An optical immunosensor based on surface plasmon resonance for determination of bFGF

An optical immunosensor based on surface plasmon resonance (SPR) has been developed for immunosensing. The sensor is designed on the basis of fixing incident angle of light and measuring the reflected intensities in the wavelength range of 400-800 nm simultaneously. The SPR spectrum was shown in terms of reflected light intensities versus wavelengths of incident light. The intensity of the reflected light reaches the minimum at the resonant wavelength. Molecular self-assembling in solution is used to form the sensing membrane on gold substrate. The kinetic processes of sensing monolayer formation were studied. The basic fibroblast growth factor, a kind of basic polypeptide, was determined in the concentration range of 0.24-9.6 μg/ml. Under optimum experimental conditions, the sensor has a good repeatability, reversibility and selectivity.     

Fabrication of a novel immunosensor using functionalized self-assembled monolayer for trace level detection of TNT by surface plasmon resonance

We have developed a new immunosensor based on self-assembly chemistry for highly sensitive and label-free detection of 2,4,6-trinitrotoluene (TNT) using surface plasmon resonance (SPR). A monolayer of amine terminated poly(ethylene glycol) hydrazinehydrochloride (PEG-NH₂) thiolate was constructed on an activated gold surface and immobilized with trinitrophenyl-β-alanine (TNPh-β-alanine) by amide coupling method. The binding interaction of a monoclonal anti-TNT Ab (M-TNT Ab) with TNPh-β-alanine immobilized thiolate monolayer surface was monitored and evaluated for detection of TNT based on the principle of indirect competitive immunoreaction. Here, the competition between the self-assembled TNT derivative and the TNT in solution for binding with antibody yields in the response signal that is inversely proportional to the concentration of TNT in the linear detection range. With the present immunoassay format, TNT could be detected in the concentration range from 0.008 ng/ml (8 ppt) to 30 ng/ml (30 ppb). The response time for an immunoreaction was 2 min and one immunocycle could be done with in 4 min including surface regeneration. Bound antibodies could be easily eluted from the self-assembled immunosurface at high recoveries (more than 100 cycles) using pepsin solution without any damage to the TNT derivatives immobilized on the surface. The compact self-assembled monolayer was highly stable and prevented the non-specific adsorption of proteins on the surface favoring error free measurement.     

Fabrication of novel molecular recognition membranes by physical adsorption and self-assembly for surface plasmon resonance detection of TNT

Recent concern on international terrorism and weapons of mass destruction demands the development of novel analytical methods for identification and quantification of explosive molecules. In this article, we describe the development of high-performance immunosensors for detection of 2,4,6-trinitrotoluene (TNT), a prime component of the landmines and bombs used by terrorist and military forces. The immunosensors were constructed by physical adsorption and self-assembly methods, and their binding interactions with a monoclonal anti-TNT antibody were evaluated for TNT detection using the surface plasmon resonance technique. A home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanine conjugate was used for physical adsorption. A poly(ethylene glycol) hydrazine hydrochloride thiolate was used in the construction of self-assembled monolayer surface and was immobilized with trinitrophenyl-β-alanine by the amide coupling method. The immunosensors were highly selective, regenerable, rapid, and exhibited remarkable sensitivity down to the parts-per-trillion level for TNT by the indirect competitive inhibition principle.     

表面等离子体共振免疫传感器在蛋白质检测中的应用及其研究进展

表面等离子体共振(SPR)技术是20世纪90年代发展起来的一种新型技术,应用SPR原理可检测生物传感芯片上配位体与分析物之间的相互作用情况,在生命科学、医疗检测、药物筛选、食品检测及环境监测等领域具有广泛的应用需求.SPR技术可与免疫传感器结合,利用抗原抗体的特异性反应可用于各种蛋白质抗原的检测.本文重点总结了SPR免疫传感器在食品及医疗领域蛋白质检测的应用,综述了近年来SPR免疫传感技术在这该领域的研究热点及进展.     

Immunosensor Based on Surface Plasmon Resonance for Antigen Recognition

A novel immunosensor based on surface plasmon resonance（SPR） has been developed for the recognition of antigen. The sensor was designed on the basis of the fixed angle of incidence and measuring the reflected intensities in a wavelength range of 430--750 nm in real-time. An ultra-bright white light-emitting diode（LED） was used as the light source. Molecular self-assembling in solution was used to form the sensing membrane on gold substrate. It has been seen that the sensitivity of the SPR sensor with 3-mercaptopropionic acid（MPA）/protein A（SPA） sensing membrane is considerably higher than that with MPA or SPA modified sensing membrane. The kinetic processes on the sensing membrane were studied. The human B factor（Bf）, an activator of complement 3（C3）, was recognized among the other antigens. This sensor can also be used for other antigen/antibody or adaptor/receptor recognition. Under optimized experimental conditions, the sensor has good selectivity, repeatability, and reversibility.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

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     

Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance

Biosurface fabrication using the Fab′ fragment of immunoglobulin (IgG) was carried out by self-assembly (SA) technique. The pepsin-digested monoclonal antibody (Mab) against bovine insulin containing the F(ab′)₂ fragment and residual proteins was separated using affinity chromatography and dialysis. To prevent the nonspecific binding of F(ab′)₂ onto gold (Au) substrate, the native disulfide bridge was reduced using dithiothreitol (DTT) to convert F(ab′)₂ into Fab′, which made the immobilization to be carried out via the native thiol (–SH) group. The fabricated biosurface using SA technique showed the formation of stable thin film through AFM topography. Through the concentration change of DTT and Fab′, the absorption characteristics against the Au surface were investigated using surface plasmon resonance (SPR) with the flow cell. The amount of immobilized antibody fragment and the antigen binding capacity were regulated with respect to the reduction state and concentration of F(ab′)₂. Based on the biosurface of the fabricated Fab′, the insulin-detection was carried out by the measurement of SPR. The proposed antibody surface could successfully detect the bovine insulin at the concentration from 100 ng/mL to 10 μg/mL.     

A review on novel developments and applications of immunosensors in food analysis

The present review deals with novel developments in immunosensors destined for final application in food analysis. In this perspective particular emphasis will be given to the most important approaches which recently have been used for immunosensor construction and assembling. For this reason, electrochemical, surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) techniques will be explored in detail and recent and practical examples on food matrices will be reviewed. Objective of this survey is to give a general overview of the possible application of immunosensors to the food analysis field.     

Surface plasmon resonance immunosensor for histamine based on an indirect competitive immunoreaction

The use of a surface plasmon resonance immunosensor for the analysis of histamine (β-imidazole ethylamine) is described. The method is based on an indirect competitive reaction of an anti-histamine antibody in a sample solution with histamine immobilized on a sensor chip and with histamine in the sample solution. A sensor chip immobilized with histamine was prepared using a self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA) as an anchor membrane, followed by an amino-coupling reaction with histamine after activation of the 11-MUA layer on the sensor chip by treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide. The sensor chip can be reused, after regeneration with a 10 mM HCl solution, which dissociates the anti-histamine antibody complex from histamine on the sensor chip. The affinity constants for the immunocomplex of the anti-histamine antibody with histamine in the solution and for that of the anti-histamine antibody with histamine immobilized on the sensor chip were calculated to be 1.5 × 10⁷ and 7.2 × 10⁵ M⁻¹, respectively, by assuming a Langmuir-type adsorption of the anti-histamine antibody to histamine immobilized on the sensor chip. The detection limit of the method was determined to be 3 ppb.     

A capacitive immunosensor for detection of cholera toxin

Contamination of food with biological toxins as well as their potential use as weapons of mass destruction has created an urge for rapid and cost effective analytical techniques capable of detecting trace amounts of these toxins. This paper describes the development of a sensitive method for detection of cholera toxin (CT) using a flow-injection capacitive immunosensor based on self-assembled monolayers. The sensing surface consists of monoclonal antibodies against the B subunit of CT (anti-CT), immobilized on a gold transducer. Experimental results show that the immunosensor responded linearly to CT concentrations in the range from 1.0 × 10⁻¹³ to 1.0 × 10⁻¹⁰ M under optimized conditions. The limit of detection (LOD) was 1.0 × 10⁻¹⁴ M. Two more analytical methods were employed for detection of CT using the same antibody namely, sandwich ELISA and surface plasmon resonance (SPR)-based immunosensor. The former had an LOD of 1.2 × 10⁻¹² M and a working range from 3.7 × 10⁻¹¹ to 2.9 × 10⁻¹⁰ M whereas, the later had an LOD of 1.0 × 10⁻¹¹ M and a linearity ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ M. These results demonstrate that the developed capacitive immunosensor system has a higher sensitivity than the other two techniques. The binding affinity of CT to the immobilized anti-CT was determined using the SPR-based immunosensor and an association constant (K_A) of 1.4 × 10⁹ M⁻¹ was estimated.     

Present and future of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves—surface plasmon-polaritons—to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.     

表面等离子体子共振技术实时研究蛋白质在修饰表面的静电吸附行为

研究蛋白质在固相表面的静电吸附特性,进而控制蛋白质在修饰表面的静电吸附尤为重要,表面等离子体子共振可以检测金属表面吸附物质厚度和折射率的变化^[1]。这种技术已在研究生物分子相互作用^[2]和考察自组装单层的形成^[3]及蛋白质在固体表面吸附行为^[9-11]等方面得到广泛的应用。对蛋白质在固体表面吸附行为的研究多为考察不同的蛋白质在不同的修饰表面的吸附行为。然而,对蛋白质在修饰表面静电吸附的本质影响因素的研究却少有报道^[4]。本文使用表面等离子体子共振技术实时研究了蛋白质在甲羧基化葡聚糖修饰表面的静电吸附与溶液pH值及离子强度的依赖关系。     

Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate

A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25 ppt to 25 ppb, and the coefficient of variation of the SPR signals for the 25 ppb TNP solution was determined to be 13% (n = 4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations.     

基于表面等离子体子共振的B因子传感器

以分辨率较高的一米光栅单色仪为分光系统,光电倍增管为检测系统,改进了自行组装的表面等离子体子共振(SPR)传感装置,提高了仪器的检测能力约50倍,对于发展改变波长模式的SPR传感器具有重要意义。以对金和抗体均有较强吸附作用的葡萄球菌A蛋白为基底膜,观测了人的B因子抗体和抗原之间免疫反应的动力学过程,并研究了B因子的定量测定。结果表明,B因子抗原的浓度在0.02～5μg/mL范围内与信号的响应值呈线性关系。该传感器灵敏度高,选择性和重现性均好。     

Effects of solute-matrix interaction on monitoring the conformational changes of immobilized proteins by surface plasmon resonance sensor

A real-time and labeling-free surface plasmon resonance (SPR) sensor was used to monitor the conformational changes of immobilized globule proteins (RNase A and lysozyme) in chemical unfolding and refolding. The effects of chemical denaturants on the protein structures were investigated. The methodology in protein conformational study on the solid surface is refined through the theoretic calculations and the conformational information of native/denatured proteins in solution. Additionally, our observation illustrates that the ambient buffer solution is merit to influence the refractive index of immobilized protein films and directly be observed from the SPR resonance angle shifts.     

Investigation of SPR and electrochemical detection of antigen with polypyrrole functionalized by biotinylated single-chain antibody: A review

An electrochemical label-free immunosensor based on a biotinylated single-chain variable fragment (Sc-Fv) antibody immobilized on copolypyrrole film is described. An efficient immunosensor device formed by immobilization of a biotinylated single-chain antibody on an electropolymerized copolymer film of polypyrrole using biotin/streptavidin system has been demonstrated for the first time. The response of the biosensor toward antigen detection was monitored by surface plasmon resonance (SPR) and electrochemical analysis of the polypyrrole response by differential pulse voltammetry (DPV). The composition of the copolymer formed from a mixture of pyrrole (py) as spacer and a pyrrole bearing a N-hydroxyphthalimidyl ester group on its 3-position (pyNHP), acting as agent linker for biomolecule immobilization, was optimized for an efficient immunosensor device. The ratio of py:pyNHP for copolymer formation was studied with respect to the antibody immobilization and antigen detection. SPR was employed to monitor in real time the electropolymerization process as well as the step-by-step construction of the biosensor. FT-IR demonstrates the chemical copolymer composition and the efficiency of the covalent attachment of biomolecules. The film morphology was analyzed by electron scanning microscopy (SEM).Results show that a well organized layer is obtained after Sc-Fv antibody immobilization thanks to the copolymer composition defined with optimized pyrrole and functionalized pyrrole leading to high and intense redox signal of the polypyrrole layer obtained by the DPV method. Detection of specific antigen was demonstrated by both SPR and DPV, and a low concentration of 1 pg mL⁻¹ was detected by measuring the variation of the redox signal of polypyrrole.     

Label-free detection of cancer biomarker candidates using surface plasmon resonance imaging

In this work, we present an antibody array for the detection of cancer biomarker candidates by a surface plasmon resonance (SPR) imaging sensor with polarization contrast. Responses from the SPR imaging sensor are shown to be similar to those from a conventional spectroscopy-based SPR sensor. Antibodies are spotted onto a self-assembled monolayer (SAM) composed of oligo(ethylene glycol) (OEG)-containing alkanethiol chains. Detection of two cancer biomarker candidates, activated leukocyte cell adhesion molecule/CD 166 (ALCAM) and transgelin-2 (TAGLN2), is demonstrated. Limits of detection for ALCAM and TAGLN2 are established at 6 ng/mL and 3 ng/mL, respectively, in buffer. No cross-reactivity is observed between immobilized antibodies and nonspecific antigen. Biomarker candidates are also detected in a 10% human serum solution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

中性红作为DNA作用方式光谱探针的研究

以溴乙锭（ＥＢ）为探针在研究吩噻嗪药物与ＤＮＡ间相互作用的基础上,用中性红（Ｎｅｕｔｒａｌ Ｒｅｄ,ＮＲ）代替ＥＢ为ＤＮＡ作用方式对光谱探针的可行性进行了研究。结果表明,ＮＲ作为ＤＮＡ作用方式光谱探针与ＥＢ具有可比性,且由于其低毒性和高稳定性而具有较好的应用潜力。     

Double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor

A double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor was developed. As a proof-of-concept, a designed alkyne functionalized human IgG was used as a capture antibody and a HRP-labeled rabbit anti-goat IgG was used as signal antibody for the determination of the anti-human IgG using the sandwich model. The immunosensor was fabricated by electrochemically grafting a phenylazide on the surface of a glassy carbon electrode, and then, by coupling the alkyne functionalized human IgG with the phenylazide group through an electro-click chemistry in the presence of Cu(II). The amperometric measurement for the determination of the anti-human IgG was performed after the fabricated immunosensor was incubated with the target anti-human IgG and then with the HRP-labeled anti-goat IgG at −0.25 V in 0.10 M PBS (pH 7.0) containing 0.1 mM hydroquinone and 2.0 mM H₂O₂. The results showed that the increased current was linear with the logarithm of the concentration of the anti-human IgG in the range from 1.0 × 10⁻¹⁰ g mL⁻¹ to 1.0 × 10⁻⁸ g mL⁻¹ with a detection limit of 3 × 10⁻¹¹ g mL⁻¹. Furthermore, the feasibility of the double electrochemical covalent coupling method proposed in this work for fabricating the amperometric immunosensor array was explored. This work demonstrates that the double electrochemical covalent coupling method is a promising approach for the fabrication of the immunosensor and immunosensor array.