cell disruption. The complex demonstrated significant biofilm inhibition and eradication, addressing the challenge of biofilm-related antibiotic tolerance, which often necessitates high antibiotic doses. Additionally, it maintained stability and efficacy across varying pH conditions.
研究领域:
微生物学
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生物技术
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医药科学
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DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
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2024
·
IF 13.3
Xiao-Lin Hou,Lin-Fang Tan,Xiao-Ting Xie,Bin Zhang,Qiong Wang,Kai Cheng,Jin-Xuan Fan,Tian-Cai Liu,Bo Liu
The peroxisome is an important organelle for H 2 O 2 metabolism and O 2 redistribution in organisms. Drawing inspiration from this biochemical pathway, a T4-Ce6-Cat-Lox (TCCL) simpler biomimetic peroxisome was constructed using the T4 phage’s Soc and Hoc sites to enhance photodynamic therapy (PDT). The cascading catalytic system of lactate oxidase (Lox) and catalase (Cat) was displayed on the phage architecture through in vitro phage display technology. 720 Lox molecules and 95 Cat molecules displayed on the T4 phage can be precisely controlled by the feed concentration. The enzymatic reaction of Lox, triggered by lactic acid in the tumor microenvironment, generated H 2 O 2 at the Soc site. Subsequently, the H 2 O 2 was decomposed into O 2 by Cat at the Hoc site. By manipulating the cascading catalytic system ratio of Lox and Cat to 7.5:1, the ROS generation efficiency of Ce6 was as high as 99.2 %, and the tumor inhibition rate reached 91.25 %. More importantly, the TCCL was constructed by natural T4 nanocarrier, exhibiting good biocompatibility and low immunogenicity in vivo . This study explored the construction of a simplified biomimetic peroxisome using the T4 phage display technology and provided a promising strategy for cascading catalytic system construction.
研究领域:
生物医学工程
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肿瘤学
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酶工程
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DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2022
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IF 9.221
Ran Luo,Guowei Lian,Hengxuan Li,Houyu Han,Dianming Zhou,Xiaoqun Gong
Signal amplification plays a significant role in nucleic acid analysis, especially in trace nucleic acid detection. Transcription-based signal amplification is a very important component in the field of signal amplification, and has received extensive attention depending on outstanding cyclic transcription ability. However, the low efficiency of transcription caused by limited template length usually restricts their further applications significantly. Herein, we developed a transcription-based signal amplification method with high sensitivity and selectivity . The approach we proposed couples the advantageous features of long template synthesis from phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) with self-amplification from cell-free transcription system. The results demonstrate that this approach can be applied to quantify T4 Polynucleotide Kinase (T4 PNK) phosphatase activity in a broad range from 0.00001 to 0.01 U/mL, with a detection limit of 8.1 × 10 -6 U/mL. Moreover, this device can be further employed as a superior signal amplification platform for the detection of other analytical targets after moderate modifications.
研究领域:
分子生物学
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分析化学
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生物技术
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DOI
高影响因子
FOOD CHEMISTRY
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2025
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IF 9.8
Qiaoli Yang,Yaping Wang
This research developed an electrochemical biosensor based on T4 bacteriophages assembled with beta-galactosidase (β-gal) for Escherichia coli K12 ( E. coli K12) detection. The β-gal Soc fusion proteins were affinity-assembled on the Hoc − Soc − T4 phage capsid, exhibiting superior substrate affinity and enhanced stability compared to the free enzyme. The antibodies were immobilized on the multiwalled carbon nanotube/gold nanoparticle modified electrode, and then the engineered β-gal T4 was bonded the bacterial cells captured by the antibodies, constructing the electrochemical biosensor. The biosensor can detect E. coli K12 as low as 6 CFU/mL within 90 mins utilizing catalytic events of β-gal T4. The biosensor exhibited high specificity, distinguishing live target bacteria from non-target bacteria or dead target bacteria, which is attributed to its dual recognition mechanism involving antibody and β-gal T4. The biosensing platform can be extended to other bacteria by customizing phage-enzyme combinations, enabling bacterial detection in food, environment and other fields.
研究领域:
微生物学
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生物传感技术
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分子生物学
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产品关联
DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2025
·
IF 3.3
Wu Dan,Khan Sulaiman,Zhang Shujie,Wang Huan,Chen Wei,Wang Shenqi
Enzymes, key catalysts in biochemical reactions, are prone to denaturation under harsh conditions, leading to reduced stability and higher costs. Enzyme immobilization, using carriers like magnetic nanoparticles, metal–organic frameworks, and viruses, is a common solution. T4 bacteriophage, a virulent E. coli phage containing 155 Hoc and 870 Soc proteins, offers a cost-effective and highly stable platform for enzyme immobilization. In this study, Soc-β-galactosidase (Soc-β-gal) was immobilized on the surface of T4 bacteriophage via affinity fixation and further encapsulated with a metal-polyphenol network (MPN) coating. Comparative analysis of the biochemical properties revealed that the immobilized enzyme, β-gal T4, retained over 85% activity after 6 h at 50 °C, while free Soc-β-gal retained only 40.63%. Moreover, β-gal T4@TA-Ti demonstrated superior stability, retaining 92.88% of its activity after 6 h of UV exposure, compared to 10.21% for β-gal T4 and 7.23% for Soc-β-gal. The MPN coating also enhanced resistance to proteolytic degradation, with β-gal T4@TA-Ti retaining 9.48% of its activity after exposure to proteinase K, in contrast to 4.62% for β-gal T4. Overall, these results demonstrate that enzyme immobilization significantly enhances stability, while the MPN coating further improves resistance to extreme pH, ultraviolet radiation, and other environmental stressors, highlighting the potential of this approach for biocatalytic applications.
研究领域:
生物化学
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酶工程
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材料科学
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DOI
PubMed
高影响因子
Analytical Methods
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2016
·Vol. 8
·期 20
·4119-4126
·
IF 1.915
Lin Lin,Dongmin Shi,Quanfa Li,Guangfeng Wang,Xiaojun Zhang
Determination of nucleotide kinase activity is valuable due to its importance in regulating nucleic acid metabolism. In this work, a novel and simple colorimetric method for discriminative detection of T4 polynucleotide kinase (PNK) through a MnO2 nanosheet-3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric system is developed. In aqueous solution at pH 7.4, the MnO2 nanosheet, a mimic enzyme, rapidly converts colourless TMB to a blue oxidized form. In the presence of T4PNK, a hairpin DNA could be phosphorylated and further cleaved by λ exonuclease (λ exo) and the ssDNA fragments would adsorb on the surface of the MnO2 nanosheet, resulting in the inhibition of the catalytic effect of the MnO2 nanosheet to the oxidation of TMB and weakening of the blue color of oxidized TMB with the absorption peak centred at 652 nm. The presence of T4PNK can in this way easily be detected with the naked eye, based on the obvious colour change. The experimental results revealed that the advanced strategy was sensitive for detecting T4PNK in the concentration range from 0.005 to 10 U mL−1 with a detection limit of 0.005 U mL−1. In addition, the relative standard deviation was 3.92% in 3 repetitive assays of 10 U mL−1 T4PNK, indicating that the reproducibility of this strategy was acceptable. By comparison with other relevant fluorogenic assays, the sensitivity of the developed chromogenic assays was satisfactory. The present approach opens the possibility for the design of simple and sensitive colorimetric assays for polynucleotide kinase using inexpensive and available TMB as a universal chromogenic compound. Therefore, besides desirable sensitivity and selectivity, due to the colorimetric system, the developed method for T4PNK detection also showed cost-effective, reliable and simplified operations and strategy, which has considerable potential for biological process research.
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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产品关联
DOI
高影响因子
JOURNAL OF ELECTROANALYTICAL CHEMISTRY
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2020
·
IF 3.807
Jingxiu Mao,Xin Chen,Huahuang Xu,Xueqin Xu
T4 polynucleotide kinase (T4 PNK), a bifunctional repair enzyme, catalyzes the transfer of γ-phosphate residues from adenosine triphosphate (ATP) to 5′- hydroxyl termini in nucleic acids，which plays an essential role in regulating DNA phosphorylation modes during the repair of damaged DNA. Herein, a sensitive DNAzyme-driven DNA walker biosensor was developed for amplified electrochemical detection of T4 PNK activity and inhibition based on T4 PNK-triggered phosphorylation reaction and λ exonuclease (λ exo) cleavage. A novel functional hairpin-shaped swing arm DNA (SA) containing a DNA sequence of the Pb 2+ -dependent DNAzyme is designed to specifically bind to and hydrolyze the substrate strand (rSS) sequence that can drive DNA walker. Upon the reaction of SA， T4 PNK and ATP, λ exo removes the mononucleotides from the stem of the phosphorylated SA, unfolding the hairpin structure and initiating DNAzyme-driven DNA walker. Subsequently, the DNA walker can repeatedly bind and cleave rSS with the aid of Pb 2+ , releasing the methylene blue (MB) - labeled fragment of rSS. The T4 PNK activity can be facilely monitored by recording the electrochemical signal change of MB. The proposed biosensor exhibited a broad linear range from 0.01 U mL −1 to 15 U mL −1 with a low detection limit of 0.001 U mL −1 . The proposed biosensor has also been utilized to screen the T4 PNK inhibitors and exhibited good performance in real sample analysis, making such a strategy hold potential promise in biological fields and clinical diagnosis.
研究领域:
生物化学
·
分子生物学
·
分析化学
·
临床诊断
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产品关联
DOI
高影响因子
BIOCHIMIE
·
2025
·
IF 3
Lin Zhang,Lvping Wu,Yiwei Guo,Enjie Wang,Jiaxing Zhang,Shengping You,Rongxin Su,Wei Qi
Recombinase polymerase amplification (RPA) is a prominent isothermal nucleic acid amplification method widely applied in molecular diagnostics. The stability and functionality of the single-stranded DNA-binding protein T4 gene 32 (gp32) crucial for pre-synaptic filament formation and D-loop stabilization, play a key role in determining RPA efficiency. In this study, V62C/T80C and Y186R mutants with improved performance were screened by rational disulfide bond construction and virtual saturation mutagenesis, respectively. The structural changes in V62C/T80C and the altered ssDNA-binding capacity in Y186R both contribute to RPA amplification by enhancing the formation of UvsX-ssDNA presynaptic filaments and stabilizing the D-loop structure during homologous recombination, respectively. The two mutants each demonstrated unique advantages in the RPA process. V62C/T80C significantly accelerates the amplification process, reducing the RPA reaction time by 47%, while Y186R showed a 123% increase in efficiency across the entire amplification cycle. Totally, this study applied a rational strategy on gp32 optimization, shortening RPA reaction times, enhancing the RPA reaction efficiency, and advancing its application in clinical and point-of-care diagnostics.
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
TALANTA
·
2022
·
IF 6.556
Jinpeng Tao,Zaiqiong Liu,Zhenyu Zhu,Yanli Zhang,Hongbin Wang,Pengfei Pang,Chun Yang,Wenrong Yang
An ultrasensitive electrochemical detection of the activity and inhibition of T4 polynucleotide kinase (T4 PNK) was developed by using magnetic Fe 3 O 4 @TiO 2 core-shell nanoparticles , which was triggered by a rolling circle amplification strategy (Fe 3 O 4 @TiO 2 -RCA). We used Fe 3 O 4 @TiO 2 as a substrate to anchor a DNA primer . DNA S1 with 5′-OH termini was phosphorylated in the presence of T4 PNK and ATP, which was adsorbed on the surface of Fe 3 O 4 @TiO 2 NPs and served as the primer for subsequent RCA reactions. After adding circular template DNA S2, RCA was initiated in the presence of phi29 DNA polymerase and dNTPs. Then, Fc-labeled DNA S3 (Fc-S3) was hybridized with RCA. The obtained Fe 3 O 4 @TiO 2 -RCA was adsorbed on the surface of a magnetic gold electrode (MGE) by magnetic enrichment, resulting in an enhanced electrochemical signal. The T4 PNK activity can be monitored by measuring the electrochemical signal generated. This electrochemical assay is sensitive to the activity of T4 PNK with a dynamic linear range of 0.00001–20 U/mL and a low detection limit of 3.0 × 10 −6 U/mL. The proposed strategy can be used to screen the T4 PNK inhibitors, so it has great potential in the discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.
研究领域:
生物化学
·
分析化学
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Jingjing Xu,Qiaoli Yang,Pei Wang,Dan Wu,Xiaohan Yang,Wei Chen,Song Gao,Shenqi Wang
Due to the highly efficient and specific catalysis , enzymes have been extensively applied in various fields. In the development of enzyme-based sensing devices, the immobilization of enzyme is critical for achieving superior performance which is drastically dependent on enzyme stability and activity. In this study, we construct and assemble the beta-galactosidase( β -gal)-Soc fusion proteins onto biologically produced nanoparticle – T4 phage in vitro as a specific sensing probe for the rapid colorimetric detection of Escherichia coli K12. The stability and substrate affinity of β -gal assembled on T4 phage were largely enhanced in comparison with that of free β -gal due to this affinity-based immobilization, and the assay based on this β -gal T4 phage was able to quantify bacteria at concentration of 10 3 cfu mL −1 in drink water within 2 h. This affinity binding based enzyme immobilization strategy employed T4 bacteriophage capsid as the supporting nanoparticle and exhibited enhanced enzyme activity and stability which are highly favored for the future development of sensing applications.
研究领域:
生物技术
·
酶学
·
微生物学
·
纳米技术
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产品关联
DOI
高影响因子
MICROCHIMICA ACTA
·
2018
·Vol. 186
·期 1
·1-9
·
IF 5.705
Zhang Xingxing,Liu Qiang,Jin Yan,Li Baoxin
A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3′-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570 nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07 U mL −1 to 15 U mL −1 and a detection limit of 0.06 U mL −1 .
The sequence-dependent fluorescence of DNA-templated copper nanoclusters, which are in-situ synthesized through the reduction of CuSO 4 by ascorbate (Vc) in the presence of dsDNA template, is utilized to obtain the method for sensitive detection of T4 PNKP activity with near-zero background.
研究领域:
生物化学
·
分子生物学
·
分析化学
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DOI
PubMed
高影响因子
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