伴刀豆球蛋白-糖蛋白特异性识别作用构筑的多层膜及其在生物传感器制备中的应用

研究了伴刀豆球蛋白-糖蛋白特异性分子识别作用构建的多层膜及其在葡萄糖生物传感器制备中的应用.结果表明,含有糖残基的葡萄糖氧化酶可以通过识别作用组装进入多层膜,不含糖基的过氧化氢酶经过化学修饰引入麦芽糖残基,也可以通过分子识别作用组装到多层膜中.对于多层膜厚度研究的结果表明,膜的厚度随层数的增加呈线性关系.研究了多层膜的表面形貌,并讨论了膜结构与功能的关系.伴刀豆球蛋白-酶多层膜修饰的金电极的电化学实验表明,多层膜中的酶具有催化活性.过氧化氢酶/葡萄糖氧化酶串联组装的生物传感器能够有效消除样品中过氧化氢对葡萄糖测定带来的干扰.     

辣根过氧化物酶电极在H2O2分析中的应用

辣根过氧化物酶(HRP)能催化过氧化氢与氢供体间的氧化还原反应,是当今生物传感器研究的热点之一.HRP分子内含有α-D-葡萄糖和α-D-甘露糖,是一种糖蛋白,在pH 7.0下,能与具有识别α-D-葡萄糖和α-D-甘露糖功能的外源植物凝集素伴刀豆球蛋白(Con A)结合.通过Con A与HRP之间的识别作用在半胱氨酸修饰的金表面构造HRP多层自组装膜电极,以亚甲蓝(MB)溶液为介体,对电极进行了电化学表征,并用该酶电极测定了过氧化氢浓度.     

糖蛋白-凝集素自组装构筑有序膜及在酶电极的应用

利用糖蛋白-凝集素的识别作用交替沉积伴刀豆球蛋白（Con A）与辣根过氧化物酶(HRP)制备酶自组装多层膜，用原子力显微镜（AFM）观测了自组装膜的表面形貌、表面粗糙度； AFM和椭圆偏振研究测定了自组装膜的厚度.结果表明, Con A和HRP膜厚分别为9.0和4.6 nm，与两者的晶体衍射结果一致，说明生物识别自组装方法能很好地保持分子的原有形态.以亚甲蓝（MB）溶液为介体，用循环伏安法测定了表面修饰了三层（Con A/HRP）自组装膜的金电极对H2O2的电化学催化还原作用，在H2O2浓度为0.2～1.0 mmol•L－1时，响应电流对H2O2浓度变化成线性，酶电极灵敏度为24.0 mA•mol－1•L，表观米氏常数为4.2 mmol•L－1.     

以伴刀豆球蛋白为固定基质的脲酶生物传感器

本文制备了一种以溶剂聚合膜pH电极作原电极,利用伴刀豆球蛋白(Con A)与糖蛋白间的特异性识别作用,将Con A和脲酶表面的麦芽糖残基结合,采用交替沉积Con A和脲酶,进行多层脲酶膜组装的脲酶生物传感器。研究了酶固定化条件的影响,优化了实验条件,测试了传感器对尿素的生物电化学响应。在6.9×10-5~1.0×10-3mol.L-1的浓度范围内传感器响应的电极电位与尿素浓度的对数成正比,检出限为4.5×10-5mol.L-1。将传感器用于牛奶样品中回收率的测定,结果满意。     

基于二硫代氨基甲酸盐自组装的糖芯片制备与表征

糖基传感芯片是定量研究糖-蛋白相互作用的有力工具。传统糖基传感芯片的制备过程通常涉及糖基硫醇衍生物的合成,过程复杂且产率较低。本文采用脱氧氨基糖与二硫化碳温和条件下一步反应合成了一类新型糖基自组装功能分子-糖基二硫代氨基甲酸盐(DTC)化合物,进而在金衬底芯片上构筑了糖基传感功能膜。采用X射线光电子能谱(XPS)分析了该糖基传感功能膜的元素组成和元素化学环境;采用表面等离子体共振(SPR)和酶联凝集素分析(ELLA)技术定量分析了其在蛋白质水平的糖生物学活性。通过混合自组装的方法,制备了一系列表面葡萄糖密度不同的糖基传感功能膜并测定了伴刀豆球蛋白(Con A)吸附的热力学和动力学数据。通过调控表面密度,我们观察到了蛋白在葡萄糖表面吸附的多价态现象。当自组装溶液中葡萄糖-DTC摩尔分数低于1%时,Con A呈现单价态吸附,其解离平衡常数(Kd)为(39.10±0.12)μmol?L-1;当自组装溶液中葡萄糖-DTC摩尔分数高于2%时,Con A呈现多价态吸附,解离平衡常数降至(1.17±0.18)μmol?L-1。本文所发展的糖基自组装功能分子合成方法快速便捷、适用范围广,通过混合自组装可以实现蛋白结合价态的调控,是一种深入研究基于糖-蛋白相互作用的诸多生物过程的有效工具。     

基于表面等离子体子共振的白蛋白免疫传感器的研究

采用自行组装的全波长表面等离子体子共振(SPR)传感装置,以对金和蛋白抗体均有较强吸附作用的A蛋白作为基底膜,测试了A蛋白在金膜表面结合的动力学常数,其结果为2.3×10⁵L/(mol·s).研究了在A蛋白基底膜上白蛋白抗体的定向自组装程度和速率.观测了白蛋白抗体与抗原之间免疫反应的动力学过程,并优化了实验条件.结果表明,白蛋白抗原的浓度在0.02～10mg/mL范围内与信号的响应值呈良好的线性关系.将此SPR传感器用于糖尿病肾病患者尿蛋白的检测,结果较好.     

基于聚多巴胺/铜微粒自组装多层膜的无酶葡萄糖传感器

利用多巴胺易于在电极表面发生自聚反应,且聚多巴胺膜中富含邻苯二酚等反应性基团,可通过二次反应实现电极表面的进一步功能化修饰的特点,在玻碳电极(GCE)表面,将多巴胺自聚膜(PDA)与铜微粒(Cu)进行层-层自组装,构建了无酶葡萄糖电化学传感器(GCE/(PDA/ Cu) n )。传感器的灵敏度可通过控制多层膜的组装层数进行调控。采用紫外-可见光谱跟踪表征了多层膜的组装过程,结果表明,多层膜的生长是逐步且均匀的过程。采用循环伏安法和电流-时间曲线法研究了修饰电极对葡萄糖的电催化氧化性能。对于GCE/(PDA/ Cu)4,检测葡萄糖的线性范围为0.5~9.0 mmol/ L,检出限为5.8μmol/ L(S/ N=3)。本传感器具有良好的重现性、稳定性和较强的抗干扰能力。将本传感器用于血清中葡萄糖的测定,结果令人满意。     

基于层-层自反应的葡萄糖氧化酶有序多层膜电极

以胱胺修饰的金电极为基础电极, 利用席夫碱反应使经高碘酸根氧化的葡萄糖氧化酶在该电极表面进行自身的层-层有序组装. 用电化学交流阻抗法对多层酶膜形成过程的跟踪结果表明, 该多层酶膜的生长是一个逐步形成的均匀过程. 用循环伏安法和I-t曲线法研究了该酶电极对葡萄糖的电催化氧化. 实验结果表明, 当采用羟基二茂铁作为人工电子转移媒介体时, 该酶电极对葡萄糖具有很好的电催化氧化功能. 该传感器制作简便, 响应迅速, 性能稳定, 催化电流与葡萄糖浓度在一定范围内成正比, 并且可以通过控制葡萄糖氧化酶的组装层数来调节该生物传感器的灵敏度与检测限.     

表面等离子体共振成像法用于糖蛋白分析

建立了一套表面等离子体共振成像方法, 用于筛选糖蛋白和非糖蛋白, 并可以区分不同糖蛋白与伴刀豆凝集素的识别强度. 波长调制的表面等离子体共振与成像法所得结果一致. 伴刀豆凝集素的洗脱成像实验为识别强度提供了补充验证方案.     

利用层层累积技术制备基于纳米碳管的葡萄糖生物传感器

以多壁纳米碳管(MWNTS)为电子媒介体和酶的吸附载体,利用层层累积的自组装技术固定葡萄糖氧化酶(GOX)的多层(MWNTa/GOx).复合薄膜修饰电极,制备了一种新型葡萄糖生物传感器.结果表明,传感器对葡萄糖的响应电流值随着MWNTa//GOx复合薄膜层数的不同而变化,当MWNTa//GOx复合薄膜的层教为6时,响应电流值迭到最大.(MWNTs/GOx).复合薄膜修饰的葡萄糖生物传感器对30mmol/L葡萄糖的响应电流为1.63μA,响应时间仅为6.7 s.该生物传感器检测的线性范围为0.5～15 mmol/L,最低检测浓度可达0.09 mmol/L.     

A comparative analysis of localized and propagating surface plasmon resonance sensors: the binding of concanavalin a to a monosaccharide functionalized self-assembled monolayer

A comparative analysis of the properties of two optical biosensor platforms: (1) the propagating surface plasmon resonance (SPR) sensor based on a planar, thin film gold surface and (2) the localized surface plasmon resonance (LSPR) sensor based on surface confined Ag nanoparticles fabricated by nanosphere lithography (NSL) are presented. The binding of Concanavalin A (ConA) to mannose-functionalized self-assembled monolayers (SAMs) was chosen to highlight the similarities and differences between the responses of the real-time angle shift SPR and wavelength shift LSPR biosensors. During the association phase in the real-time binding studies, both SPR and LSPR sensors exhibited qualitatively similar signal vs time curves. However, in the dissociation phase, the SPR sensor showed an approximately 5 times greater loss of signal than the LSPR sensor. A comprehensive set of nonspecific binding studies demonstrated that this signal difference was not the consequence of greater nonspecific binding to the LSPR sensor but rather a systematic function of the Ag nanoparticle's nanoscale structure. Ag nanoparticles with larger aspect ratios showed larger dissociation phase responses than those with smaller aspect ratios. A theoretical analysis based on finite element electrodynamics demonstrates that this results from the characteristic decay length of the electromagnetic fields surrounding Ag nanoparticles being of comparable dimensions to the ConA molecules. Finally, an elementary (2 x 1) multiplexed version of an LSPR carbohydrate sensing chip to probe the simultaneous binding of ConA to mannose and galactose-functionalized SAMs has been demonstrated.     

Highly Sensitive Determination of Concanavalin A Lectin Based on Silver-Enhanced Electrogenerated Chemiluminescence of Luminol

A highly sensitive bioassay based on silver-enhanced luminol electrogenerated chemiluminescence (ECL) is reported for the determination of concanavalin A lectin. A gold electrode modified with the mixed self-assembled monolayer of thiolated mannoside and mercaptohexanol was used to selectively capture a target lectin, concanavalin A, through the specific interaction between mannoside and concanavalin A. Mannoside-functionalized gold nanoparticles were further introduced to the opposite binding sites of the tetrameric concanavalin A to form a sandwich-type complex. Silver enhancement step was performed to coat the surface of mannose-stabilized gold nanoparticles with silver. The deposited silver was dissolved in an acidic solution and further neutralized. The resulting silver ions were finally detected with luminol electrogenerated chemiluminescence, in which the silver ions greatly enhanced the chemiluminescence intensity. The present electrogenerated chemiluminescence bioassay detected concanavalin A from 0.190 to 10.0?µg/mL (r²?=?0.999) with a detection limit of 0.146?µg/mL (signal to noise ratio?=?3), which is much lower compared to previously reported methods such as microgravimetry, surface plasmon resonance, and colorimetry. Furthermore, the present bioassay showed good selectivity over possible interfering lectin proteins.     

Sensing capabilities of colloidal gold modified with a self-assembled monolayer of a glucose-carrying polymer chain on a glass substrate

A disulfide-carrying polymer with pendent glucose residues (poly(2-methacryloyloxyethyl D-glucopyranoside)) was obtained by using a benzyl N,N-diethyldithiocarbamoyl derivative which shows the abilities of initiation, chain transfer, and termination (iniferter). The disulfide-carrying polymer was accumulated on a colloidal Au-immobilized glass substrate, and the usefulness of the polymer as a sensing element of concanavalin A (Con A) was examined by using a UV-visible spectrophotometer with the help of surface plasmon resonance. The sensor showed a concentration-dependent specific binding of Con A with a detection limit of 1.9 nM, and furthermore, it had a very high stability at high ionic strength. The polymer-coated device examined here was not only useful as a simple biosensor chip but is also expected to expand our knowledge of interfacial phenomena by introducing various functional polymers on colloidal Au.     

Bi-functionalization of a patterned Prussian blue array for amperometric measurement of glucose via two integrated detection schemes

A novel amperometric sensor that integrates two independent measurement schemes into a single chip for detection of glucose is fabricated. The sensor uses micro-patterned Prussian blue (PB) and ferrocene modified glucose oxidase covered by a thin Nafion membrane. We have developed an amperometric sensor for the detection of glucose that integrates two measurement schemes into a single chip. For fabrication of the sensing interface, micro-contact printing was used to transfer a self-assembled monolayer template onto a gold substrate, allowing selective electrochemical deposition of a PB array. The protective layer of the PB array was subsequently removed and replaced with a layer of redox-functionalized glucose oxidase (GOx), while the entire surface was finally covered with a perm-selective GOx-Nafion membrane. A variety of surface analytical techniques, including atomic force microscopy, surface plasmon resonance imaging and spectroscopic ellipsometry were employed to characterize the composite PB array electrode. The hybrid sensing interface allowed amperometric measurements of glucose to be carried out with two independent schemes at different potentials. The cathodic current was obtained with the PB array functioning as the electrocatalyst, while the anodic current was measured at a higher potential via a mediation mechanism using the ferrocene-modified GOx. For the quantitative detection of glucose, flow-injection analysis was used, and both the operating conditions and the design parameters were optimized. Linear responses were obtained for both anodic and cathodic signals over a concentration range from 0.1 to 50 mM, with a detection limit of 75 microM. The specificity of the sensor was demonstrated with respect to ascorbic and lactic acid. The implementation of integrated detection mechanisms allows the independent measurement of amperometric signals at two separate potentials. This improves the information gathering and opens up new avenues for developing novel methods that potentially eliminate false signal readings.     

On-Line Monitoring 1H-Indole-3-Acetic Acid in Plant Tissues Using Molecular Imprinting Monolayer Techniques on a Surface Plasmon Resonance Sensor

A surface plasmon resonance (SPR)-based detection methodology, which allows the identification and quantification of the plant hormone, ¹H-indole-3-acetic acid (IAA) in Peach, Rosa flower, and Crape myrtle bud is described. To achieve high selectivity, a molecularly imprinted monolayer (MIM) was prepared from alkanethiols self-assembled on an SPR sensor chip with preadsorbed template (IAA). The association constant of the MIM to IAA was calculated to be 2.8 × 10¹¹ M^?1 by analysis of the SPR wavenumber changes induced by different concentrations of IAA. A good calibration curve was obtained in a low concentration range of IAA. Moreover, the MIM revealed impressive selectivity with the selectivity factors of 1.0, 0.12, and 0.20 for IAA and other related analogues. Compared to the MIM sensor chip, the affinity of the self-assembled monolayer (SAM) control sensor chip for IAA was significantly low. The single SPR sensor decorated with the MIM was used to determine IAA concentrations in peach, Rosa flower, and Crape myrtle bud, and satisfactory results were obtained. Based on a signal to noise ratio of 3, the detection limits were 0.23 pM for Crape myrtle, 0.20 pM for Peach, and 0.32 pM for Rosa. The method showed good recoveries and precision, 95.7% (RSD 0.54%) for Peach, 98.1% (RSD 3.4%) for Rosa, and 98.1% (RSD 2.5%) for Crape myrtle, spiked with 0.4171 µg, respectively. These results indicate that a combination of SPR sensing and the MIM provides a simple, promising alternative method for determining endogenous IAA in plant tissue in a real-time manner.     

Enhanced sensitivity of self-assembled-monolayer-based SPR immunosensor for detection of benzaldehyde using a single-step multi-sandwich immunoassay

This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde–ovalbumin conjugate (BZ–OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ–OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ–OVA bound on the mixed SAM. The BZ–OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ–OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL⁻¹) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin–bovine serum albumin (Bio–BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response time of no more than ca. 10 min. Figure A single-step multi-sandwich immunoassay step increases SPR sensor signal by ca. 12 times affording a low detection limit for benzaldehyde of 5 ppt     

Recognition of sugars on surface-bound cap-shaped gold particles modified with a polymer brush

A dithiolated random copolymer with pendent phenylboronic acid residues (Cys-Poly(3-acrylamidophenylboronic acid-co-2-dimethylaminopropyl methacrylamide), Cys-Poly(APBA-co-DMAPMA)) obtained by photo-iniferter method was accumulated as a polymer brush on a cap-shaped gold particles deposited on a vacuum-evaporated gold film, and the usefulness of the polymer brush as a sensing element for glycoprotein, ovalbumin (OVA), was examined by using UV-vis spectroscopy with a help of surface plasmon resonance. A similar system was constructed with a dithiolated mannose-carrying polymer, dithiolated-poly(2-methacryloyloxyethyl-D-mannopyranoside) (DT-PMEMan), prepared by the atom transfer radical polymerization (ATRP). The brush composed of this polymer was examined as a sensing element for lectin, concanavalin A (Con A). The sensor cells modified with Cys-Poly(APBA-co-DMAPMA) and DT-PMEMan showed a concentration-dependent binding of OVA and Con A, respectively, with a comparable detection limit to those with a monolayer of polymer brush-coated gold particle deposited on a glass substrate. Using this system, it can be expected to open a new perspective to various functional polymer brushes fixed to the cap-shaped gold particle on a solid substrate.     

Part per trillion determination of atrazine in natural water samples by a surface plasmon resonance immunosensor

A new immunoassay for continuously monitoring atrazine in water has been developed. It uses a portable biosensor platform based on surface plasmon resonance (SPR) technology. This immunoassay is based on the binding inhibition format with purified polyclonal antibodies, with the analyte derivative covalently immobilized on a gold sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed on the gold-coated sensor surface in order to obtain a reusable sensing surface. The low detection limit for the optimized assay, calculated as the concentration that produces a 10% decrease in the blank signal, is 20 ng/L. A complete assay cycle, including regeneration, is accomplished in 25 min. Additionally, a study of the matrix effects of different types of wastewater was performed. All measurements were carried out with the SPR sensor system (β-SPR) commercialised by the company Sensia, S.L. (Spain). The small size and low response time of the β-SPR platform would allow it to be used in real contaminated locations. The immunosensor was evaluated and validated by measuring the atrazine content of 26 natural samples collected from Ebro River. Solid-phase extraction followed by gas chromatography coupled to mass spectrometric detection (SPE–GC–MS) was used to validate the new immunoassay.     

Application of polypyrrole/GOx film to glucose biosensor based on electrochemical-surface plasmon resonance technique

A surface plasmon resonance (SPR) based biosensor for glucose is presented in which a thin gold film modified with polypyrrole and glucose oxidase (PPy-GOx) acts as the sensor chip. It is based on SPR response to the change of refractive index of PPy-GOx film by the enzymatic catalytic reaction. The co-electropolymerization of pyrrole and GOx was carried out under cyclic voltammetric conditions, and simultaneously monitored by in-situ SPR. It has been revealed that the enzymatic reaction between GOx and PPy in the presence of glucose can lead to distinct changes in the SPR signal. From the experiments, a linear relationship was obtained in the range 1–100 μmol L^?1 between glucose concentration and the rate of redox transformation of PPy. The detection limit was 0.5 μmol L^?1 (S/N?=?3) and recoveries were 95.2–102.7%.     

Surface characterization and efficiency of a matrix-free and flat carboxylated gold sensor chip for surface plasmon resonance (SPR)

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM–MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen–antibody interaction. For both interactions, the well characterized densely functionalized SAM–MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.