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Jie Feng Yonglei Chen Yangxia Han Juanjuan Liu Cuiling Ren Xingguo Chen 《Analytica chimica acta》2016
In recent years, extensive researches are focused on the fluorescent carbon nanoparticles (CNPs) due to their excellent photochemical, biocompatible and water-soluble properties. However, these synthesis methods are generally suffered from tedious processes. In this paper, fluorescent carbon nanoparticles are synthesized by a facile, one-pot, low-temperature method with trypsin and dopamine as precursors. The synthesis process avoids any heating operation and organic solvent, which provides a “green” and effective preparation route. The obtained CNPs exhibit excellent water-solubility, salt-tolerance and photostability. Based on the synergistic action of the inner filter effect and static quenching mechanism, the CNPs are exploited as a “turn-off” fluorescence sensor for sensitive and selective detection of Fe3+ ions. The probe shows a wide linear range from 0.1 to 500 μM, with a limit of detection of 30 nM. Furthermore, the as-fabricated fluorescent sensing system is successfully applied to the analysis of Fe3+ in biological samples such as human urine and serum samples with satisfactory recoveries (92.8–113.3%). 相似文献
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Abdennour Abbas Dominique Vercaigne-Marko Philippe Supiot Bertrand Bocquet Cline Vivien Didier Guillochon 《Colloids and surfaces. B, Biointerfaces》2009,73(2):315-324
This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV–vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process. 相似文献
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Gong Cheng Ping Chen Zhi-Gang Wang Xiao-Jing Sui Ji-Lin Zhang Jia-Zuan Ni 《Analytica chimica acta》2014
A simple, fast, efficient, and reusable microwave-assisted tryptic digestion system which was constructed by immobilization of trypsin onto porous core-shell Fe3O4@fTiO2 microspheres has been developed. The nanostructure with magnetic core and titania shell has multiple pore sizes (2.4 and 15.0 nm), high pore volume (0.25 cm3 g−1), and large surface area (50.45 m2 g−1). For the proteins, the system can realize fast and efficient microwave-assisted tryptic digestion. Various standard proteins (e.g., cytochrome c (cyt-c), myoglobin (MYO), β-lactoglobulin (β-LG), and bovine serum albumin (BSA)) used can be digested in 45 s under microwave radiation, and they can be confidently identified by mass spectrometry (MS) analysis; even the concentration of substrate is as low as 5 ng μL−1. Furthermore, the system for the 45 s microwave-assisted tryptic digestion is still effective after the trypsin-immobilized microspheres have been reused for 5 times. Importantly, 1715 unique proteins from 10 μg mouse brain proteins can be identified with high confidence after treatment of 45 s microwave-assisted tryptic digestion. 相似文献
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氢键和极性相互作用在抑制剂-蛋白结合专一性识别过程中起到重要作用.抑制剂Benzamidine(BEN)与胰岛素trypsin相互作用机制的阐明有助于胰岛素高效抑制剂的研发.本文采用分子动力学模拟和MM-PBSA(molecular mechanics-Poisson Boltzmann surface area)从原子层次上研究BEN与胰岛素的结合模式.结果表明抑制剂BEN的脒基不仅与Asp189的羰基产生静电相互作用,而且与残基Ser190和Gly214形成氢键相互作用.基于残基能量分解的计算表明抑制剂的苯基与残基His58,Cys191,Gln192,Trp211,Gly212和Cys215形成有利于抑制剂结合的疏水性相互作用.期望当前的研究能为胰岛素有效抑制剂的研发提供重要的理论指导. 相似文献
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Inhibitory activity and conformation changes of soybean trypsin inhibitors induced by ultrasound 总被引:1,自引:0,他引:1
The inhibitory activities, sulfhydryl bonds and far-UV circular dichroism (CD) spectra of Kunitz and Bowman–Birk soybean trypsin inhibitors (KTI and BBTI) were measured before and after ultrasound treatments. The differences between KTI and BBTI in conformation changes and resistance to ultrasound were observed. The secondary structures of KTI were found to be composed of β-sheet (22.5%), β-turn (16.2%) and random coils (61.4%) while that of BBTI composed of only β-sheet (52.6%) and random coils (47.4%). KTI lost its inhibitory activity more quickly than BBTI, proportionally to the ultrasound amplitudes and sonication durations used. Relevant synchronous phenomena observed included the inactivation of KTI, significant rise in sulfhydryl content and corresponding conformation changes, in which there were dramatic decreases in both β-turn and random coil contents and increase in the β-sheet structure over the entire sonication duration and ultrasonic amplitude scale used by the study. Ultrasound affected the activities and conformations of KTI and BBTI by exerting an influence on their disulfide bonds. For KTI, up to 55% of inhibitory activity could be inactivated, at which about 71.5% of disulfide bonds were altered and the [θ]200nm value was changed from native −2545 deg cm2 dmol−1 to −1827 deg cm2 dmol−1. Whereas for BBTI, far-UV CD spectra, β-sheet and random structures were barely affected, only about 5.29% of disulfide bonds were found altered and the [θ]200nm value was changed only from native −797 deg cm2 dmol−1 to −700 deg cm2 dmol−1. It is concluded that ultrasound inactivates KTI by inducing a reduction in the disulfide bonds and then changes the conformations. 相似文献
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This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein
digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column
was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence
of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution
through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC.
The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein
digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution
through the column, and 1.0 μL min−1 was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required
pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and
the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple
and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation
of proteins. 相似文献
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WANG Hua ZHAO Mi-feng PU Li-mei DING Wei HUANG Yi-bing HOU Rui-zhen ZENG Hong-bin XU Li ZHANG Xue-zhong 《高等学校化学研究》2006,22(6):721-726
IntroductionA characteristic tripeptide sequence Arg-Gly-Asp(RGD) that is found within fibronectin and other rela-ted adhesion molecules in extracellular matrices(ECM)has received considerable attention from researcherssince it was proved to be a recognit… 相似文献
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Bossi A Guizzardi L D'Acunto MR Righetti PG 《Analytical and bioanalytical chemistry》2004,378(7):1722-1728
In the present paper, the covalent immobilisation of the digesting enzyme trypsin has been achieved through photo-immobilisation on a portion of a silica capillary, thus leading to the construction of a capillary electrophoretic (CE)-microreactor for peptide mapping. The CE-microreactor is characterised by being a single piece, thus ensuring no fluidic or electrical leakage. The enzyme was immobilised with a surface density of 15.8 g/cm2, the stability was high (80% after 38 days) and the rate of conversion was 0.2 ng/s. On-line protein mapping was tested with proteins of different dimensions, showing competitiveness in terms of time (completed map within 15 min) and exhaustive maps of small proteins. The results of the CE-microreactor and the potential to immobilise biocomponents easily on a desired portion of the capillary indicate further developments towards the construction of a variety of miniaturised enzymatic screening devices for high-throughput screening analysis. 相似文献