Preparation of Chitosan-coated Nylon Membranes and their Application as Affinity Membranes

Chitosan-coated nylon membranes which possess a large number of reactive groups of-CH2OH and -NH2 were prepared by coupling chitosan onto the nylon membrane. Then polylysine as ligand was also immobilized onto the composite membranes by 1, 1＇-carbonyl-diimidazole activation to prepare affinity membranes for bilirubin adsorption. The results showed that these membranes exhibited high binding affinity capacities for bilirubin and the adsorption isotherm fitted the Freundlich model well.     

Adsorption of Human Serum Albumin onto PVA-coated Affinity Microporous PTFE Capillary

Affinity dye-ligand Cibacron Blue F3GA（CB F3GA） was covalently coupled with poly（vinyl alcohol）（PVA） coated on the inner surface of microporous poly（tetra-fluoroethylene）（MPTFE） membranous capillary. The PVA-coated PTFE capillary surface was characterized by XPS and FESEM. The grafting degree of PVA and the amount of CB F3GA immobilized onto the membranous capillary were 23.5 mg/g and 89.6 pmol/g, respectively. These dyed membranous capillaries were chemically and mechanically stable, and could be reproducibly prepared. Human serum albumin（HSA） was selected as model protein. The saturation adsorbance of the dye attached membranous capillary was 85.3 mg HSA/g, while the capacity of non-specific adsorption for HSA was less than 0.3 mg/g.     

Immobilization and Properties of Lipase from Candida rugosa on Electrospun Nanofibrous Membranes with Biomimetic Phospholipid Moities

Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly（acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine）s（PANCMPCs） were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile（PAN） nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%（molar fraction） in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.     

Preparation of polyacrylamide based monolith with immobilized pH gradient and its application for protein analysis

Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate and N,N′-memylenebisacrylamid in the presence of trinary porogens, including 1,4-butanediol, dodecanol and dimethyl sulphoxide. With Ampholine immobilized on the monolith by chemical bonding according to their pIs, the monolithic immobilized pH gradient (M-IPG) was prepared, and applied to the separation of four standard proteins. Compared with polyacrylate based M-IPG, the hydrophilicity of the new material was improved. It could not only avoid the adsorption of proteins, but also make the synthesized procedure simple, which showed great potential in the analysis of proteins.     

Selective Pre-concentration and Solid Phase Extraction of Mercury（Ⅱ） from Natural Water by Silica Gel-loaded （E）-N-（1-Thien-2＇-ylethylidene）-1,2-phenylenediamine Phase

Silica gel-loaded （E）-N-（1-thien-2＇-ylethylidene）-1,2-phenylenediamine （TEPDA） phase was synthesized based on physical adsorption approaches. The stability of a chemically modified TEPDA especially in concentrated hydrochloric acid that was then used as a recycling and preconcentration reagent allowed the further uses of silica gel-loaded immobilized TEPDA phase. The application of this silica gel-loaded phase to sorption of a series of metal ions was performed by using different controlling factors such as the pH of the metal ion solution and the equilibration shaking time by the static technique. This difference was interpreted on the basis of selectivity incorporated in these sulfur containing silica gel-loaded TEPDA phases. Hg（Ⅱ） was found to exhibit the highest affinity towards extraction by these silica gel-loaded TEPDA phases. The pronounced selectivity was also confirmed by the determined distribution coefficients （Kd） of all the metal ions, showing the highest value reported for mercury（Ⅱ） extraction by the silica gel immobilized TEPDA phase. The potential applications of the silica gel immobilized TEPDA phase to selective extraction of mercury（Ⅱ） from aqueous solution were successfully accomplished and preconcentration of low concentration of Hg（Ⅱ） （30 pg·mL^-1） from natural tap water with a preconcentration factor of 200 for Hg（Ⅱ） off-line analysis was conducted by cold vapor atomic absorption analysis.     

The application of immobilized liquids for open tubular liquid chromatography

Summary The applicability of immobilized silicone phases, as reversed phase retentive layers in 5–25μm fused silica capillaries for open tubular liquid chromatography (OTLC) has been investigated. Various types of silicone phases have been tested, of which vinyl containing gums show the most promising features, to create a stable retentive layer in fused silica capillaries. Diffusion coefficients of solutes in the immobilized silicone phases were determined by static measurement and found to be in the order of 5–27 · 10⁻¹² m²/s. These relatively small diffusion coefficients form the main drawback of the immobilized silicone phases, because this seriously hinders the use of thick layers of the stationary phase which is preferred in OTLC to avoid mass overload.     

Photocurrent generation by recombinant allophycocyanin trimer multilayer on TiO2 electrode

A recombinant allophycocyanin trimer was successfully immobilized on a mesoporous TiO₂ electrode. The formation of the immobilized surface was confirmed by multilayer adsorption of protein complexes. The key biophotovoltaic parameters were obtained,which showed that the recombinant allophycocyanin trimer could be a candidate for photosensitizer materials.The values of short-circuit current, open-circuit voltage,fill factor,and conversion efficiency were up to 0.73 mA/cm²,0.52 V,0.69,and 0.26%,respectively.     

Suzuki Reaction of Aryl Bromides Using a Phosphine-Free Magnetic Nanoparticle-Supported Palladium Catalyst

A palladium catalyst immobilized on superparaganetic nanoparticles was prepared with a palladium loading of 0.30 mmol/g. The catalyst was characterized using X-ray diffraction, scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry, thermogravimetric analysis, Fourier transform infrared, atomic absorption spectrophotometry, and nitrogen adsorption. The immobilized palladium catalyst was an efficient catalyst without added phosphine ligands for the Suzuki cross-coupling reaction of several aryl bromides with phenylboronic acid. The recovery of catalyst was simply by magnetic decantation in the presence of a magnet. The immobilized palladium catalyst can be reused many times without significant degradation in catalytic activity. No leaching of active palladium species into the reaction solution was detected.     

Analysis of Ascorbic Acid in Single Human Neutrophils by Electrochemical Detection

Ascorbic acid in individual human neutrophils was determincd by capillary zone clectrophoresis with electrochemical detection. In order to overcome the influence of the adsorption of the substances in cells on the inner surface wall of the capillary on the migration time and the unmber of theorctical plates, a proccdure for treating capillaries has bccn described.     

Immobilization of acetylcholinesterase on functionalized SBA-15 mesoporous molecular sieve for detection of organophosphorus and carbamate pesticide

A novel method for the immobilization of acetylcholinesterase (AChE) on amino modified SBA-15 mesoporous molecular sieves was developed via electrostatic adsorption and glutaraldehyde crosslinking. The immobilized AChE could be exploited as a fast, sensitive and low-cost biocatalyst towards the detection of pesticides residues which could be stored at room temperature for a long time.     

Preparation approaches of the coated capillaries with liposomes in capillary electrophoresis

The use of liposomes as coating materials in capillary electrophoresis has recently emerged as an important and popular research area. There are three preparation methods that are commonly used for coating capillaries with liposomes, namely physical adsorption, avidin–biotin binding and covalent coupling. Herein, the three different coating methods were compared, and the liposome-coated capillaries prepared by these methods were evaluated by studying systematically their EOF characterization and performance (repeatability, reproducibility and lifetime). The amount of immobilized phospholipids and the interactions between liposome or phospholipid membrane and neutral compounds for the liposome-coated capillaries prepared by these methods were also investigated in detail. Finally, the merits and disadvantages for each coating method were reviewed.     

新型亲和膜色谱用于去除胆红素的研究

 以纤维素膜为亲和基质 ,接枝后分别以聚赖氨酸和季铵盐为配基 ,制备了两种亲和膜介质 ,用于去除磷酸缓冲液 (pH 7 4)及人血白蛋白 (HSA)溶液中的胆红素。实验结果表明 ,该方法对磷酸缓冲液中胆红素的去除率能达到 70 %以上 ;对HSA溶液中的胆红素 ,其去除率稍低 ,但在较低浓度的HSA溶液中 ,也能获得高于 5 0 %的去除率。并对亲和膜与胆红素间的亲和作用进行了研究 ,考察了温度、流速、HSA浓度及胆红素初始浓度等相关条件对去除效果的影响。结果表明 ,在较高的温度条件下 ,去除效果较佳 ;在较高的初始浓度情况下 ,胆红素的吸附速率较大。     

含氨基聚甲基丙烯酸羟乙酯树脂对胆红素的吸附性能研究

以交联聚甲基丙烯酸羟乙酯树脂为载体,以己二胺和多乙烯多胺为功能基制备了一系列胆红素吸附剂,研究了它们在不同吸附温度、离子强度和胆红素浓度等条件下,对胆红素的吸附性能的影响.研究表明,该类吸附剂对胆红素具有良好的吸附性能,其中以己二胺和质子化己二胺为功能基的吸附剂对胆红素的吸附作用最佳.     

Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm⁻² trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL⁻¹ horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.     

Study of adsorption behavior of bilirubin on human-albumin monolayer using a quartz crystal microbalance

The quartz crystal microbalance was employed to study the adsorption behavior of bilirubin on human-albumin layer, which was chemically bound to the self-assembled monolayer of 4-aminothiophenol on the surface of a gold electrode of the crystal via glutaraldehyde. A long-time adsorption process of bilirubin that took place on a human-albumin-modified surface was observed, and the adsorption kinetic parameters were estimated from the in situ frequency measurements. The amount of adsorbed bilirubin increased with increasing of both hydrogen ions and bilirubin concentration and was larger than that estimated based on the conclusion that there are two affinity sites for bilirubin per albumin molecule. With the present method, the displacement of bilirubin from an albumin layer caused by aspirin was also examined. QCM measurement provides a facile method for in situ monitoring of the adsorption/desorption of bilirubin on proteins layers.     

Quartz crystal microbalance studies on bilirubin adsorption on self-assembled phospholipid bilayers

Bilirubin adsorption on self-assembled phospholipid bilayers was studied using quartz crystal microbalance, and factors influencing its adsorption such as pH, temperature, and solution ionic strength were discussed in detail. The results show the amount of adsorbed bilirubin on self-assembled phospholipid bilayers is small at higher temperature and large at higher pH and solution ionic strength, and the adsorption kinetic parameter estimated from the in situ frequency measurement is (1.8+/-0.27)x10(6) M(-1) (mean +/- S.D.). With the present method, the desorption of adsorbed bilirubin caused by human serum albumin and the photoinduced decomposition of adsorbed bilirubin under light illumination were also examined. QCM measurement provides a useful method for monitoring the adsorption/desorption process of bilirubin on self-assembled phospholipid bilayers.     

β-环糊精交联聚合物对胆红素吸附性能的研究

考察了β-环糊精交联聚合物对胆红素的动力学吸附曲线,以及温度、pH值、胆红素初始浓度和牛血清白蛋白对去除率的影响。实验结果表明这种聚合物材料对胆红素具有较快的吸附速度,并且在较高的温度下有利于胆红素的吸附。当溶解胆红素的磷酸盐缓冲液的pH为7.8,胆红素起始质量浓度为40 mg/L时,β-环糊精聚合物对胆红素的去除率高达92.6%。随着胆红素初始质量浓度的增加,胆红素的去除率下降,但吸附量增加。     

壳聚糖微球负载的己二胺型聚酰胺-胺树状大分子的制备及对胆红素的吸附

通过反相悬浮反应制备了戊二醛交联的壳聚糖微球。以所制备的壳聚糖微球为载体,合成了己二胺型低代数聚酰胺-胺（Polyamidoamine,简称PAMAM）树枝状大分子（Genaration≤3）。考察了该微球在生理条件下对水溶液中胆红素的吸附行为,以及溶液的pH值,离子强度,温度,胆红素初始浓度,牛血清白蛋白等因素对吸附的影响。结果表明,吸附剂对胆红素具有良好的吸附性能,CS-G2.0,CS-G3.0,CS-G1.0,CS-G0和CS微球的平衡吸附率分别为94.61%,93.44%,92.97%,86.47%,52.38%,CS-G1.0-G3.0微球在0.5h吸附率已经超过70%,1h基本接近平衡,对胆红素的吸附量高达42.78mg/g。     

CE-LIF analysis of mitochondria using uncoated and dynamically coated capillaries

This report is the first demonstration of the use of uncoated and dynamically coated capillaries for the separation of individual mitochondria via CE. Currently, the analysis of individual mitochondria relies upon fused-silica capillaries coated with a hydrophilic polymer (e.g. poly(acryloylaminopropanol)), which is used to minimize adsorption to the capillary surface. Both uncoated fused-silica capillaries and 0.2% w/w poly(vinyl alcohol) dynamic coating solutions are used to successfully analyze isolated individual mitochondrial particles using CE-LIF. While it was possible to separate mouse liver mitochondria on an uncoated capillary, rat liver mitochondria proved to have strong adsorption characteristics that only allowed them to be adequately separated with a PVA dynamic coating or a poly(acryloylaminopropanol) (AAP) capillary. The possible causes for this adsorption are analyzed and discussed. This study shows that uncoated and dynamically coated capillaries can be used in place of AAP-coated capillaries to analyze mitochondria and suggests the use of these capillaries for the analysis of other organelles, offering a greatly simplified method for the analysis of individual organelles.