新型内表面反相限进填料的制备与评价

通过在硅烷化硅胶内表面和外表面分别键合己胺和聚乙烯醇,制备了能够在线直接进样分析生物样品的新型内表面反相限进填料。采用元素分析、电镜观察对该限进填料的结构进行了表征。以普萘洛尔、阿替洛尔、苯巴比妥、卡马西平作溶质探针,并以Merck公司生产的限进填料柱作参比,对合成的限进填料的色谱性能进行了研究。研究结果表明,所制备的限进填料有较好的蛋白质排阻能力、富集能力和反相色谱性能,能同时实现排阻生物大分子杂质和富集小分子被分析物的功能,可作为在线、快速直接进样检测分析生物样品的预处理柱,适用于普萘洛尔血浆的直接进样分析。     

Spectroscopic and theoretical characterization of the A2delta-X2pi transition of CH-Ne

The A2delta-X2pi transition of CH-Ne was examined using laser-induced fluorescence and fluorescence depletion techniques. The spectrum was found to be particularly congested due to the large number of bound states derived from the CH(A,n=2)+Ne interaction, and the small energy spacings between these states resulting from the relatively weak anisotropy of the van der Waals bond. High-level ab initio calculations were used to generate two-dimensional potential energy surfaces for CH(X)-Ne and CH(A)-Ne. The equilibrium structures from these surfaces were bent and linear for the X and A states, respectively. Variational calculations were used to predict the bound states supported by the ab initio surfaces. Empirical modification of the potential energy surfaces for the A state was used to obtain energy-level predictions that were in good agreement with the experimental results. Transitions to all of the optically accessible internal rotor states of CH(A,n=2)-Ne were identified, indicating that CH performs hindered internal rotations in the lowest-energy levels of the A and X states. The characteristics of the potential energy surfaces for CH-Ne in the X,A,B, and C states suggest that dispersion and exchange repulsion forces dominate the van der Waals interaction.     

Enzyme immobilization to ultra‐fine cellulose fibers via amphiphilic polyethylene glycol spacers

Ultra‐high specific surface cellulose fibers with an average diameter of 500 nm were generated from electrospinning and alkaline hydrolysis of cellulose acetate and used as porous supports for enzyme immobilization. The cellulose fiber surfaces were reacted with polyethylene glycol (PEG) diacylchloride to simultaneously add amphiphilic spacers and reactive end groups for coupling with a lipase enzyme. The quantity of reactive carboxylic acid on the fiber surfaces could be readily controlled by COCl/OH molar ratios and PEG lengths. The highest free acid (COOH) content of 1.0 mmol per gram of cellulose was obtained at 10 COCl/OH ratio with the 600‐Da PEG diacylchloride. Enzyme coupling on such PEG‐attached cellulose was optimal in the presence of a water‐soluble carbodiimide [1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC)] at a very low EDC/COOH molar ratio of 0.2 under acidic condition and at ambient temperature. Whereas the free lipase retained only 25% of its original activity, the fiber‐bound lipase possessed much superior retention of catalytic activity after exposure to cyclohexane (81%) and toluene (62%) and hexane (34%). The fiber‐bound lipase also exhibited significantly higher catalytic activity at elevated temperatures than the free form, that is, 10 times at 70 °C. The ultra‐fine, fibrous, and porous structures were retained throughout alkaline hydrolysis, activation, coupling, and activity assays. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4289–4299, 2004     

From the plateau problem to periodic minimal surfaces in lipids,surfactants and diblock copolymers

A novel method is presented for generating periodic surfaces. Such periodic surfaces appear in all systems which are characterized by internal interfaces and which additionally exhibit ordering. One example are systems of AB diblock copolymers, where the internal interfaces are formed by the chemical bonds between the A and B blocks. In these systems at least two bicontinuous phases are formed: the ordered bicontinuous double diamond phase and the gyroid phase. In these phases the ordered domains of A monomers and B monomers are separated by a periodic interface of the same symmetry as the phases themselves. Here we present a novel method for the generation of such periodic surfaces based on the simple Landau-Ginzburg model of microemulsions. We test the method on four known minimal periodic surfaces, find two new surfaces of cubic symmetry, and show how to obtain periodic surfaces of high genus and n-tuply continuous phases (n > 2). So far only bicontinuous (n = 2) phases have been known. We point out that the Landau model used here should be generic for all systems characterized by internal interfaces, including the diblock copolymer systems.     

Covalent binding of biorecognition groups to solids using poly(hydromethylsiloxane) as linkage

By activating SiH bonds, poly(hydromethylsiloxane) can be covalently bound in a first step to various metal or polymer surfaces. In a second step, unreacted SiH bonds can be brought to react with organic compounds having adequate functional groups such as double or triple bonds, carbonyl or hydroxyl groups. This scheme is used to bind biorecognition groups to solids. The novel concept is demonstrated by attaching a newly synthesized biotin derivative to Au. It is shown that the immobilized biotin is capable of binding streptavidin.     

Fluorinated Boron-Dipyrromethene (BODIPY) Dyes: Bright and Versatile Probes for Surface Analysis

A family of bright boron-dipyrromethene-type fluorophores with a high number of fluorine atoms (F-BODIPYs) has been developed and characterized by X-ray crystallography and optical spectroscopy. The introduction of 3,5-bis(trifluoromethyl)phenyl and pentafluorophenyl moieties significantly enhances the photostability of such dyes, yielding for instance photostable near-infrared (NIR) fluorophores that show emission maxima>750 nm, when the BODIPY’s π system is extended with two (dimethylamino)styryl and (dimethylamino)naphthastyryl moieties, or green-emitting BODIPYs with fluorescence quantum yields of unity. When equipped with a suitable group that selectively reacts for instance with amines, F-BODIPYs can be used as potent dual labels for the quantification of primary amino groups on surfaces by X-ray photoelectron spectroscopy (XPS) and fluorescence, two powerful yet complementary tools for the analysis of organic surface functional groups. The advantage of reactive F-BODIPYs is that they allow a fast and non-destructive mapping of the labelled supports with conventional fluorescence scanners and a subsequent quantification of selected areas of the same sample by the potentially traceable XPS technique. The performance is exemplarily shown here for the assessment of the amino group density on SiO₂ supports, one of the most common reactive silica supports, in particular, for standard microarray applications.     

Surface modification. II. Functionalization of solid surfaces with vinylic monomers

A new method to functionalize surfaces of solid substrates such as glass, silicon crystals, and silica microspheres with appropriate vinylic monomers, i.e., methyl vinyl ketone, methyl acrylate, methacrolein, and acrolein, is described. The surface modification process was performed through the following sequence of reactions: (a) derivatization of the surfaces with to-nitrile groups by interacting the substrates with SiCl₃(CH₂)₃CN; (b) subsequent reduction of the a)-nitrile groups with diborane to w-amine groups; (c) binding of the vinylic monomers to the surfaces via the to-amine groups. pK_1/2 of the surface primary amine groups, as determined by contact angle titration, was found to be 2–4 units lower than the pK_1/2 values of primary amine analogous in solution. Methyl vinyl ketone and methyl acrylate were covalently bound to the amine surfaces only under basic conditions via the Michael addition reaction. Methacrolein and acrolein were covalently bound to the amine surfaces under both acidic and basic conditions via two major reactions: the Michael addition reaction and Schiff base bond formation. The concentration of the aldehyde groups of the surfaces obtained by the reaction with methacrolein and acrolein was significantly higher than that obtained using the common, published method in which glutaraldehyde interacts with the amine surfaces.     

On/off chemical determination of reactive amino groups immobilized on silica surfaces

Reaction of silica frameworks with aminosilanes to generate reactive amino groups on the surface of solid supports is a widely used step in the chemical modification of such surfaces. These reactive amino groups can aid in the chemical attachment ofproteins (enzymes and/or antibodies)_or chelating agents. Analytical accounting of the reactive groups generated is of interest in method optimization and in reactor design when these materials are used. A nondestructive method for the determination of reactive amino groups after attachment to silica surfaces is presented; it can be used for in-situ determinations in reactors. The method involves on/off chemistry based on the attachment of a chromophoric probe (p-dimethylaminoinnamaldehyde), its subsequent detachment under different experimental conditions, and spectrophotometric measurement of the released probe at 390 nm. Aspects of covalent attachment of amino groups to silica surfaces are also discussed.     

Hemocompatible polyurethane surfaces

With the aim of improving the hemocompatibility of blood‐contacting devices, antithrombotic or fibrinolytic biological molecules‐containing polyurethane (PU) materials have been developed. Cationic PU surfaces were prepared by grafting poly(dimethylaminoethyl methacrylate) and quaternizing the tertiary amino groups with iodomethane. The surfaces were characterized by water contact angles and X‐ray photoelectron spectroscopy. The materials (PU‐CH₃I) were treated with antithrombotic or fibrinolytic drugs, such as hirudin or tissue plasminogen activator (tPA) in Tris‐buffered saline (pH 9.0) to yield hirudin‐loaded or tPA‐loaded PU surfaces. The hirudin and tPA quantity of the surfaces was observed using a radiolabeling method. The quantities of hirudin and tPA taken up by the cationic surfaces were significantly greater than those on the unmodified PU: approximately 200‐fold greater for hirudin and 10‐fold for tPA. The release of the bound hirudin and tPA from the materials in contact with plasma was slow, and at 48 h, ~78% of the initial hirudin and ~26% of the initial tPA remained bound. The activity of the bound hirudin and tPA, as measured by a plasma recalcification assay, was largely preserved. This approach may have potential for the development of surfaces having antithrombotic or fibrinolytic properties. Copyright © 2011 John Wiley & Sons, Ltd.     

A simple method for controlling dendritic architecture and diversity: A parallel monomer combination approach

A novel parallel monomer combination approach to manipulating the architectural disposition of dendritic macromolecules is described. It harnesses the synthetic speed and power of the double-stage convergent growth approach and classical parallel synthesis to prepare diverse series of dendrimers that possess a predetermined number and arrangement of "internal" functional moieties. This methodology is applied for the preparation of a novel family of poly(benzyl ether) dendrimers possessing 1-15 "internal" allyloxy groups, which are displayed in a highly controlled, layer-specific, generational manner.     

Synthesis of Thiourea Derivatives on Soluble Polymeric Support

A modified poly(ethylene glycol) (PEG) has been developed as the soluble polymeric supports for liquid phase synthesis of novel thiourea derivatives.In each step of the sequence,the PEG-bound products were precipitated in cold Et2O and the unreacted materials and by-products were removed by simple filtration.The progress of reaction,purity of the isolation and the structure of the PEG-bound products were easily monitored by TLC,IR and ^1H NMR spectra.Representative thiourea derivatives were obtained in moderate yields with excellent purity from this modified PEG-bound product by the cleavage with 50% TFA/H2O.     

Planar block copolymer membranes by vesicle spreading

An easy route to planar solid-supported polymer membranes by vesicle spreading is described. Pre-organized poly(butadiene)-block-poly(ethylene oxide)(PB-PEO) assemblies were spread on two different supports, i.e. strongly hydrophilic glass surfaces and ultrasmooth gold substrates. Polymer membranes were produced on a hydrophilic support by spreading hydroxyl-functionalized polymer vesicles, while covalently immobilized polymer membranes were obtained by spreading LA-functionalized polymer vesicles on gold substrates. Covalently bound membranes were further incubated with the peptide polymyxin B. Interactions with the polymer membrane were detected by EIS. These systems are of great interest to fundamental membrane science and have potential in technological applications, such as drug screening and (bio)sensing.     

Use of a novel cation-exchange restricted-access material for automated sample clean-up prior to the determination of basic drugs in plasma by liquid chromatography

A new kind of silica-based restricted-access material (RAM) has been tested in pre-columns for the on-line solid-phase extraction (SPE) of basic drugs from directly injected plasma samples before their quantitative analysis by reversed-phase liquid chromatography (LC), using the column switching technique. The outer surface of the porous RAM particlescontains hydrophilic diol groups while sulphonic acid groups are bound to the internal surface, which gives the sorbent the properties of a strong cation exchanger towards low molecular mass compounds. Macromolecules such as proteins have no access to the internal surface of the pre-column due to their exclusion from the pores and are then flushed directly out. The retention capability of this novel packing material has been tested for some hydrophilic basic drugs, such as atropine, fenoterol, ipratropium, procaine, sotalol and terbutaline, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The elution profiles of the different compounds and the plasma matrix as well as the time needed for the transfer of the analytes from the pre-column to the analytical column were determined in order to deduce the most suitable conditions for the clean-up step and develop on-line methods for the LC determination of these compounds in plasma. The cationic exchange sorbent was also compared to another RAM, namely RP-18 ADS (alkyl diol silica) sorbent with respect to retention capability towards basic analytes.     

Bound state spectroscopy of NH-He

The NH-He van der Waals complex was characterized via laser excitation of bands associated with the NH A (3)Pi-X (3)Sigma(-) transition. It was demonstrated that the ground state supports a bound level with a rotational constant of B"=0.334(2) cm(-1). These results are in agreement with the predictions of recent high-level theoretical calculations. Spin-orbit predissociation of the excited complex was observed, and the spectra yield insights regarding the NH(A)+He potential energy surfaces.     

Detecting the chemoselective ligation of peptides to silicon with the use of cobalt-carbonyl labels

While fluorescent-based methods are generally used to detect the immobilization and the interactions of biomolecules to solid supports, recent studies have shown their limitations in the case of silicon surfaces. As an alternative, we investigated the synthesis of peptides labeled with a metal transition complex and their subsequent immobilization to the silicon surfaces. The feasibility of using such probes has been explored by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). By starting with hydrogen-terminated or oxidized silicon surfaces, we functionalized those surfaces with semicarbazide groups and showed the site-specific linkage of glyoxylyl peptides labeled with a Co2(CO)6 moiety.     

Synthesis and characterization of SPUU–PEO–heparin graft copolymers

A new synthetic approach for the preparation of segmented polyurethaneurea (SPUU)–PEO–Heparin graft copolymers (B–PEO–Hep) has been developed. The procedure involved the coupling of hexamethylene diisocyanate (HMDI) to soluble Biomer® (B) through an allophanate/biuret reaction. The free isocyanate (NCO) groups attached to Biomer® were then coupled to PEO terminal hydroxyl groups to form PEO grafted Biomer® (B–PEO). B–PEO free hydroxy groups were modified with HMDI to introduce terminal isocyanate groups. The NCO functionalized B–PEO was then coupled to heparin (Hep) functional groups (? OH, ? NH₂) producing B–PEO–Hep graft copolymer. Synthetic intermediates were confirmed by FTIR, NCO group determination, and toluidine blue heparin assay. Physical characterization techniques, such as contact angle measurements, water swelling, light scattering measurements, and DSC thermal analysis, detailed properties of the graft copolymer containing covalently bound heparin. This new heparinized copolymer can be applied as a coating on other existing blood contacting surfaces without changing bulk properties. The heparin bioactivity observed attests to the usefulness of this new procedure as a coating to improve the blood compatibility of blood-contacting surfaces.     

Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.     

Interactions between chitosan-modified particles and mucin-coated surfaces

Lipid-based particles (Cubosome particles) were surface-modified by chitosan and the ratio between particles and chitosan was optimized to minimize the free chitosan concentration in the dispersion. The modified particles were characterized by electrophoretic measurements and the pH dependence of the zeta potential could be directly related to the protonation of chitosan. Interaction between the modified particles and mucin-coated silica surfaces were subsequently investigated in situ by ellipsometry to assess the mucoadhesive properties at physiologically relevant conditions. The result showed that a substantial amount of modified particles was adsorbed to mucin-coated silica surfaces at both pH 4 and pH 6, probably due to electrostatic interactions between amino groups in chitosan and negatively charged groups in mucin. Furthermore, the amount of bound particles decreased by less than 15% upon rinsing indicating relatively strong interactions. This investigation demonstrates that ellipsometry is a useful tool to study mucoadhesive properties of particles in the submicrometer range. Moreover, the novel chitosan-modified particles may be of interest for mucosal drug delivery applications.     

The Internal Surface Reverse Phase. Concepts and Applications

Abstract

Patented in 1985, introduced commercially in 1986, internal-surface reversed phase (ISRP) supports have attracted wide attention. ISRP supports allow the analysis of serum and plasma samples by high pressure liquid chromatography (HPLC) without requiring the prior removal of protein. Proteins cannot enter the pores of ISRP supports and are not adsorbed by ISRP outer surfaces; proteins pass right through ISRP HPLC columns. Therefore, the number of serum injections that given ISRP-guarded ISRP columns can receive runs into the thousands; ISRP columns nicely lend themselves to automation. ISRP indifference to proteins is complemented by the remarkable selectivity toward drugs of its stationary phase, glycine-phenylalanine-phenylalanine (GFF), a selectivity that recently has been shown to extend to peptides. More recently still, it has been shown that ISRP columns can be used to analyze both the free and the bound forms of drugs, even distinguishing among different bound forms. A potential new intrinsically monomeric GFF shows improved retention and surprisingly high chromatographic efficiency.     

Mesomorphous structure and properties of non-equimolar complexes of poly(ethylenimine) and perfluorooctanoic acid

A series of solid complexes, PEI-PFAO, made of poly(ethylenimine) (PEI) and perfluorooctanoic acid (PFOA) with different compositions were prepared through a "starving addition" method, where PFOA was fed into PEI solution at the molar ratio, phi(feed), of acid group to the amino group of PEI, never beyond unity. Wide-angle X-ray diffraction diagrams confirmed amorphous structure of these complexes. Small-angle X-ray scattering indicated two ordered mesomorphous structures of alpha and beta lamellar phases, with respective long periods of 2.29 and 1.15 nm in the complexes. By increasing the actual molar ratio, phi, of PFOA to the amino group of PEI, the complex structure was altered from alpha-phase dominant to beta-phase dominant. All complexes exhibited two thermal degradation processes induced by decomposition of the bound PFOA below 230 degrees C and PEI backbone at about 350 degrees C. The initiating degradation temperature, Tid, decreases with increasing phi due to the preferential degradation of the PFOA chain bound to the tertiary amino groups. The glass transition temperature, Tg, of the complex increases with phi up to the degradation of the complex of phi = 1. This increase in Tg with phi also supports an ordered alignment of the bound PFOA chains, which greatly restricts the PEI mobility. The solid surface tension, gammaS, and critical surface tension, gammac, of the complex are between 15.4 and 16.8 mN/m and between 13.5 and 15.4 mN/m, respectively. The latter is very close to or even smaller than gammac of PTFE (15 mN/m), suggesting the enrichment of CF2 and CF3 groups at the complex surfaces. The fact that the PEI-POFA complex combines high hydrophobicity with selective thermal degradation of bound fluorinated chains promises a potential of selective change and local functionalization of the surface in a well-controlled manner.