AuAg bimetallic nanoparticles film fabricated based on H2O2-mediated silver reduction and its application

A method to fabricate AuAg bimetallic nanoparticles film by H₂O₂-mediated reduction of silver was reported. Gold nanoparticles (Au NPs) were first adsorbed onto the surface of a self-assembled 2-aminoethanethiol monolayer-modified gold film or 3-aminopropyltriethoxysilane (APTES) monolayer-modified quartz slide. Upon further treatment of this modified film with the solution containing silver nitrate (AgNO₃) and H₂O₂, silver was deposited on the surface of Au NPs. The size of the AuAg bimetallic particles could be readily tuned by manipulating the concentration of H₂O₂. Surface plasmon resonance (SPR) was used to investigate the process, the deposition of silver on Au NPs modified gold film resulted in an obvious decrease of depth in the SPR reflectance intensity and minimum angle curves (SPR R-θ curves), which may be utilized for the quantitative SPR detection of the analyte, H₂O₂. Combination of the biocatalytic reaction that could yield H₂O₂ by using the enzyme, glucose oxidase, with the deposition of silver may enable the design of a glucose biosensor by SPR technique. Furthermore, we evaluated the AuAg bimetallic nanoparticles film for their ability to be an effective substrate for surface-enhanced Raman scattering (SERS).     

Improvement of surface plasmon resonance biosensor with magnetic beads via assembled polyelectrolyte layers

The conjugates of magnetic beads coupled with an antibody can be trapped on the Au film firmly due to the magnetic force for the immunoassay of a surface plasmon resonance (SPR) biosensor. However, this approach exhibits significant limitations in robustness and sensitivity due to incomplete dissociation of magnetic beads from the Au film. The incorporation of a polyelectrolyte film on the Au surface can prevent the magnetic beads from the direct contact with the Au film. The layer-by-layer assembly of polyelectrolyte was used as spacer between the gold surface and the magnetic bead. Different layers of polyelectrolyte can be assembled onto the Au film based on an electrostatic force between polycations and polyanions. After the polyelectrolyte film was fabricated on the Au film, the deposition of the magnetic beads was maintained effectively on the film, which favors the sensitivity of the biosensor and the regeneration of the sensing membrane. When the polyelectrolyte layers of (PAH/PSS)₃ were constructed on the Au film, the SPR biosensor with magnetic beads exhibited a satisfactory response to human IgG in the concentration range from 0.25 to 30.00 μg mL⁻¹, and the determination limit obtained is eight times lower than that obtained with (PAH/PSS)₁ layer.     

The preparation and characterization of poly(o-phenylenediamine)/gold nanoparticles interface for immunoassay by surface plasmon resonance and electrochemistry

The poly-o-phenylenediamine (PoPD) nonconducting film and gold nanoparticles (AuNPs) were combined to fabricate AuNPs/PoPD film, which is used as a novel biocompatible interface for the immobilization of antibody and develop a simple and sensitive label-free immunoassay for the detection of the related antigen (human immunoglobulin G (IgG)). Surface plasmon resonance (SPR) and electrochemical methods were used to provide the real-time information about the polymer film growth, assembling of various sizes of gold nanoparticles, anti-human IgG antibody (anti-hIgG) immobilization and the antigen–antibody interaction. The microstructures of the PoPD and AuNPs/PoPD films were characterized by atomic force microscopy (AFM). These results demonstrated that AuNPs were uniformly dispersed on the porous surface of PoPD film, which formed a nano-structure biocompatible AuNPs/PoPD interface. The use of gold nanoparticles and PoPD film could enhance the immunoassay sensitivity and anti-nonspecific property of the resulting immunoassay electrode. Additionally, the reproducibility and preliminary application of anti-hIgG/AuNPs/PoPD/Au electrode for SPR detection of hIgG was also evaluated.     

Continuous Immunoassay for Sulfamethazine by Surface Plasmon Resonance-Based Biosensor

Regeneration of the sensor chip surface is difficult in many surface plasmon resonance (SPR) biosensor assays. Improper regeneration will reduce life span of the sensor chip and decrease the quality of the data. Considering the advantages of reducing the regeneration frequency, a theoretically feasible continuous SPR biosensor immunoassay for sulfamethazine (SMT) was developed. In the continuous inhibitive immunoassay, the sensor chip surface is regenerated only once after a definite number of tests instead of every test. The SMT-bovine serum albumin (BSA) conjugate was covalently immobilized to a carboxymethyldextran modified gold film. The immobilization conditions of the antigen were studied and the working dilution of the antibody was optimized. The antibody was mixed with SMT of different concentrations prepared with PBS buffer to construct the calibration curve. The limit of detection was 0.5 ng mL^?1. The continuous SPR biosensor assay was proved to be simpler and more practical than a normal one.     

A novel electrochemical SPR biosensor

In this paper, we demonstrate for the first time that upon electrochemical oxidation/reduction, the transition in the conductivity of polyaniline (PAn) film on gold electrode surface leads to a large change of surface plasmon resonance (SPR) response due to a change in the imaginary part of dielectric constant of PAn film. Based on the amplifying response of SPR to the redox transformation of PAn film as a direct result of the enzymatic reaction between horseradish peroxidase (HRP) and PAn in the presence of H₂O₂, a novel PAn-mediated HRP sensor has been fabricated. The electrochemical SPR biosensor, unlike a usual binding assay with SPR, can afford a larger SPR response, and can also be reused by reducing the PAn film electrochemically to its reduced state. This method opens up a new route to the fabrication of SPR biosensor.     

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.     

Sensitivity enhancement of SPR biosensor with silver mirror reaction on the Ag/Au film

Based on well-known silver mirror reaction the Ag film was formed on Au film modified by self-assembled monolayer (SAM) of 1,6-hexanedithiol (HDT). The sensitivity of the biosensor based on this Ag/Au film is enhanced compared to that based on Au film. When the surface plasmon resonance (SPR) biosensor based on this Ag/Au film was used to determine human IgG, the range of concentrations of human IgG that could be determined is 0.30-40.00 μg mL⁻¹. The lowest concentration (0.30 μg mL⁻¹) that could be detected was about 8 times lower than that obtained by the biosensor without modification by Ag film (2.50 μg mL⁻¹), which demonstrated that the biosensor based on Ag/Au film could make the resonant wavelength move to longer wavelength following with the sensitivity enhancement of the SPR biosensor.     

表面等离子体共振法检测人血清白蛋白抗体活性

表面等离子体共振法是研究生物大分子间相互作用的有效方法之一,和非直接方法（如酶联免疫）相比,具有实时、快速和免标记等特点。我们在甲羧基化葡聚糖修饰的传感片表面直接交联固一人血清白蛋白（ＨＳＡ）,用于ａｎｔｉ－ＨＳＡ抗体活性的检测,并用Ｈ３ＰＯ４（０．１ｍｏｌ／Ｌ）溶液再生。结果表明表面等离子体共振（ＳＰＲ）生物传感器能快速实时检测ａｎｔｉ－ＨＳＡ抗体的活性,且传感片能够重复使用１００次以上。     

Au nanoparticle-enhanced surface plasmon resonance sensing of biocatalytic transformations

N-(3-Aminopropyl)-N'-methyl-4,4'-bipyridinium is coupled to tiopronin-capped Au nanoparticles (diameter ca. 2 nm) to yield methyl(aminopropyl)viologen-functionalized Au nanoparticles (MPAV(2+)-Au nanoparticles). In situ electrochemical surface plasmon resonance (SPR) measurements are used to follow the electrochemical deposition of the bipyridinium radical cation modified Au nanoparticles on an Au-coated glass surface and the reoxidation and dissolution of the bipyridinium radical cation film. The MPAV(2+)-functionalized Au nanoparticles are also employed for the amplified SPR detection of NAD(+) and NADH cofactors. By SPR monitoring the partial biocatalyzed dissolution of the bipyridinium radical cation film in the presence of diaphorase (DP) NAD(+) is detected in the concentration range of 1x10(-4) M to 2x10(-3) M. Similarly, the diaphorase-mediated formation of the bipyridinium radical cation film on the Au-coated glass surface by the reduction of the MPAV(2+)-functionalized Au nanoparticles by NADH is used for the amplified SPR detection of NADH in the concentration range of 1x10(-4) M to 1x10(-3) M.     

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%.     

Preparation and application of triangular silver nanoplates/chitosan composite in surface plasmon resonance biosensing

This paper reports the utilization of triangular silver nanoplates (TSNPs) to enhance the sensitivity of surface plasmon resonance (SPR) biosensor. TSNPs modified with 3-mercaptopropinic acid (MPA) were simply mixed with chitosan and glutaraldehyde to form TSNPs/chitosan composite. The composite was deposited on Au film as immobilization substrate for SPR biosensor. The novel structures of TSNPs are preserved against etching by MPA and chitosan polymer. Moreover, chitosan cross-linked by glutaraldehyde enables antibody to be immobilized on fabricated substrate directly via Schiff alkali reaction. In the optimized conditions, the resulting biosensor based on TSNPs/chitosan composite shows a satisfactory response to bovine IgG in the concentration range of 0.075–40.00 μg mL⁻¹. While the biosensor based on chitosan without TSNPs shows a response in the concentration range of 0.6–40 μg mL⁻¹ and the biosensor based on Au film shows a response in the concentration range of 2.5–40 μg mL⁻¹. The experiment results show that the sensitivity of SPR biosensor based on TSNPs/chitosan composite was significantly enhanced and the immobilization procedure of antibody was simplified.     

Surface plasmon resonance biosensor for genetically modified organisms detection

The development of a surface plasmon resonance (SPR) affinity biosensor based on DNA hybridisation is described. This biosensor has been applied to genetically modified organisms (GMOs) detection. Single stranded DNA (ssDNA) probes were immobilised on the sensor chip of an SPR device and the hybridisation between the immobilised probe and the complementary sequence (target) was monitored. The probe sequences were internal to the sequence of 35S promoter and NOS terminator which are inserted sequences in the genome of GMO regulating the transgene expression. The system has been optimised using synthetic oligonucleotides, then applied to real samples analysis. Samples, containing the transgenic target sequences, were amplified by polymerase chain reaction (PCR) and then detected with the SPR biosensor.     

纳米金颗粒增强信号的表面等离子体共振生物传感器用于甲氧檗因高灵敏检测的研究

报道了一种基于表面等离子体共振(SPR)生物传感器的高灵敏检测抗癌药物甲氧檗因的新方法. 分别在纳米金颗粒和金膜表面修饰富含腺嘌呤(A)的DNA链, 当存在甲氧檗因时, 由于一个甲氧檗因分子可与4个A碱基相结合, 从而使得修饰在纳米金颗粒和金膜表面的DNA形成稳定的双链结构, 进而将功能化纳米金颗粒捕获在金膜表面. 由于纳米金颗粒与金膜之间的电场耦合效应可增强SPR信号, 从而可实现对小分子甲氧檗因的高灵敏、特异性检测. 本方法的检测下限低至0.07 pmol/L, 相对比色法和荧光法而言, 降低了约5～6个数量级. 以4种药物(盐酸小檗碱、青霉素G、硫酸庆大霉素、5-氟尿嘧啶)作为对照考察了该传感器的选择性, 结果表明该传感器具有较好的选择性.     

核壳型Fe_3O_4/Au粒子的制备及其在表面等离子体子共振传感器中的应用

合成了核壳型Fe3O4/Au复合粒子,并对其形貌、光学性质进行了表征.通过外加磁场将Fe3O4/Au复合粒子与兔抗人IgG的偶联体固定于表面等离子体子共振(SPR)传感器的金基底膜上,形成了Fe3O4/Au/抗IgG敏感膜.与传统的通过巯基丙酸连接蛋白的方式相比,磁场作用固定的Fe3O4/Au/抗IgG敏感膜制备简单,易洗脱,具有良好的再生性,且在一定程度上提高了传感器的灵敏度.并对人IgG进行了测定,结果表明,传感器对于浓度范围在1.25～20.00μg·mL-1的人IgG有良好的信号响应.     

An optical surface plasmon resonance biosensor for determination of tetanus toxin

Surface plasmon resonance (SPR) has been successfully applied for the simple, rapid, and label-free assay of various biomolecules. This assay evaluates a novel wavelength modulation SPR biosensor for the detection of tetanus toxin. The wavelength modulation SPR biosensor is designed based on fixing the incident angle of light and measuring the reflected intensities in the resonance wavelength range spanning 400-800 nm simultaneously. Tetanus toxin (TeNT), one of the most potent toxins known, is synthesized as a 150 kDa single polypeptide chain. The SPR biosensor has been shown to be capable of directly detecting concentration of tetanus toxin as low as 0.028 Lf ml⁻¹. Under selected experimental conditions, the SPR biosensor has a good reproducibility, sensitivity and reversibility. The results illustrate how wavelength modulation SPR biosensor can be used to detect biomolecular interactions.     

An SPR biosensor for the detection of microcystins in drinking water

A surface plasmon resonance (SPR) biosensor for the detection of microcystins (MCs) in drinking water has been developed. Several assay formats have been evaluated. The selected format is based on a competitive inhibition assay, in which microcystin-LR (MCLR) has been covalently immobilized onto the surface of an SPR chip functionalized with a self-assembled monolayer. The influence of several factors affecting sensor performance, such as the nature and concentration of the antibody, the composition of the carrier buffer, and the blocking and regeneration solutions, has been evaluated. The optimized SPR biosensor provides an IC₅₀ 0.67 ± 0.09 μg L⁻¹, a detection limit of 73 ± 8 ng L⁻¹, and a dynamic range from 0.2 to 2.0 μg L⁻¹ for MCLR. Cross-reactivity to other related MCs, such as microcystin-RR (88%) and microcystin-YR (94%), has also been measured. The SPR biosensor can perform four simultaneous determinations in 60 min, and each SPR chip can be reused for at least 40 assay–regeneration cycles without significant binding capacity loss. The biosensor has been successfully applied to the direct analysis of MCLR in drinking water samples, below the provisional guideline value of 1 μg L⁻¹ established by the World Health Organization for drinking water.     

Surface plasmon resonance-based immunoassay for procalcitonin

A surface plasmon resonance (SPR) biosensor has been developed for rapid immunoassay of procalcitonin (PCT) with high detection sensitivity and reproducibility. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A (PrA), diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES) was dispensed on a KOH-treated Au-coated SPR chip, resulting in the covalent binding of PrA in 30 min. This “single-step” PrA immobilization strategy led to the oriented binding of the anti-PCT antibody (Ab) on a PrA-functionalized gold (Au) chip. The leach-proof immobilization procedure is five-fold faster than conventional counterparts, enabling high detection specificity and reproducibility. The IA detects 4–324 ng mL⁻¹ of PCT with a limit of detection (LOD) and a limit of quantification (LOQ) of 4.2 ng mL⁻¹ and 9.2 ng mL⁻¹, respectively. It was capable of detecting PCT in real sample matrices and patient samples with high precision. The Ab-bound SPR chips were stable for more than five weeks.     

表面等离子体共振技术在蛋白质-蛋白质相互作用研究中的应用

表面等离子共振(SPR)近年来迅速发展为用于分析生物分子相互作用的一项技术.该技术无需标记、特异性强、灵敏度高、样品用量小,可实现在线连续实时检测.目前SPR已被广泛应用于免疫学、蛋白质组学、药物筛选、细胞信号转导、受体/配体垂钓等领域.该文阐述了基于表面等离子体共振技术生物传感器的基本原理和技术流程,综述了SPR在蛋白质-蛋白质相互作用动力学研究、蛋白质结构及功能研究、蛋白质突变和碎片分析、信号转导中的应用以及SPR在蛋白质-蛋白质相互作用研究中的多项关键技术.指出SPR通过与光谱、电化学等多技术联用后,可以获得更加详实的信息.     

纳米金增强SPR检测产毒赭曲霉中PKS基因的特异性碱基

基于双通道表面等离子体子共振(surface plasmon resonance,SPR)传感器,分别采用直接法和金纳米粒子作为传感层的方法,通过检测赭曲霉毒素A(ochratoxin A,OTA)合成初期阶段表达的聚酮合成酶(polyketide synthase,PKS)基因的25个特异性碱基的寡核苷酸链,建立了一种高灵敏、间接检测OTA的新方法.同时考察了6-巯基己醇(6-mercapto-1-hexanol,MCH)作为封闭液对SPR响应信号的影响.结果表明,直接法的检测限为12.5 nmol/L,MCH的加入可使响应信号有所增强,使用金纳米粒子作为传感层的检测下限为0.25 nmol/L,与直接法相比较灵敏度提高了50倍,与以往使用金纳米粒子标记抗体或抗原相比,其作为传感层也能大大提高SPR检测灵敏度,且操作简单易行.     

Detecting interferon-gamma release from human CD4 T-cells using surface plasmon resonance

Cytokine secretion by leukocytes is an important indicator of immune response to pathogens and therefore has significant implications in disease diagnostics. Given heterogeneity of leukocyte subsets and the ability of multiple cell subsets to secrete the same cytokines, connecting cytokine production to a specific leukocyte subset is a distinct challenge. In the present paper we describe a strategy combining antibody (Ab)-based affinity cell separation and surface plasmon resonance (SPR) for capturing human CD4 T-cells and for label-free detection of cell-secreted interferon (IFN)-γ – an important inflammatory cytokine. Human blood was introduced into a flow chamber modified with anti-CD4 Abs resulting in capture of CD4⁺ T-cells. After mitogenic activation of cells inside the flow chamber, culture medium was routed onto an SPR chip modified with monoclonal IFN-γ Abs. SPR signal observed in this experiment correlated with cytokine production by T-cells. The strategy of combining SPR detection with cell purification may be used in the future for label-free, sensitive detection of multiple cytokines or proteins secreted by the desired cell subset.