动态固定化胰蛋白酶条件的优化

以固定化人工膜（immobilized artificial membrane，IAM）为基质动态固定胰蛋白酶，研究缓冲液种类、浓度、pH值对胰蛋白酶在IAM上的固载率和酶活性的影响。本课题选择硼酸盐、磷酸盐、Tris？HCl三种缓冲液体系，考察了缓冲液浓度及pH值对IAM固载胰蛋白酶的影响，并建立以酪蛋白为底物测定固定化胰蛋白酶的活性的方法，优化了动态固定化胰蛋白酶的条件，为进一步研究在线固定化酶解反应器奠定基础。     

双适配体夹心微阵列芯片在凝血酶检测中的应用

核酸适配体不仅具备抗体分子的专一性识别性能,而且还具备其独特的优异特性。凡涉及到抗体的识别范畴,几乎都可以用适配体来取代抗体。微阵列芯片具有高通量、微量化和自动化等优点。本论文以凝血酶为检测对象,在优化氨基化适配体（T29）在醛基化玻璃基片上的固定条件,并对固定结果进行表征的基础上,设计制作了用于凝血酶检测的基于双适配体夹心原理的微阵列芯片。结果表明,氨基化适配体T29．1在醛基化玻璃基片上的固定浓度为5gM,最佳的固定缓冲液为含有37．5％DMSO的3＊SSC,在微阵列芯片加工制作的基础上,利用双适配体夹心法的原理初步实现了凝血酶的检测,这为利用微阵列芯片实现多种物质的同时检测奠定了方法学基础。     

核酸适配体介导无机纳米递药系统在肿瘤靶向治疗中的研究进展

核酸适配体和靶物质具有良好的亲和力、高特异性等特性,在药物靶向递送系统研究领域具有很大的应用价值。到目前为止,不同种类的基于核酸适配体药物系统已被报道。本综述总结了核酸适配体介导无机纳米材料在靶向抗肿瘤的最新研究进展,并对其存在的问题和未来的研究方向进行了展望。     

核酸适配体及其在纸上纳米传感器研究中的应用

核酸适配体是一种能识别目标物质并与之结合的单链DNA或者RNA,被越来越多地应用于纸上纳米生物传感器的研究中。本文从核酸适配体出发,在综述其特点及筛选原理的基础上,结合纸上纳米生物传感器的特点,综述了核酸适配体在纸上纳米生物传感器中的应用,不难发现,除利用适配体与抗体相似的识别与捕获能力之外,这些传感器还创新性的利用了诸如,适配体易于修饰的特点设计新的信号转换方式,碱基互补配对规则设计适配体与其他分子之间的交联以及适配体可以扩增的特点进行信号放大等等开展相关的研究工作。     

核酸适配体在液相色谱和毛细管电泳中的应用

指数富集配体的系统进化(SELEX)技术是一种新的组合化学技术,应用人工合成的随机寡核苷酸文库,通过筛选、分离、富集获得能与氨基酸、蛋白、药物、有机小分子、无机离子等靶分子特异性结合的寡核苷酸适配子,对于靶分子具有高亲和力。本文从核酸适配体分子识别特性出发,综述了核酸适配体在液相色谱和毛细管电泳中应用的研究进展。     

酶法恒温有机磷农药快速测定仪

本文介绍了一种用固定化酶在恒温下快速检测有机磷农药的仪器。仪器主要由带固定化酶的比色皿、恒温室、半导体制冷器、光电传感器等组成;比色皿架及比色皿底部由热良导体材料制成,固定化酶柱粘贴在比色皿底面并置于恒温室中;恒温室的温度可调节,在检测时调节到最佳反应温度;平常设置为低温状态以保存酶柱,维持酶的活性。     

固定化酶及其在食品和生物领域的研究进展

固定化酶技术是酶工程的核心,渗透在现代各种工艺之中,成为不可或缺的工具。本文对其发展、优缺点和固定技术做了介绍,并简述了其在生物和食品方面的一些研究和应用。     

溶胶-凝胶固定纳米光纤探针上生物活性分子的研究

为提高纳米光纤生物传感器的性能,研究了以溶胶-凝胶法将生物活性分子固定于纳米光纤探针上。首先以正硅酸乙酯(TEOS)为前驱体,制备了以葡萄糖氧化酶(GOD)为生物活性分子的溶胶,然后利用提拉法将GOD凝胶固定于纳米光纤探针上;讨论了固定化GOD活性及凝胶膜在纳米光纤探针上成膜的条件;最后通过扫描电子显微镜检测到薄膜均匀连续,生物探针尺寸小于1μm,并利用电子探针验证了GOD被固定于纳米光纤探针上。     

酶电极中酶固定化方法研究进展

酶电极是最常用也是最早开发的生物传感器,而固定化酶作为酶电极的关键也得到了广泛的关注。本文介绍了酶电极及固定化酶的特点,重点讨论了制作酶电极的关键技术即酶固定化的方法,包括吸附法、包埋法、共价键合法和交联法等传统方法和静电纺丝法、纳米技术处理等一些新型的固定化方法,并进一步探讨了各种固定化方法的优缺点。     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱联用鉴定椴树蜂蜜中的抗氧化活性肽

应用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱联用法(UPLC-Q-Exactive)鉴定椴树蜂蜜蛋白经胰蛋白酶水解后获得的肽类物质的组成和结构,并探讨椴树蜂蜜酶解多肽与其抗氧化活性的关系。椴树蜂蜜经三氯乙酸溶液沉淀,所得蛋白用胰蛋白酶酶解,酶解肽段经C18固相萃取柱富集净化后,采用0.1%甲酸(A)-乙腈(B)作为流动相进行梯度洗脱,以高分辨质谱(Q-Exactive)的Full MS/ddMS2模式对其进行鉴定。采用MaxQuant软件分析质谱结果,所得肽段在Uniprot上进行Blast序列对比,共鉴定出52种椴树蜂蜜胰蛋白酶解多肽,其中71.15%来自蜂王浆主要蛋白(MRJPs),鉴定出的肽段归属于10种蛋白质,其中6种(60%)属于MRJP家族蛋白。对椴树蜂蜜酶解多肽质谱鉴定结果进行重复性分析,2次以上重复质谱分析可实现对椴树蜂蜜多肽(87.1%)的可靠鉴定。结合文献报道的蜂蜜多肽抗氧化活性的构效关系,筛选出11条可能具有抗氧化活性的多肽。化学合成11条多肽后,进行4种不同的抗氧化活性实验。结果表明,这11条多肽的抗氧化活性存在较大差异,其中1 g/L VIYEWK多肽显示出较强的ABTS自由基清除活性(33.8%±0.6%)、DPPH自由基清除活性(96.5%±0.0%),并表现出一定的还原力(0.010±0.001)和Fe2+螯合活性(4.0%±0.2%)。该方法能快速有效鉴定椴树蜂蜜多肽结构,可为揭示椴树蜂蜜多肽结构与抗氧化活性的关系提供依据。     

Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy

We describe a technique for imaging enzyme activity through steady‐state fluorescence anisotropy measurements on a per‐pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 µm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy‐FL‐labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five‐fold. Fluorescence lifetime and time‐resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy‐FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy‐containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.     

Label-free target DNA recognition using oligonucleotide-functionalized polypyrrole nanotubes

Conjugated polymers for oligonucleotide immobilization offer extraordinary potential as transducers for detecting DNA duplex formation, because the electrical, optical, and electrochemical properties are strongly affected by relatively small perturbations. Moreover, carboxylated conducting polymer supports are an attractive alternative due to their versatile immobilization with DNA, protein, and enzyme using various pendant groups: -SH, -NH(2), and -COOH. Therefore, we report the fabrication of carboxylic acid-functionalized polypyrrole nanotubes (CPPy NTs) using oxidant-impregnated template synthesis. The diverse number of carboxylates on the surface of CPPy NTs was applied to binding sites for amino-terminal oligonucleotides. Conductance of single DNA strands (ssDNA) and hybridized DNA helix were readily measured by means of depositing DNA-functionalized nanotubes on gold leads, and indicated high sensitivity (DeltaR/R(0)=1.7) even at low concentration (1nmol) of target DNA. In addition, target DNA concentration was distinguished up to a narrow difference of 2nmol. The successful DNA immobilization on polymer nanotubes was confirmed and visualized by the photoluminescence of fluorescein isothiocyanate (FITC)-tagged target DNA using confocal laser scanning microscopy.     

Direct immobilization of cupredoxin azurin modified by site-directed mutagenesis on gold surface

For making efficient bioelectronic device, we have developed novel immobilization method of cupredoxin azurin modified on gold (Au) surface. A recombinant protein with cysteine residue by using site-directed mutagenesis was designed and then directly immobilized on Au surface without any chemical linker. The immobilization of the functionalized protein is confirmed by surface plasmon resonance (SPR) and its surface morphology is analyzed by scanning tunneling microscopy (STM). The immobilization efficiency has been increased about 75.6%, as compared to that of wild-type azurin. The electrochemical property of the fabricated thin film was investigated by the cyclic voltammetry (CV). As a result, cysteine-modified azurin can be used for making high-quality protein film, and applied to the fabrication of nano-scale bioelectronics.     

Fabrication of self-assembled oligophenylethynylenethiol monolayer for electrochemical glucose biosensor

An electrochemical glucose biosensor was developed using a gold (Au) electrode, which was composed of self-assembled oligophenylethynylenethiol monolayer and glucose oxidase (GOx) structure. Oligophenylethynylenethiol was used as a chemical linker for the immobilization of GOx on Au electrode, which facilitates the transfer of electron produced by enzyme reaction to the Au electrode. The electrical property of self-assembled oligophenylethynylenethiol monolayer was investigated by using cyclic voltammetry (CV). The formation of self-assembled oligophenylethynylenethiol monolayer and GOx layer on Au surface was verified by using atomic force microscopy (AFM) and surface plasmon resonance (SPR). The electrochemical glucose biosensor exhibited a linear relationship between target concentration and oxidation current in the range of 2–30 mM and its detection limit was 2 mM.     

Optimization of the linear quantification range of an online trypsin digestion coupled liquid chromatography–tandem mass spectrometry (LC–MS/MS) platform

Tandem mass spectrometry (MS/MS)-based proteomic workflows with a bottom-up approach require enzymatic digestion of proteins to peptide analytes, usually by trypsin. Online coupling of trypsin digestion of proteins, using an immobilized enzyme reactor (IMER), with liquid chromatography (LC) and MS/MS is becoming a frequently used approach. However, finding IMER digestion conditions that allow quantitative analysis of multiple proteins with wide range of endogenous concentration requires optimization of multiple interactive parameters: digestion buffer flow rate, injection volume, sample dilution, and surfactant type/concentration. In this report, we present a design of experiment approach for the optimization of an integrated IMER-LC–MS/MS platform. With bovine serum albumin as a model protein, the digestion efficacy and digestion rate were monitored based on LC–MS/MS peak area count versus protein concentration regression. The optimal parameters were determined through multivariate surface response modeling and consideration of diffusion controlled immobilized enzyme kinetics. The results may provide guidance to other users for the development of quantitative IMER-LC–MS/MS methods for other proteins.     

用于黄曲霉素B1检测的表面增强型荧光光学传感器

黄曲霉素B1(aflatoxin B1,AFB1)是一种常见于农作物中的真菌毒素,是所有真菌霉素中毒性最强且具有致癌性。因此,快速、有效地检测出食品中AFB1对于食品安全来说具有重要意义。设计了一种基于表面增强荧光(surface-enhanced fluorescence,SEF)技术的光学芯片用于AFB1灵敏检测。该光学芯片以纳米多孔金(nanoporous gold,NPG)作为荧光增强基底,通过在其表面先后组装适体SH-DNA2和互补适体Cy5-DNA1构建针对AFB1的功能芯片。该芯片利用AFB1和Cy5-DNA1与SH-DNA2之间竞争结合,释放Cy5-DNA1引发来自Cy5荧光信号的衰减,通过监测Cy5的荧光强度的变化实现对AFB1的检测,检测极限可达到10^?7μg/L且线性动态范围有4个量级。     

Fungal assembly of L-asparaginase using solid-state fermentation: a review

Because of its antitumor therapeutic-activity, as well as its application in food industries to improve the quality, L-asparaginase has attracted considerable attention from several investigators. In recent years, fungi have occupied advanced rank among microorganisms in the production process of the enzyme. This review is spotting the light on the advantages of fungal enzyme and its applications in the food industry and medications. The solid-state fermentation was discussed as the wide alternative and most accepted biosynthesis technique. However, some lights were also spotted to the statistical experimental design of the fermentation process, mainly on the methodology of the response surface for L-asparaginase biosynthesis by fungi. Finally, the immobilization of the enzyme and the features of the widely used solid substrates for the maximization of the production process were explored.     

Electrochemical DNA biosensor with nanometer scale using nano-patterning lithography machine

Major challenges in the field of electrochemical DNA hybridization biosensors are the immobilization of DNA and the detection of hybridization signals. The method of DNA immobilization using the nano-patterning machine and detection for DNA hybridization signals has been proposed. Here, two gold electrodes were deposited on SiO₂ layer and the gap between the electrodes was fabricated by electron beam lithography. 3-aminopropyltriethoxysilane (APTES) solution was selectively treated to immobilize the amino-modified oligonucleotides onto the SiO₂ layer between the electrodes. The recognition of DNA hybridization was accomplished by metallic aggregation of nano-particles. The results showed that DNA is immobilized with nanometer scales and the method for detecting hybridization signals is useful. The experimental results were verified by I-V curves. The conductance between two electrodes changed with the density of the Au-nanoparticles immobilized onto the oxide layer. These results can be applied to the DNA chip and the multi-functional sensors which will be researched in the further study.     

Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen.

Acrosin activity is associated with normal fertility in human and bovine spermatozoa. The aim of the study was to determine the variation of the enzyme activity in the proacrosin-acrosin system in capacitated and acrosome reacted cryopreserved bovine sperm. Enzyme activity was assessed spectrophotometrically using N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) as specific substrate for acrosin at pH 8. Capacitation with heparin and quercitin failed to induce conversion of proacrosin to acrosin. An increase in acrosin activity produced by the presence of progesterone, in a dose-dependent manner, was related with the induction of true acrosome reaction. The total level of acrosin activity registered showed that 96% of acrosin of capacitated sperm samples and control is present in the zymogen form. Moreover, progesterone is capable of duplicating the level of active enzyme, indicating that enzyme activity changes are related to acrosome reaction, suggesting that only a minor proportion of the total of proacrosin-acrosin system is required in the exocytotic process on cryopreserved bovine sperm.