固定化乙酰胆碱酯酶的制备及其特性研究

本研究以期研制出能重复使用的固定化乙酰胆碱酯酶(AChE),为天然产物复杂体系中AchE抑制剂筛选新方法的发展奠定基础.以氨基化硅胶(APS-Si)微球为载体,戊二醛为交联剂对乙酰胆碱酯酶进行交联固定化,并研究了酶的最佳固定化条件和固定化酶的性质.结果表明,0.05 g氨基化硅胶微球载体,用戊二醛溶液活化6 h后,在给酶量5 U,28℃固定16 h条件下,得到固定化酶的活性最大.固定化酶在常温(20～40℃),以及较宽pH范围内(pH 6～10)均具有较高的活性,并且具有良好的保存稳定性和可重复利用率,为基于固定化靶酶亲和-色谱质谱联用分析快速筛选乙酰胆碱酯酶抑制剂新方法的发展奠定了基础.     

戊二醛合成跟踪分析

用气相色谱法对液 液相转移反应萃取法合成戊二醛（ＧＡ）反应进行跟踪分析,采用φ３ｍｍ×４ｍ［ｍ（ＳＥ ３０）∶ｍ（硅烷化１０１白色担体）＝１５∶１００］＋φ３ｍｍ×２ｍ［ｍ（ＰＥＧ ２０Ｍ）∶ｍ（硅烷化１０１白色担体）＝１０∶１００］不锈钢柱,双柱双氢焰程序升温,升温程序为７０℃（４ｍｉｎ）１０℃／ｍｉｎ１４０℃（６ｍｉｎ）。Ｎ２４５ｍＬ／ｍｉｎ,Ｈ２６５ｍＬ／ｍｉｎ,空气３００ｍＬ／ｍｉｎ,汽化温度１８５℃,检测温度１８５℃。所建立的方法能较好地分离环戊烯、溶剂、氧化中间产物、ＧＡ、内标、氧化副产物。     

Capturing sodium dodecyl sulfate-treated protein antigens by antibody affinity electrophoresis

Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.     

Flow injection analysis for L-lactate with immobilized lactate dehydrogenase

Immobilized lactate dehydrogenase (LDH) is used for determination of L-lactate in a continuous flow system. The LDH is immobilized by reaction with glutaraldehyde onto the surface of alkylamino-bonded silica gel and packed into a column in the flow system. The reduction of NAD⁺ occurs simultaneously, and the NADH formed is detected amperometrically. The peak current is linearly related to the L-lactate concentration in the range 1–80 × 10^-6 M; 30 samples h^-1 can be analyzed with a r.s.d. of 0.5–1.5%. The immobilized LDH retains over 90% of its initial activity after repetitive use for 3 months.     

Continuous-flow analysis for ethanol with immobilized alcohol dehydrogenase

Alcohol dehydrogenase is immobilized by reaction with glutaraldehyde onto silica gel, which is packed in a column in a flow system. Ethanol (0.1–5 mmol) is determined by catalysis of the reaction with NAD⁺, the NADH produced being detected amperometrically. The reproducibility is ca. 1%.     

Electrophoresis and detection of tin-labeled DNAs on open-faced gels.

Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar or to better than radioisotopes but allows automated, high-speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub-attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance-ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter-initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open-faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by-products, dried, and stored until use. Various concentrations of an Sn-labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 microns thick gel. Gels as thin as 60 microns lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open-faced gels by employing discontinuous buffers systems and DNA mobility modifiers.     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Amide bond formation with a new fluorous carbodiimide: separation by reverse fluorous solid-phase extraction

A new fluorous carbodiimide is introduced along with a convenient procedure for amide coupling reactions. Reactions of acids and amines under standard conditions for carbodiimide couplings, followed by simple reverse fluorous solid-phase extraction (FSPE) over standard silica gel, provide the target amide products in good yields and purities. The use of HFE-7100 as a fluorous solvent is crucial for the success of the reverse FSPE.     

Direct astatination of a tumour-binding protein, human epidermal growth factor, using nido-carborane as a prosthetic group

We have investigated a method for direct astatine labeling of proteins. Binding sites for astatine were created by coupling of a nido-carborane derivative to a protein, the human epidermal growth factor (hEGF), using two different conjugation methods - by glutaraldehyde cross-linking or by introduction of sulfohydryl groups by Traut's reagent with subsequent linking of ANC-1 with m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester. The conjugates were astatinated using the Chloramine-T method in high yield. The best labeling was obtained by the glutaraldehyde conjugate with an average yield of 68±9%. In vitro stability tests indicated that the glutaraldehyde conjugated label was as stable as hEGF labeled with astatobenzoate.     

Automation of fluorous solid-phase extraction for parallel synthesis

An automatic fluorous solid-phase extraction (F-SPE) technique is developed by using FluoroFlash SPE cartridges on the RapidTrace workstation. A 10-module workstation has the capability to complete a maximum of 100 SPEs each round in 1-2 h. Another important feature of the RapidTrace system is that it has the capability to load slurry samples onto the F-SPE cartridges. The F-SPE cartridge charged with 2 g of fluorous silica gel is used to purify up to 200 mg of crude sample. Sample loading, elution solvent, cartridge reuse, and SPE reproducibility are evaluated. The automatic SPE system is used for purification of a small urea library generated from amine-scavenging reactions using fluorous dichlorotriazine, a 96-membered amide library generated using 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine as the coupling agent, and another 96-membered library generated from fluorous Mitsunobu reactions. Approximately 90% of the products have > 90% purity after F-SPE.     

Modification of tricine–SDS–PAGE for online and offline analysis of phosphoproteins by ICP-MS

This study describes a modification to tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis to make it more effective for the separation of low molecular mass proteins and for coupling with inductively coupled plasma mass spectrometry (ICP-MS). The modified method employs low-percentage polyacrylamide gels (7–10%) (w/v) and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. Using phosphopeptides as a model system, and offline analysis, we obtained recoveries of 73% (w/v) in a 9% gel compared with 55% in a conventional 16% gel. Online coupling was achieved by modification of a standard commercially available gel electroelution apparatus and casting of the gel into a 7.3-cm-long tube. Online separation of a digest of β-casein was demonstrated with recovery of the mono- and tetraphosphopeptides, which were identified by comparison with peptide standards. A mass balance study with the standards yielded recoveries of 95% for tetraphosphopeptides and 48% for monophosphopeptides. The factors affecting the separations and recoveries are discussed in detail. The detection limits for 10-μL samples of the mono- and tetraphosphopeptides were 0.7 μM (7 pmol) and 0.2 μM (2 pmol) respectively.     

以硅胶为载体通过γ-胺丙基三甲氧基硅烷偶联剂制备季铵盐类水不溶性杀菌剂的方法研究(II)

在利用硅胶表面上的羟基通过g -胺丙基硅烷把季铵盐固载到硅胶载体上制成水不溶性杀菌剂的基础上,考察了原料硅胶对制备产品杀菌性能的影响。在相同操作条件下,使用同一种原料硅胶,随着原料粒度的减小,所制备杀菌剂产品的杀菌率有所增加,但不明显;选用不同种原料硅胶合成的水不溶性季铵盐类杀菌剂的杀菌结果表明,原料比表面积越大,孔体积越小,合成产品的杀菌率越高。红外光谱证明了 -胺丙基三甲氧基硅烷已通过与硅胶表面羟基的反应键合到硅胶表面,叔胺化的硅胶中间体和产物上存在的很强的烷基峰也证明了硅胶上确实固载上了烷基官能团。交叉极化的硅核磁共振谱有效地表征了硅胶上表面羟基的反应情况,为-106处峰的基本消失证明了原料硅胶表面的硅羟基与胺丙基硅烷的键合反应比较完全。使用后的杀菌剂再生结果表明,其再生性能良好,具备重复使用的特点。     

Reductive Decarboxylative Alkynylation of N‐Hydroxyphthalimide Esters with Bromoalkynes

A new method for the synthesis of terminal and internal alkynes from the nickel‐catalyzed decarboxylative coupling of N‐hydroxyphthalimide esters and bromoalkynes is presented. This reductive cross‐electrophile coupling is the first to use a C(sp)−X electrophile, and appears to proceed via an alkynylnickel intermediate. The internal alkyne products are obtained in yields of 41–95 % without the need for a photocatalyst, light, or a strong oxidant. The reaction displays a broad scope of carboxylic acid and alkyne coupling partners, and can tolerate an array of functional groups, including carbamate NH, halogen, nitrile, olefin, ketone, and ester moieties. Mechanistic studies suggest that this process does not involve an alkynylmanganese reagent and instead proceeds through nickel‐mediated bond formation.     

EPR Spectroscopic Studies of Lipoxygenases

Polyunsaturated fatty acids are sources of diverse natural, and chemically designed products. The enzyme lipoxygenase selectively oxidizes fatty acid acyl chains using controlled free radical chemistry; the products are regio‐ and stereo‐chemically unique hydroperoxides. A conserved structural fold of ≈600 amino acids harbors a long and narrow substrate channel and a well‐shielded catalytic iron. Oxygen, a co‐substrate, is blocked from the active site until a hydrogen atom is abstracted from substrate bis‐allylic carbon, in a non‐heme iron redox cycle. EPR spectroscopy of ferric intermediates in lipoxygenase catalysis reveals changes in the metal coordination and leads to a proposal on the nature of the reactive intermediate. Remarkably, free radicals are so well controlled in lipoxygenase chemistry that spin label technology can be applied as well. The current level of understanding of steps in lipoxygenase catalysis, from the EPR perspective, will be reviewed.     

Effect of polymer-precursor molecular mass on the formation and properties of covalently crosslinked chitosan cryogels

Features of cryotropic gelation in moderately frozen solutions of chitosan samples that have various molecular masses and that are covalently crosslinked with glutaraldehyde are studied. It is shown that the chain length of the macromolecular precursor, chitosan, affects the yield of the gel fraction; the swelling ability of the polymer phase of wide-pore cryogels; the dimensions of the gross capillary pores of cryogels; and, as a consequence, the hydrodynamic parameters of columns packed with a permeable continuous matrix based on the spongy gel material. The characteristics of the latter depend in a sophisticated manner on the molecular mass of the polymer used for formation of cryogels, a circumstance that is associated with competition of differently directed factors, specifically cryoconcentration effects, a strong increase in the viscosity of the reaction medium in the unfrozen liquid microphase, kinetic features of chain crosslinking at a high concentration of reagents, etc. Therefore, in each particular case, individual conditions for specific combination of the above-mentioned parameters of the process arise. As a result, a complex dependence corresponding is observed for the efficiency of cryostructuring of the chitosan-glutaraldehyde system and for the properties of the resulting cryogels.     

壳聚糖修饰纳米纤维膜表面对氧化还原酶行为的影响

采用静电纺丝法制备了丙烯腈/丙烯酸共聚物(PANCAA)纳米纤维膜, 研究了纺丝液浓度对纤维形态的影响, 以扫描电子显微镜观察纤维形貌, 遴选得到最佳纺丝条件. 以1-乙基-3-(N,N-二甲基氨基丙基)碳二亚胺/N-羟基丁二酰亚胺(EDC/NHS)为偶联剂, 在纤维膜表面引入壳聚糖修饰层, 采用衰减全反射傅里叶变换红外光谱(ATR/FTTIR)、水接触角和称重法考察了修饰前后膜的变化. 通过戊二醛将过氧化氢酶固定到壳聚糖修饰的PANCAA纳米纤维膜上, 研究了壳聚糖及戊二醛浓度对固定化过氧化氢酶的影响, 结果表明, 在壳聚糖浓度为25 mg/mL及戊二醛质量分数为5%条件下, 壳聚糖修饰膜的固定化酶活性比空白膜提高了41.7%, 稳定性也得到了不同程度的提高.     

New synthetic methods for the formation of basic,polyvalent, hyperbranched grafts

A practical synthetic route to polybasic, polyamine, hyperbranched grafts using commercially available polyethyleneimine (PEI) and cyanuric chloride as a coupling agent is described. The grafting process was followed by XPS spectroscopy, TGA analysis, ATR‐IR spectroscopy, acid–base titration, and by ¹³C CP‐MAS NMR spectroscopy. In the case of silica gel, thermal gravimetric analyses showed that a 35 wt % loading of graft could be obtained. Acid–base titration of hyperbranched PEI grafts on silica gel and oxidized polyethylene powder showed the ion‐exchange capacities of these PEI‐grafted substrates were 1.00 and 0.17 mmol of base/g of solid, respectively. Although the focus of the paper is on grafting on silica gel, the influence of the kind of support and solvent on the grafting process and the ion‐exchange capacity was examined. Water was a good solvent for PEI grafting onto silica gel, but a more hydrophobic polyethylene support required the use of dichloromethane as a solvent for PEI graft synthesis. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4654–4665. 2005     

The chemistry of enzyme and protein immobilization with glutaraldehyde

Immobilization of proteins to solid matrices has been performed for the last thirty years and has provided numerous examples of successful preparations with use in enzyme reactors, sensor preparation and immunodiagnostics. Among the arsenal of coupling reagents and procedures, glutaraldehyde plays a critical role due to its reliability and ease of use. It displays a complex chemistry that is transparent to most practitioners of immobilization. In this article we detail the structure and reactivity of glutaraldehyde protein immobilization.     

Effects of fungal laccase immobilization procedures for the development of a biosensor for phenol compounds

Fungal laccase was immobilized on carbon-fiber electrodes using classical methods: physical adsorption, glutaraldehyde, carbodiimide and carbodiimide/glutaraldehyde. The highest biosensor response was obtained using carbodiimide/glutaraldehyde for coupling laccase to carboxyl groups on the carbon fibers. In this method, different percentages of glutaraldehyde had important effects on the sensitivity of the biosensor, the best percentage of glutaraldehyde being 10% (m/v). The behavior of the obtained biosensor was investigated in terms of sensitivity, operational range, pH and applied potential. The developed biosensor showed an optimum response at pH 5.0 and at an applied potential of -100 mV. The immobilized laccase retained a good activity for over 2 months.     

Design and application of a flow cell for carbon-film based electrochemical enzyme biosensors

A flow cell has been designed for use with an electrochemical enzyme biosensor, based on low-cost carbon-film electrodes. Three types of mediators were used: cobalt and copper hexacyanoferrates and poly(neutral red) (PNR), covered with glucose oxidase (GOx) immobilised by cross-linking with glutaraldehyde in the presence of bovine serum albumin or inside a oxysilane sol–gel network. Mixtures of sol–gel precursors were made from 3-aminopropyl-triethoxysilane (APTOS) together with methyltrimethoxysilane (MTMOS), methyltriethoxysilane (MTEOS), tetraethyloxysilane (TEOS) or 3-glycidoxypropyl-trimethoxysilane (GOPMOS), and the best chosen for encapsulation. Optimisation in batch mode, using amperometric detection at fixed potential, showed the PNR-GOx modified carbon-film electrodes to be best for flow analysis for both glutaraldehyde and sol–gel enzyme immobilisation. Both types of enzyme electrode were tested under flow conditions and the reproducibility and stability of the biosensors were evaluated. The biosensors were used for fermentation monitoring of glucose in grape must and interference studies were also performed.