Creation of biofunctionalized plasma polymerized allylamine gradients

A chemical gradient possessing gradual change of amine concentration for every 100 carbon atoms (NH_x/100 C) from 4.03 to 1.98 was prepared by plasma polymerization of allylamine on polypropylene films. Electron spectroscopy for chemical analysis and water contact angle (WCA) measurements revealed that the nitrogen incorporation resulted in the amine functionality (C? N binding) and, therefore, the formation of the wettability gradient. The gradient showed the WCAs varied from 15° to 90° as the change of amine concentration on the gradient from the nitrogen rich end to the nitrogen deprived end. Furthermore, the interactions between the gradient with mammalian cells revealed that more than twofold cell density was found at the nitrogen rich end when compared with the nitrogen deprived end. Plasma polymerization was demonstrated as an effective method to create controllable chemical gradient and the obtained allylamine gradient was useful for biomaterial applications. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2013 , 51, 1361–1367     

Immobilized chymotrypsin on reversibly precipitable polymerized liposome

A polymerized liposome (PLS) was prepared using a synthesized phosphatidylethanolamine with a diacetylene moiety that showed a reversibly precipitable property on addition and removal of salt. To prepare a soluble-insoluble immobilized enzyme, chymotrypsin was covalently immobilized on the outer surface of the PLS. The carbodiimide method was employed for the enzyme immobilization. Coupling was rapid and nearly complete at a weight ratio of enzyme to the PLS of < 0.12. The immobilized enzyme showed favorable activity yields for both low-and high-mol-wt substrates, i.e., 90 ±9% forN-benzoyl-L-tyrosine ethyl ester and 59 ±5% for casein up to an enzyme coupling density of 0.38 g/g-PLS. The immobilized enzyme was reusable and more stable at high temperature and long-term incubation than the native enzyme.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

Covalent attachment of microbial lipase onto microporous styrene-divinylbenzene copolymer by means of polyglutaraldehyde

A novel method for immobilization of Thermomyces lanuginosus lipase onto polyglutaraldehyde-activated poly(styrene-divinylbenzene) (STY-DVB), which is a hydrophobic microporous support has been successfully developed. The copolymer was prepared by the polymerization of the continuous phase of a high internal phase emulsion (polyHIPE). The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. Lipase from T. lanuginosus was immobilized covalently with 85% yield on the internal surface of the hydrophobic microporous poly(styrene-divinylbenzene) copolymer and used as a biocatalyst for the transesterification reaction. The immobilized enzyme has been fully active 30 days in storage and retained the activity during the 15 repeated batch reactions. The properties of free and immobilized lipase were studied. The effects of protein concentration, pH, temperature, and time on the immobilization, activity, and stability of the immobilized lipase were also studied. The newly synthesized microporous poly(styrene-divinylbenzene) copolymer constitutes excellent support for lipase. It given rise to high immobilization yield, retains enzymatic activity for 30 days, stable in structure and allows for the immobilization of large amount of protein (11.4mg/g support). Since immobilization is simple yet effective, the newly immobilized lipase could be used in several application including oil hydrolysis, production of modified oils, biodiesel synthesis, and removal of fatty acids from oils.     

Cellular Uptake of Covalent Poly(allylamine hydrochloride) Microcapsules and Its Influences on Cell Functions

The mechanism of the cellular uptake of polyelectrolyte microcapsules and its influences on the functions and toxicity of human SMCs are explored. The covalently assembled poly(allylamine hydrochloride)/glutaraldehyde microcapsules are easily ingested by SMCs mainly through macropinosis and caveolae‐mediated endocytosis pathways. The capsules mainly disperse in cytoplasm without colocalization in early endosomes and cell nucleus. The results of gene chips reveal substantial and profound alternation of cell phenotypes and functions. Uptake of the microcapsules cause a slight decrease of cell viability, but leads to significant changes in cytoskeleton organization, cell cycle, as well as cell adhesion and migration ability.

     

聚烯丙基胺硅胶复合材料的合成及其吸附性能

氯丙基三氯硅烷-硅胶;聚烯丙基胺硅胶复合材料的合成及其吸附性能     

Contradictory ageing behaviour and optical property of iodine doped and H2SO4 doped pulsed DC plasma polymerized aniline thin films

Polymerization of aniline in the presence of H₂SO₄ and iodine vapour is carried out using pulsed DC glow discharge plasma. The as prepared iodine doped plasma polymerized aniline films are found to be highly crystalline due to some crystal structure formed by the iodine incorporated into the film and the ex-situ H₂SO₄ doped films are found to be amorphous. However, the structure of the iodine doped films transforms to amorphous one while that of the ex-situ H₂SO₄ doped films transforms to crystalline one due to the effect of ageing. The optical band gap of the samples decreases depending upon the level of doping and annealing, having minimum optical band gap for iodine doped plasma polymerized aniline. Due to ageing, the optical property of the H₂SO₄ doped film is found to become superior while for iodine doped films a negligible change of the optical property has been noticed even after six months of ageing.     

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca²⁺ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

共价固定亲和素的磁性纳米粒子用于快速测定蛋白A含量

采用溶剂热法制备了Fe₃O₄磁性纳米粒子(MNPs), 以戊二醛为交联剂, 将亲和素共价固定于MNPs表面. 用透射电子显微镜(TEM)、 X射线衍射(XRD)、 紫外-可见吸收光谱(UV-Vis)、 傅里叶变换红外光谱(FTIR)和荧光光谱等手段对蛋白固定过程进行了监控和表征. 采用荧光光谱法评价了固定亲和素的磁性纳米粒子(Avi-MNPs)的活性, 并将Avi-MNPs应用于分光光度法测定蛋白A的含量. TEM结果表明, 功能化前后MNPs的粒度分布均匀, 粒径大小分别约为30和50 nm. XRD分析结果表明, MNPs与Fe₃O₄的特征衍射峰完全一致, 晶体纯度良好. UV-Vis, FTIR和荧光光谱结果表明, 亲和素已固定在MNPs表面. Avi-MNPs活性评价结果表明, 其结合生物素的活力为4.706 U/mg Avi-MNPs, 低于游离的亲和素活力(14.1 U/mg D-biotin). 该方法用于检测蛋白A含量比传统酶联免疫法省时、 省力, 且对检测仪器要求低.     

Covalent immobilization of an enzyme (glucose oxidase) onto a carbon sol-gel silicate composite surface as a biosensing platform

A porous organic-inorganic hybrid sol-gel carbon composite has been developed and used for surface covalent bonding of an enzyme for biosensing applications, illustrated by glucose oxidase (GOD). The composite comprises graphite powder, ferrocene, and an amino- and methyl-silicate backbone. The graphite powder provides the conductivity for the electrode and ferrocene acts as the mediator for signal transduction from the active center of the enzyme to the electron conductive surface. The presence of amine groups in the sol-gel silicate network allows for the covalent bonding sites for the enzyme via the carbodiimide reaction. The hydrophobicity and hydrophilicity properties of the electrode surface are controlled by the amine and methyl groups of the silicate network. Systematic optimization of the composite composition has been carried out and the performance of the glucose biosensor has been investigated. The optimal electrode gives a linear response range of 0.1-27 mM glucose with a sensitivity of 1.30 μA mM⁻¹ and detection limit (S/N = 3) of 26 μM.     

Covalent Immobilization of Lipase on Poly（ acrylonitrile-co-maleic acid） Ultrafiltration Hollow Fiber Membrane

IntroductionLipases are biotechnologically important enzymes,which are able to catalyze the hydrolysis/synthesis of awide range of soluble or insoluble carboxylic acid estersand amides.In this way,the enzymes have been wide-ly used biotechnologically in dairy industry,oil pro-cessing,the production of surfactants,and the prepara-tion of enantiomerically pure pharmaceuticals[1,2].However,like mostenzymes for industrial applica-tions,lipases are unstable and easy to lose their cata-lytic activit…     

Covalent immobilization of single-walled carbon nanotubes and single-stranded deoxyribonucleic acid nanocomposites on glassy carbon electrode: Preparation, characterization, and applications

Single-stranded deoxyribonucleic acid (ssDNA)-wrapped single-walled carbon nanotubes (SWNTs) were modified on the surface of glassy carbon electrode (GCE) by covalent modification technique. Field emission scanning electron microscope (FE-SEM), X-ray photoelectron spectrum (XPS), electrochemical impedance spectroscopy (EIS), and cyclic voltammetric (CV) were used to characterize the properties of this modified electrode. The results showed that SWNTs-ssDNA composites were successfully immobilized onto the surface of GCE. Moreover, this modified electrode exhibited high stability, largely active areas, and efficiently electrocatalytic activities. It had been used for the analysis of various biomolecules, such as dopamine (DA), uric acid (UA), and ascorbic acid (AA), and the results were satisfactory.     

Construction of Tetrathiafulvalene-based Covalent Organic Frameworks for Superior Iodine Capture

The effective capture of radioiodine species during nuclear fuel reprocessing and nuclear accidents is of primary importance but remains challenging for the sustainable development of nuclear energy. Herein, we report two newly designed two-dimensional(2D) and three-dimensional(3D) covalent organic frameworks by introducing tetrathiafulvalene functional groups into the building units for the simultaneous physisorption and chemisorption capture of iodine molecules. Remarkably, the obtained 3D TTF-TAPT-COF material exhibited a superior iodine vapor adsorption capacity of up to 5.02 g/g at 348 K and under ambient pressure and an adsorption kinetics of 0.515 g/(g∙h), surpassing most of other materials reported so far. The strong physiochemical interactions between iodine molecules and the frameworks of the obtained COFs were revealed by a set of experimental techniques. This study provides a feasible approach for the rational design and the construction of novel and effective COF-based adsorbents for iodine enrichment and related environmental remediation.     

Composite polymer particles with stimuli-responsive surface properties and specific activity of adsorbed/released trypsin

Micron-sized monodispersed stimuli-responsive polys’ (PS)/poly(2-dimethylaminoethyl methacrylate-N-isopropyl acrylamide-ethylene glycol dimethacrylate) [P(DM-NIPAM-EGDM)] composite polymer particles were prepared by seeded copolymerisation of DM, NIPAM and EGDM with PS seed particles. Adsorption behaviour of trypsin suggested that composite particles surface has both temperature- and pH-responsive swelling–deswelling characteristics. The performance of composite polymer particles as a carrier for biomolecules in adsorption/release experiments was evaluated by measuring the specific activities of adsorbed trypsin as a function of temperature and pH.     

Comparison of Physical Adsorption and Covalent Coupling Methods for Surface Density-Dependent Orientation of Antibody on Silicon

The orientation of antibodies, employed as capture molecules on biosensors, determines biorecognition efficiency and bioassay performance. In a previous publication we demonstrated for antibodies attached covalently to silicon that an increase in their surface amount Γ, evaluated with ellipsometry, induces changes in their orientation, which is traced directly using Time-of-Flight Secondary Ion Mass Spectroscopy combined with Principal Component Analysis. Here, we extend the above studies to antibodies adsorbed physically on a 3-aminopropyltriethoxysilane (APTES) monolayer. Antibodies physisorbed on APTES (0 ≤ Γ ≤ 3.5 mg/m²) reveal the Γ ranges for flat-on, side-on, and vertical orientation consistent with random molecular packing. The relation between orientation and Γ is juxtaposed for silicon functionalized with APTES, APTES modified with glutaraldehyde (APTES/GA) and N-hydroxysuccinimide-silane (NHS-silane). Antibody reorientation occurs at lower Γ values when physisorption (APTES) is involved rather than chemisorption (APTES/GA, NHS-silane). At high Γ values, comparable proportions of molecules adapting head-on and tail-on vertical alignment are concluded for APTES and the NHS-silane monolayer, and they are related to intermolecular dipole–dipole interactions. Intermolecular forces seem to be less decisive than covalent binding for antibodies on the APTES/GA surface, with dominant head-on orientation. Independently, the impact of glutaraldehyde activation of APTES on vertical orientation is confirmed by separate TOF-SIMS measurements.     

Covalent immobilization of Candida antarctica lipase B on nanopolystyrene and its application to microwave-assisted esterification

Nanopolystyrene was used as a solid support for the covalent immobilization of Candida antarctica lipase B (CalB) using the photoreactive reagent 1-fluoro-2-nitro-4-azido benzene (FNAB) as a coupling reagent. The obtained derivative was then used as a biocatalyst in a microwave assisted esterification experiment. Factors such as contact time, pH, and enzyme concentration were investigated during immobilization. The hydrolytic activity, thermal, and operational stability of immobilized-CalB were determined. The maximum immobilized yield (218 μg/mg support) obtained at pH 6.8 exhibited optimum hydrolytic activity (4.42 × 10³ mU p-nitrophenol/min). The thermal stability of CalB improved significantly when it was immobilized at pH 10, however, the immobilized yield was very low (93.6 μg/mg support). The immobilized-CalB prepared at pH 6.8 and pH 10 retained 50% of its initial activity after incubation periods of 14 and 16 h, respectively, at 60 ℃. The operational stability was investigated for the microwave assisted esterification of oleic acid with methanol. Immobilized-CalB retained 50% of its initial activity after 15 batch cycles in the microwave-assisted esterification. The esterification time was notably reduced under microwave irradiation. The combined use of a biocatalyst and microwave heating is thus an alternative total green synthesis process.     

Modification of polysulfone membranes 5. Effect of n-butylamine and allylamine plasma

This paper describes some properties of microwave plasma polymers of n-butylamine and allylamine deposited on the surface of polysulfone substrate. Contact angle evaluation, ATR-FTIR spectroscopy, X-ray photoelectron spectroscopy analysis and estimation of pore size distribution of ultrafiltration polysulfone membranes were used. It was found that addition of Ar to the amine vapor significantly stabilized the plasma and converted it to the ablation mode. The surface became more hydrophilic and the surface groups were enriched in oxygen. Both amines gave deposits of various compositions: the n-butylamine polymer was not as enriched in amines as the polymer formed from allylamine. However, the amounts of nitrogen in both deposits indicated allylamine to be the precursor for the preparation of membranes with weakly basic functionalities. When porous membranes are modified, the ultrafilters obtained may be named `fouling protected' as they do not foul so intensively with proteins as their unmodified analogues. To a lesser extent, similar behavior was shown by membranes modified by deposition of plasma-polymerized n-butylamine.     

Synthesis,polymerization, and thermal properties of benzoxazine based on bisphenol‐S and allylamine

A bifunctional benzoxazine monomer, 6,6′‐bis(3‐allyl‐3,4‐dihydro‐2H‐benzo[e][1,3]oxazinyl) sulfone (BS‐ala), was synthesized from bisphenol‐S, allylamine and formaldehyde via a solution method. The chemical structure of BS‐ala was confirmed by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and elemental analysis. The polymerization behavior of BS‐ala was investigated by FTIR, solid‐state ¹³C NMR, and differential scanning calorimetry (DSC). The oxazine ring opening polymerization is prior to the addition polymerization of allyl group, and the exothermic peaks corresponding to the two reactions appear partially overlapped in the DSC curve. The storage modulus of the resultant polybenzoxazine at 25°C is about 3.9 GPa, and the glass transition temperature is 254°C. The 5% and 10% weight loss temperatures of the polybenzoxazine are about 335°C and 361°C in both air and nitrogen, respectively. The char yield is about 58% at 800°C in nitrogen, whereas almost no residue is remained at 700°C in air. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies of electroosmotic flow and the effects of protein adsorption in plasma‐polymerized microchannel surfaces

This paper presents a study of EOF properties of plasma‐polymerized microchannel surfaces and the effects of protein (fibrinogen and lysozyme) adsorption on the EOF behavior of the surface‐modified microchannels. Three plasma polymer surfaces, i.e. tetraglyme, acrylic acid and allylamine, are tested. Results indicate EOF suppression in all plasma‐coated channels compared with the uncoated glass microchannel surfaces. The EOF behaviors of the modified microchannels after exposure to protein solutions are also investigated and show that even low levels of protein adsorption can significantly influence EOF behavior, and in some cases, result in the reversal of flow. The results also highlight that EOF measurement can be used as a method for detecting the presence of proteins within microchannels at low surface coverage (<1 ng/cm² on glass). Critically, the results illustrate that the non‐fouling tetraglyme plasma polymer is able to sustain EOF. Comparison of the plasma‐polymerized surfaces with conventionally grafted polyelectrolyte surfaces demonstrates the stabilities of the plasma polymer films, enabling multiple EOF runs over 3 days without deterioration in performance. The results of this study clearly demonstrate that plasma polymers enable the surface chemistry of microfluidic devices to be tailored for specific applications. Critically, the deposition of the non‐fouling tetraglyme coating enables stable EOF to be induced in the presence of protein.