An enzyme-immobilization method for integration of biofunctions on a microchip using a water-soluble amphiphilic phospholipid polymer having a reacting group

A water-soluble phospholipid polymer having an active ester group in the side chain, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)(PMBN), was used for the immobilization of an enzyme on a plastic microchip. The MPC polymers with BMA units were adsorbed onto the poly(methyl methacrylate)(PMMA) microchip, and the active ester group in the MEONP unit reacted with the amino groups of the proteolytic enzyme, trypsin. Trypsin was immobilized on the sample reservoir, and catalyzed the hydrolysis of the fluorescently labeled ArgOEt to Arg. The consequent separation of product from the substrate, and their detection, were integrated on the microchip and this meant that all procedures from the enzymatic activity to product detection were completed in less than three minutes.     

Stabilization of phospholipid polymer surface with three-dimensional nanometer-scaled structure for highly sensitive immunoassay

A phospholipid polymer platform and an antibody as a bioaffinity ligand were used to construct a biointerface for a highly sensitive immunoassay. The platform had a nanometer-scaled particle deposition surface and it was constructed with poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN) by an electrospray deposition (ESD) method. The PMBN surface could immobilize specific antibodies through covalent chemical bonding by the reaction between MEONP units and amino groups in the antibody. In addition, the PMBN could prevent nonspecific protein adsorption from an analyte. However, the nanometer-scaled structure of the PMBN lost its shape after immersion in an aqueous medium. To stabilize the nanometer-scaled structure in an aqueous medium, the PMBN was cross-linked with 1,4-butylenediamine and then heat-treated. These treatments effectively improved the stability of the nanometer-scaled structure, that is, the structure had a high porosity even after immersing in an aqueous medium. The stabilization affected the specific signal in the enzyme-linked immunosorbent assay (ELISA), that is, the specific signal in ELISA was enhanced.     

Feasibility Study of Introducing Redox Property by Modification of PMBN Polymer for Biofuel Cell Applications

In this study, the feasibility of introducing redox property to an amphiphilic phospholipid polymer (PMBN) was investigated. The active ester group in the side chain of the polymer was used to react with pyrroloquinoline quinine (PQQ). Redox peaks that corresponded to PQQ redox potentials were observed after the modification. Glucose oxidase was immobilized to the modified polymer. Electrochemical oxidation of glucose was carried out with the polymer electrode. The oxidation current increased with elevating glucose concentration indicating electron transfer established between the electrode and enzyme. It suggests that by modification, PMBN is possible to use for enzyme electrode for bioelectronics.     

Dual mode bioreactions on polymer nanoparticles covered with phosphorylcholine group

We investigated the preparation of polymer nanoparticles covered with phosphorylcholine (PC) groups and the immobilization of proteins in order to observe dual mode bioreactions on the nanoparticles. For the surface modification on the nanoparticles, a water-soluble amphiphilic phospholipid polymer with PC groups as a hydrophilic moiety was synthesized. In this polymer, an active ester group, which can immobilize proteins, was introduced. Using the phospholipid polymer as a solubilizer, poly(L-lactic acid) nanoparticles were prepared from its methylene chloride solution in an aqueous medium by the solvent evaporation method. The diameter of the nanoparticles was ca. 200 nm and the surface was covered with the PC groups and active ester groups. Proteins could immobilize on the nanoparticles under mild conditions by the reaction between the active ester group and amino group in the proteins. Both an antibody and enzyme were immobilized on the nanoparticles and bioreactions such as the antigen/antibody reaction and enzymatic reaction were observed. When an antigen was added to the suspension of the nanoparticles, aggregation of the nanoparticles occurred and then they precipitated. Also, the enzymatic reaction proceeded well when the enzyme substrate was added to the suspension. Based on these results, we provided polymer nanoparticles functionalized with both the antibody and enzyme, and the dual mode bioreactions could occur. We concluded that the novel polymer nanoparticles could be used for nano-/micro-scaled diagnostic and medical treatment systems.     

Structuring of supported hybrid phospholipid bilayers on electrodes with phospholipase A2

The effect of a lipolytic enzyme, pork pancreatic phospholipase A(2), on hybrid bilayer membranes was monitored using voltammetry, impedance spectroscopy and surface plasmon resonance. The hybrid bilayers were prepared by Langmuir-Schaefer transfer of lipid monolayers onto gold electrodes modified with self-assembled alkanethiol monolayers, or by liposome spreading. The electrodes were immersed in the phospholipase aqueous solution to allow adsorption of the enzyme and cleavage of the ester bond in the sn-2 position of phospholipids in the outer leaflet of the hybrid layers. The action of phospholipase A(2) led to perforation of the lipid films. Impedance spectroscopy and surface plasmon resonance were used for monitoring enzyme adsorption, phospholipid hydrolysis and product desorption. The results obtained show that transport efficiency of an electroactive probe, ferrocyanate, and of an electroactive drug, doxorubicin, through the bilayer depends on the action of the enzyme; the state of the lipid layer covering the electrode surface depends on the latter as well. Cyclic voltammetry and electrochemical impedance spectroscopy were used to study this effect. The doxorubicin reduction/oxidation signals appearing at potentials close to those observed using a bare gold electrode indicated facilitated penetration of the drug into the layer. The results obtained were interpreted in terms of pore formation in the lipid matrix; phospholipase A(2) can be considered as a nano-device for high precision perforation of the lipid layer.     

具有内皮细胞选择性的细胞膜仿生支架材料的研究

利用AIBN引发自由基反应,由单体2-(甲基丙烯酰氧基)乙基-2-(三甲基氨基)乙基磷酸酯(MPC)、甲基丙烯酸十八酯(SMA)、对硝基苯氧羰基聚乙二醇甲基丙烯酸酯(MEONP)合成了一种新型类细胞膜仿生涂层材料.MPC可以阻抗非特异性吸附;MEONP可以结合抗体或多肽促进特异性识别.通过表面固定的方法引入多肽序列Arg-Glu-Asp-Val(REDV),使涂层具有内皮细胞选择性.核磁、紫外吸收、红外光谱表征证实聚合物的组成以及REDV多肽在表面的固定;并通过血浆复钙化实验表征涂层的血液相容性.细胞黏附与增殖实验反映REDV多肽构建的涂层表面具备良好的特异性识别并结合内皮细胞的能力.     

Phosphorylcholine (PC)-bonded protein A affinity chromatographic medium for high-throughput purification with reduced non-specific protein adsorption

A protein A affinity chromatographic medium based on porous silica modified with phosphorylcholine (PC) groups and amino groups (PNSP) was synthesized. The PC groups functioned as suppressors of non-specific protein adsorption. Recombinant protein A was bound to the amino groups on PNSP with a glutaraldehyde used as a spacer (PNSP-PA). The PC groups and amino groups were immobilized on porous-silica particles using two silane coupling reagents, PC-bound silane, and 3-aminopropyltrimethoxysilane. After optimizing various factors in the synthetic process, the resultant protein A medium showed improvements in non-specific protein adsorption, dynamic binding capacity, and chemical stability under basic conditions compared with conventional protein A affinity media.     

An electrochemical impedance immunoanalytical method for detecting immunological interaction of human mammary tumor associated glycoprotein and its monoclonal antibody

An electrochemical impedance immunosensor has been developed for the specific detection of immunological interaction between human mammary tumor associated glycoprotein and its monoclonal antibody (GP1D8). Antibody proteins were immobilized by spontaneous adsorption of antibody on gold. Consequently, electrochemical impedance spectroscopy (EIS) measurements of a gold electrode coated with the antibody showed changes in a.c. current response after the addition of the specific antigen. The successful immunological reaction between the immobilized antibody–antigen at the electrode surface could be monitored.     

Investigation of the Antifouling Properties of Phenyl Phosphorylcholine‐Based Modified Gold Surfaces

Low impedance, antifouling coatings on gold electrodes based on three new zwitterionic phenyl phosphorylcholine (PPC)‐based layers namely 1) reductively adsorbed PPC diazonium salt, 2) dithiocarbamate PPC SAM and 3) lipoamide PPC SAM (PPC coupled to α‐lipoic acid) were evaluated. The layers were assessed for their ability to limit nonspecific adsorption of proteins to electrode surface with some significant differences observed compared with previously studied PPC diazonium salts reductively adsorbed on glassy carbon. Fluorescence microscopy and electrochemical impedance spectroscopy results suggest that protein adsorption is sensitive to the difference in the structure of the PPC molecules and the charge neutrality of the layers. The lipoamide PPC SAM was shown to be the most effective at resisting nonspecific protein adsorption and this layer was as effective as the ‘gold standard’ of oligo(ethylene oxide) SAMs on gold and PPC diazonium salts reductively adsorbed on glassy carbon.     

The electroactive multilayer films of polyelectrolytes and Prussian blue nanoparticles and their application for H2O2 sensors

Prussian blue (PB) nanoparticles were immobilized in polyelectrolyte (PE) multilayers of various compositions and thickness. Films containing nanoparticles and poly(allylamine hydrochloride) (PAH) were formed using the layer-by-layer adsorption method. A layer of branched poly(ethyleneimine) (PEI) was used to anchor the multilayer structure at the surface of a gold electrode. The films exhibited electroactive properties, increasing with the number of deposited PB layers. The properties of PEI/(PB/PAH) _n multilayers were then compared with the ones containing additionally the conductive polymer poly(3,4-ethylenedioxythiophene)–poly(styrenesulfonate) (PEDOT:PSS). We found that the addition of the conductive, water-soluble polymer enhances the electroactive properties of the multilayer films. It also increased sensitivity of the multilayer-covered electrodes for electrochemical detection of hydrogen peroxide.     

血清髓过氧化物酶电流型免疫传感器的研究

将Nafion吸附到玻碳电极表面,并通过静电吸附和共价键合作用将硫堇和纳米金层层自组装到Nafion修饰的电极表面,然后通过形成的纳米金单层吸附髓过氧化物酶(MPO)抗体,最后用辣根过氧化物酶(HRP)封闭电极上的非特异吸附位点,同时起到放大响应电流信号的作用,研制了一种检测MPO的新型电流型免疫传感器.实验结果表明,该免疫传感器对MPO的响应特性良好,其线性检测范围为2.5～100 μg/L; 检出限为1.425 μg/L,达到95%稳态响应时间<30 s,批间、批内的平均RSD(n=20) <2.94%和4.15%.电极的稳定性良好,在连续30 d内进行10次测量后,响应电流开始下降,平均测量值为初始的85.6%.探讨了抗体浓度、底物浓度、pH、温度及其它干扰物质等对该传感器的影响.使用本方法和经典的酶联免疫吸附实验(ELISA)同时对40份人血清标本MPO进行测试,结果表明: 两者相关性良好(r=0.9971, p<0.0001).该电流型免疫传感器具有灵敏度高、特异性好、不需标记和可以重复测量等优点.     

Covalent immobilization of antibody fragments on well-defined polymer brushes via site-directed method

Well-defined polymer brushes and block copolymer brushes consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC) and glycidyl methacrylate (GMA) were prepared by surface-initiated atom transfer radical polymerization (ATRP). The polymer brushes were used for the immobilization of antibody fragments in a defined orientation. Pyridyl disulfide moieties were introduced to the polymer brushes via a reaction of epoxy groups in GMA units. Fab’ fragments were then immobilized onto these surfaces via a thiol-disulfide interchange reaction and the reactivity of antibodies with antigens was investigated. Antigen/antibody binding on the polymer brushes was more preferable than that on epoxysilane films as a control surface. Furthermore, the activity of the antibodies immobilized on the block copolymer brushes having biocompatible PMPC was greater than that on other surfaces that did not have PMPC in their structures.     

Individually addressable recessed gold microelectrode arrays with monolayers of thio-cyclodextrin nanocavities

Four, individually addressable 30 microm diameter, e-beam deposited, gold microelectrodes recessed by 6 microm were suitably spaced on a single substrate to avoid diffusional overlap between each microelectrode. The single substrate device was functionalised with thiolated alpha-, beta-, and gamma-cyclodextrin nanocavities without spacer groups to ensure close proximity of the cavities to the electrode surface. The microelectrodes were assessed in two stages. The e-beam deposited micron sized electrodes were characterized using models for recessed and inlaid microdisk electrodes. Subsequently, each individually addressable, atomically flat, micro-patterned gold electrode with thiolated CD ensembles was treated as a nanoporous electrode assembly. Theoretical and experimental results were compared using cyclic voltammetry. Atomic force microscopy was also used to characterise the modified microelectrodes. Comparisons were made with thiolated CDs deposited on macroelectrodes. This is the first report of the behaviour of immobilized CD nanocavities ensembles on atomically flat gold microelectrodes.     

A piezoelectric immunoassay based on self-assembled monolayers of cystamine and polystyrene sulfonate for determination of Schistosoma japonicum antibodies

A piezoelectric immunosensor based on an improved immobilization strategy combining self-assembled monolayers (SAM) of cystamine (Cys) and polystyrene sulfonate (PSS) has been developed for the determination of Schistosoma japonicum antibodies (SjAb) in rabbit serum. Cys SAM were first applied to the gold electrode surface of the crystal, serving as a positively-charged base. Schistosoma japonicum antigen (SjAg) was then electrostatically immobilized on the crystal by means of a negatively-charged PSS layer. When sealed by use of an appropriately selected blocking reagent for BSA and normal rabbit serum (NRS), non-specific adsorption could be substantially reduced.The immunosensor was used to determine SjAb in optimized buffer medium with addition of poly(ethylene glycol) (PEG), which served as an immunoreaction enhancer. It was shown experimentally that SjAg immobilized by the Cys-PSS adsorption procedure had higher immunological activity or binding efficiency than those immobilized by the glutaraldehyde (GLU) binding or direct attachment procedures. The immunosensor developed had satisfactory sensitivity and detection limit, and regeneration of the piezoelectric quartz-crystal was easy. Analytical results obtained with infected rabbit serum samples indicated that the proposed immunosensor is a promising alternative for qualitative and quantitative determination of SjAb in clinical diagnosis of infection with Schistosoma japonicum.     

RAFT‐mediated synthesis of poly[(oligoethylene glycol) methyl ether acrylate] brushes for biological functions

The synthesis of poly[(oligoethylene glycol) methyl ether acrylate] [poly(OEGA)] brushes was achieved via reversible addition‐fragmentation chain transfer (RAFT) polymerization and used to selectively immobilize streptavidin proteins. Initially, gold surfaces were modified with a trithiocarbonate‐based RAFT chain transfer agent (CTA) by using an ester reaction involving a gold substrate modified with 11‐mercapto‐1‐undecanol and bis(2‐butyric acid)trithiocarbonate. poly(OEGA) brushes were then prepared via RAFT‐mediated polymerization from the surface‐immobilized CTA. The immobilization of CTA on the gold surface and the subsequent polymer formation were followed by ellipsometry, X‐ray photoelectron spectroscopy, grazing angle‐Fourier transform infrared spectroscopy, atomic force microscopy, and water contact‐angle measurements. RAFT‐mediated polymerization method gave CTA groups to grafted poly(OEGA) termini, which can be converted to various biofunctional groups. The terminal carboxylic acid groups of poly(OEGA) chains were functionalized with amine‐functionalized biotin units to provide selective attachment points for streptavidin proteins. Fluorescence microscopy measurements confirmed the successful immobilization of streptavidin molecules on the polymer brushes. It is demonstrated that this fabrication method may be successfully applied for specific protein recognition and immobilization. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Resistance of zwitterionic telomers accumulated on metal surfaces against nonspecific adsorption of proteins

Telomers of N,N-dimethyl-N-(3-sulfopropyl)-3'-methacryloylaminopropanaminium inner salt (SPB), 2-methacryloyloxyethyl phosphorylcholine (MPC), and N,N-dimethyl-N-(1-carboxymethyl)-2'-methacryloyloxylethanaminium inner salt (CMB) were prepared by UV irradiation in the presence of N,N,N',N'-tetraethylthiuram disulfide and a disulfide-group-carrying iniferter (a compound which pursues initiation, chain transfer, and termination), Cys-BDC, which had been prepared by coupling N,N-diethyldithiocarbamoylmethylbenzoic acid succinimidyl ester with cystamine dihydrochloride. The telomers formed a self-assembled monolayer (SAM) on a gold electrode and a monolayer of colloidal gold deposited on a glass plate, as confirmed by the increase in potential difference (DeltaE(p)) of the voltammogram for hydroquinone as a probe using cyclic voltammetry (CV) and the increase in absorbance using localized surface plasmon resonance (LSPR) absorption spectroscopy, respectively. Nonspecific adsorption of various proteins onto the surfaces of various telomer SAMs was examined from the decrease in peak current (DeltaI) using CV and the absorption increase at 550 nm using LSPR absorption spectroscopy. The zwitterionic telomer SAM in general did not adsorb proteins significantly, suggesting the usability of zwitterionic polymer SAMs and brushes to coat various materials used in biomedical fields. The correlation between the structure of water in the vicinity of zwitterionic telomers and the resistance of the zwitterionic telomer SAMs against the nonspecific adsorption of proteins was discussed.     

Polymer-based bilayer interfaces for electrochemical rectification

In this paper, the electrochemical current rectification phenomenon exhibited at an electrochemical interface constituted by a glassy carbon electrode covered with a bilayer of polymer films is discussed. The authors have shown that Methylene Blue (MB) redox species can be confined to a very thin insulating polymer film formed from orthophenylene diamine. The poly(opd) film exhibited excellent blocking properties to redox molecules in solution. On the other hand, the insulating poly(opd) film trapped with MB could mediate electron transfer between the redox molecules in solution and the electrode. Further, a second polymeric layer (Nafion film) trapped with ferrocene redox species was formed as the outer layer over the inner poly (opd) film containing MB. This bilayer-modified electrode, due to the significant difference in the redox potentials of the MB and ferrocene species immobilized in the inner and outer layers, respectively, exhibits unidirectional current flow and the results of the voltammetric investigations on the modified electrodes are described in this communication.     

Synthesis of magnetic chelator for high-capacity immobilized metal affinity adsorption of protein by cerium initiated graft polymerization

A novel magnetic chelator with high adsorption capacity of protein by immobilized metal affinity adsorption was prepared by cerium (IV) initiated graft polymerization of tentacle-type polymer chains with iminodiacetic acid (IDA) chelating group on magnetic particles with hydroxyl groups. The micron-sized magnetic poly(vinyl acetate-divinylbenzene) (PVAc-DVB) particles were prepared by a modified suspension polymerization in the presence of oleic acid-coated magnetite nanoparticles and subsequently modified by ester exchange reaction to introduce functional hydroxyl groups. Bovine hemoglobin (BHb) was selected as a model protein to investigate the adsorption capacity of these magnetic particles. The magnetic particles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), superconducting quantum interference device (SQUID) magnetometry, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and X-ray diffraction (XRD). The results showed that the magnetic particles had an average size of 5 microm and superparamagnetism with saturation magnetization of 20.0 emu/g at room temperature. The protein adsorption indicated that the graft polymerization of tentacle type polymer chains on the magnetic particles could produce magnetic adsorbents with high adsorption capacity (1428.21 mg/g) and low nonspecific adsorption of protein. The magnetic particles with grafted tentacle polymer chains have potential application in large-scale affinity separation of proteins.     

基于网状混合自组装膜的压电免疫传感器及其应用

结合纳米金及混合自组装技术, 制备了一种新型网状混合膜, 提出了一种新的生物分子固定化方法, 研制了一种用于检测人血清抗精子抗体的压电免疫传感器. 首先, 将纳米金溶胶、巯基丙酸和1,6-二巯基己烷按一定的比例混合制得网状混合自组装膜, 然后将此膜组装到压电石英晶振的金电极表面, 经EDC/NHS活化后, 再将抗原固定到电极上, 实现对抗精子抗体的检测. 结果表明, 该方法能明显提高抗体抗原结合效率, 从而提高传感器的灵敏度, 并降低传感界面的非特异性吸附. 将此传感器应用于人血清抗精子抗体的检测, 线性范围为10~800 mU/mL, 检出限为7 mU/mL. 此传感器为抗精子抗体的临床检测提供了新平台.     

Selective electrochemical recognition of ions in solution and at self-assembled monolayers

Quinone-functionalized calix[4]arenes having carboxylic acid groups or thiol groups were prepared and their spontaneous adsorption on silver and gold surfaces, respectively, was studied. Since the cavity-like structure of calixarenes was immobilized on the noble metal electrodes, they exhibited a selective affinity towards specific hard metal ions in aqueous media. Voltammetric and spectroscopic studies showed the well-ordered deposition of organic receptors and entrapment of metal ions. It also was found that the repeated capture and removal of metal ions reversibly with chelating agents such as ethylenediaminetetraacetic acid (EDTA) was possible. This is the first example, to our knowledge, of voltammetric detection of hard metal ions in aqueous media using a chemically modified electrode with redox-active macrocyclic receptors.