Analytical Methods
·
2014
·Vol. 6
·期 8
·2499-2505
·
IF 1.938
Wenzhu Zhang,Ping Zhang,Shengzhou Zhang,Changqing Zhu
In this study, a quantum dots (QDs)-based label-free fluorescence assay has been developed for real-time monitoring of intracellular trypsin activity and screening the corresponding inhibitor. Negatively charged CdTe QDs and positively charged cytochrome c (cyt.c) first form a hybrid complex structure through an electrostatic attraction effect. The fluorescence of the QDs is well quenched because of electron transfer from QDs to cyt.c. Then, added trypsin breaks the hybrid complex structure because of the hydrolysis of cyt.c catalyzed by the trypsin. Thus, the electron transfer is switched off and a substantial fluorescence recovery is obtained. Under optimal conditions, the initial rate of the hydrolysis reaction is linearly proportional to the concentration of trypsin between 1.25 and 375 nM, and the detection limit is as low as 0.42 nM. The proposed method is effective, simple and cost-effective, demonstrating great potential for point-of-care diagnosis applications.
研究领域:
生物化学
·
分子生物学
·
分析化学
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生物医学工程
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高影响因子
Food & Function
·
2019
·Vol. 10
·期 12
·8182-8194
·
IF 3.241
Min Liu,Tingting Liu,Yabo Shi,Yanna Zhao,Hui Yan,Bin Sun,Qingpeng Wang,Zhengping Wang,Jun Han
Natural polyphenols showing a variety of beneficial effects will interact with multiple proteases after administration. The interactions of oxyresveratrol and piceatannol with trypsin and lysozyme were investigated using fluorescence spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, differential scanning calorimetry and molecular docking. Fluorescence quenching results and UV-vis absorption difference spectra revealed that the quenching process was a static mode initiated by ground-state complex formation. The different binding ability of oxyresveratrol and piceatannol with trypsin and lysozyme was discussed based on their different molecular structures. Moreover, the major driving force for the binding process was elucidated as hydrogen bonding and van der Waals forces by the negative enthalpy and entropy changes. Synchronous fluorescence, three-dimensional fluorescence and circular dichroism spectral analysis suggested that the binding of oxyresveratrol and piceatannol to trypsin and lysozyme induced some microenvironmental and conformational changes of the two enzymes. The thermal stability of the enzymes in the presence of polyphenols was studied based on the change in melting temperature by differential scanning calorimetry. The above experimental results were validated by the protein–ligand docking studies which showed the location of the two ligands in the enzymes and the surrounding amino acid residues. Furthermore, enzyme activity assays indicated that the enzymatic activity of trypsin and lysozyme was inhibited by oxyresveratrol and piceatannol. The effect of trypsin and lysozyme on the antioxidant activity and stability of oxyresveratrol and piceatannol was also investigated. In conclusion, the comparative study on the interaction of oxyresveratrol and piceatannol with trypsin and lysozyme showed that the positions of hydroxyl groups of the polyphenols had an important influence on their interaction with enzymes and their antioxidant activity and stability as well as the enzyme activities. The obtained results are expected to provide a theoretical basis for the application of polyphenols in functional foods and pharmaceuticals.
研究领域:
生物化学
·
药理学
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食品科学
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PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2018
·
IF 5.123
Fangfang Xue,Fei Qu,Wenli Han,Lian Xia,Jinmao You
Metal nanoclusters protected by glutathione (GSH) have attracted a wide attention due to the unique aggregation-induced emission (AIE) feature. However, the “trigger” effects of ethanol, temperature, pH values, and metal ions may restrict the application of these particles. In this work, the amino modified silicon nanoparticles (SiNPs) and GSH-capped gold nanoclusters (GSH-AuNCs) can self-assemble into well-defined spherical particles due to the electrostatic interaction . As a result, the unique aggregation-induced emission enhancement (AIEE) of GSH-AuNCs arises at 570 nm, and the SiNPs keep their own blue fluorescence at 450 nm, so a novel nanohybrid probe (SiNPs@GSH-AuNCs) with dual-emission property has been constructed. When protamine is added to SiNPs@GSH-AuNCs, the cationic protamine can compete with SiNPs and absorb onto the surface of GSH-AuNCs, which inhibits the self-assembly and leads to the fluorescence quenching of GSH-AuNCs; while trypsin can catalyze the hydrolysis of protamine, the self-assembly starts again, producing the AIEE recovery. In the whole process, the SiNPs act as an internal standard and their emission stays constant. By means of the fluorescence intensity ratios I 570 /I 450 , the linear range of protamine is from 0.15 to 3.00 μg mL −1 with the limit of detection (LOD) of 0.07 μg mL −1 , and trypsin shows a linear response in the range from 10 to 100 ng mL −1 with LOD of 4.50 ng mL −1 . Furthermore, this strategy exhibits good sensitivity and selectivity , and has been further validated by applying it for the determination of protamine and trypsin in serum samples.
研究领域:
生物化学
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纳米医学
·
分析化学
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Fei Qu,Zhuo Wang,Cong Li,Dafeng Jiang,Xian-en Zhao
Herein, a novel peptidyl probe is first synthesized by the amide reaction between glutathione-capped gold nanoclusters (GSH-AuNCs) and positive charged arginine-rich peptide (Arg 9 ), and it is called Arg 9 -GSH-AuNCs. Polyuridylic acid (polyU), as a polyanion , is firstly used to induce the aggregation-induced emission enhancement (AEE) of positive charged Arg 9 -GSH-AuNCs through electrostatic attraction . The peptide bond on the carboxyl terminal of the arginine residues can be recognized and cleaved by trypsin. Hence, polyU is hard to induce the aggregation of peptidyl probe again, and the AEE from GSH-AuNCs becomes weak. It is an ideal platform to detect trypsin and screen trypsin inhibitors . The fluorescent peptidyl probe exhibits excellent sensitivity for trypsin. A good linear range of trypsin is obtained from 1 to 50 ng·mL −1 with the limit of detection (LOD) 0.31 ng·mL −1 , which is lower than those of most reported works. Furthermore, the inhibitory effects of the extracts from foods (i.e., soybean seed flour, black soybean seed flour, mung bean seed flour, and azuki bean seed flour) on the trypsin activity have been studied. The half maximal inhibitory concentration (IC 50 ) values of extracts from soybean seed flour, black soybean seed flour, mung bean seed flour, and azuki bean seed flour are 4.5, 9.3, 11.6, 20.9 μg·mL −1 , respectively. The screening results show that these extracts from foods all have inhibitory effects on trypsin. Among them, the extract from soybean seed flour exhibits significant inhibitory effect.
研究领域:
分析化学
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生物化学
·
食品科学
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高影响因子
FOOD CHEMISTRY
·
2022
·
IF 9.231
Yinli Li,Zihan Yuan,Yuanhong Gao,Zhijie Bao,Na Sun,Songyi Lin
A pulsed electric field (PEF) treatment exhibits different effects on trypsin; however, the mechanism of enzyme activation remains unclear. Herein, chemical experiments combined with molecular dynamics simulations revealed the mechanism of trypsin activation by PEF treatment at the molecular level. The results indicated that compared with the values at 0 kV/cm, the enzyme activity, Vmax, and Kcat at 20 kV/cm increased by 9.30%, 4.74%, and 4.30%, respectively, and Km decreased by 11.14%, indicating an improved interaction between the enzyme and substrate. The simulation results revealed that PEF treatment increased the number of molecular hydrogen bonds and the solvent-accessible surface area, while decreasing the rotation radius and random coil content by 5.00% and 3.37%, respectively. Molecular docking indicated that PEF treatment altered the active center and increased the affinity between the enzyme and substrate. The simulation results were consistent with those of the spectroscopic experiments conducted on trypsin after PEF treatment.
研究领域:
生物化学
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食品科学
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分子生物学
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DOI
PubMed
高影响因子
Molecular Catalysis
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2022
·
IF 5.089
Xinyi Ma,Cheng Fu,Xinying Wang,Xiu Xing,Na Wang,Wei Zhang
Bovine trypsin displays a promiscuous activity to catalyze aldol reactions between isatins and ketones for easy access to 3-hydroxyindolin-2-ones. Detailed optimizations on the reaction conditions afforded the products with good yields, this system, which was compatible with various isatins. However, gradually enlarging the structure of ketones led to a significant decrease in yields. Molecular simulations were utilized to reveal the possible source of substrate selectivity. These results may provide valuable information for organic synthesis catalyzed by hydrolases.
研究领域:
生物化学
·
有机合成
·
计算生物学
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Zhenyu Ma,Jing Sun,Xiaolan Li,Wenjun Shan,Yunxiang Yan,Yongjun Liu
Trypsin, a protease produced in the pancreas, plays a crucial role in various medical conditions, including acute pancreatitis, cystic fibrosis, and malnutrition, where abnormal trypsin levels are often implicated. Consequently, trypsin detection has emerged as a valuable diagnostic tool in clinical medicine. In this study, we introduced an integrated fiber sensor that capitalizes on the amplification and transduction capabilities of liquid crystals, coupled with the high sensitivity of the whispering gallery mode, to achieve precise trypsin detection. This innovative approach offers consecutive measurement and rapid response times , typically under 200 s, and remarkably low sample consumption, as little as 3 nL, potential for detection in capillary blood sampling. Furthermore, when subjected to validation tests for trypsin detection in human serum, our method demonstrated excellent consistency and reliability, with recovery ratios falling within the range of 97–112%. Beyond trypsin, this method's adaptability and versatility make it a universal technique with potential applications in the field of biosensing and biochemistry.
研究领域:
生物传感
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医学诊断
·
光学
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DOI
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2011
·
IF 4.194
Yingzhuang Chen,Minghuo Wu,Keyi Wang,Bo Chen,Shouzhuo Yao,Hanfa Zou,Lihua Nie
A novel thiol-ene “click” strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic–inorganic monolithic capillary column with ene-functionality was fabricated by sol–gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups . Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα- p -tosyl- l -arginine methyl ester hydrochloride, TAME) and the product (Nα- p -tosyl- l -arginine, TA) with on-line capillary zone electrophoresis . After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N′-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene “click” strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals.
研究领域:
生物化学
·
分析化学
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Xiaoxia Liu,Fanghua Xia,Guojuan Zhang,Wenmei Gao,Xiaodi Guo,Jinzhong Zhao,Wuyong Zhao,Miaomiao Tian
Herein, a simple, convenient, and cost-effective distance-readout paper-based sensor is developed for the detection of trypsin. Trypsin hydrolyzes the gelatin hydrogel via the gel-to-sol transition, resulting in a change of viscosity and leading to a change in diffusion behavior. The concentration of trypsin is thus quantitatively determined by measuring the difference in diffusion diameter of the spot on the paper. The linear range for trypsin detection is from 0.01 to 10.0 mg/mL with a limit of detection of 0.0019 mg/mL. It is further applied to the quantitative detection of trypsin in human serum and the standard inhibitor aprotinin. In addition, the inhibitors screening in 10 traditional Chinese medicines has been successfully achieved. It only requires the use of the gelatin hydrogel, MCE filter paper, and a Vernier caliper. Therefore, this convenient method is very promising in point-of-care testing (POCT) applications and the development of commercial products.
研究领域:
生物传感器
·
临床诊断
·
即时检测
·
酶检测
·
纸基分析装置
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DOI
高影响因子
JOURNAL OF MOLECULAR LIQUIDS
·
2025
·
IF 5.2
Shan Huang,Xincong Tu,Shiqin Zhang,Bo Li,Chunyan Pan,Fuxiang Wei,Qi Xiao
Herein, we have pioneered a hydrothermal synthesis approach to fabricate red-emitting manganese-doped carbon dots (Mn-CDs), which exhibit a pronounced fluorescence peak at 665 nm under 525 nm excitation. This significant red-shift in emission wavelength not only minimizes autofluorescence interference but also enhances tissue penetration, making Mn-CDs highly promising for biological applications. Capitalizing on these optical properties, our study delves into the interactions between Mn-CD and trypsin, employing sophisticated spectral analysis and molecular docking techniques. Our findings reveal that Mn-CDs non-covalently bind to the catalytic center and microenvironment of trypsin through hydrogen bonds and van der Waals forces, inducing conformational changes and forming a stable complex that impedes enzymatic activity. In vitro assays further substantiate the competitive inhibitory effect of Mn-CDs on the trypsin's catalytic activity, with enzyme kinetics demonstrating a reduced Michaelis-Menten constant without altering the maximum catalytic velocity. Notably, gel electrophoresis, human cervical carcinoma cells digestion micro-imaging, and clone formation experiments collectively demonstrate that this inhibitory effect effectively shields human serum albumin and Hela cells from trypsin-mediated degradation. These insights into nanomaterial-protease interactions pave the way for innovative enzyme-regulated tumor therapies, harnessing the inhibitory properties of Mn-CDs to modulate protease activity within cancerous microenvironments. This research not only deepens our fundamental understanding of nano-bio interfaces but also propels the practical designs of diagnostic/therapeutic nanoplatforms for biomedical applications.
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高影响因子
SEPARATION AND PURIFICATION TECHNOLOGY
·
2025
·
IF 9
Hui Ye,Xiao Zhang,Xijie Wang,Chuan Gao,Shurui Han,Jing Lv,Jinke An,YuZhong Zhang,Lizhi Zhao,Qingping Xin,Ligang Lin,Xiaoli Ding
Membranes are extensively utilized for protein separation and purification; however, fouling is a significant challenge and limits further practical application. Biocatalytic membranes have attracted much attention due to their potential anti-fouling and self-cleaning properties. Herein, biocatalytic nanofibrous porous membranes were fabricated through an in-situ zeolitic imidazolate frameworks (ZIF-8) anchored and trypsin incorporation strategy. Ethylene vinyl alcohol copolymer (EVAL) nanofibers, doped with Zn(Ⅱ) or ZIF-8, were fabricated via electrospinning, and then ZIF-8 layers were anchored onto the surface of the nanofibers via in-situ growth. Continuous ZIF-8 layers were observed and almost completely covered the surface of the nanofibers, providing 2.7-fold trypsin loading than ZIF-8 crystals in solution. For the biocatalytic nanofibrous membranes, the trypsin loading were both exceeded 270 µg cm −2 and the maximum reaction rate values of membranes during filtration increased by 120 % than that of free trypsin. Enhanced hydrophilicity and protein-digestive functionality contributed to high self-cleaning properties of the membranes. The biocatalytic nanofibrous membranes demonstrated 2-fold flux recovery and improved BSA resistance compared to the pristine membrane after BSA filtration. Moreover, the membranes retained high anti-fouling properties after five reuse cycles, utilizing only a simple and cost-effective washing operation. Such biocatalytic nanofibrous membranes hold great promise for enhanced anti-fouling and self-cleaning performance in water applications.
研究领域:
材料科学
·
生物工程
·
环境工程
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高影响因子
JOURNAL OF SEPARATION SCIENCE
·
2017
·Vol. 40
·期 15
·3168-3174
·
IF 2.557
Mengxia Cheng,Zilin Chen
A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half-maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.
研究领域:
分析化学
·
制药生物技术
·
天然产物研究
·
酶抑制
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DOI
高影响因子
危险品化学品经营许可证（带存储）