基于表面等离子体共振和电化学联用的DNA传感器研究进展

表面等离子共振(surface plasmon resonance,SPR)技术旨在检测物体表面附近折射率的变化,其特点是无标记、实时、灵敏和快速,该技术多用于研究分子的相互作用,包括动力学、效率常数和大分子构象变化等。电化学(electrochemical,EC)技术是一项用于定性定量研究电子转移、物质氧化还原、界面吸附等过程的成熟技术,具有简单、低成本和设备小型化的优点。现有的DNA杂交技术,例如光学、电化学或压电转导技术,主要关注于提高DNA杂交检测系统的选择性和灵敏度。传统的SPR在DNA分析方面,由于无法测量折射率的极小变化而在超灵敏检测中的应用受到限制。因此,随着纳米材料的研发和联用技术的飞速发展,SPR与EC联用的生物传感器研究越来越成为人们关注的热点。近年来,关于SPR和EC联用在DNA检测方面的综述鲜有报道。对SPR和EC检测DNA的技术原理、联用方法、应用进展等方面作出了简要的介绍,以期为表面等离子共振和电化学联用的DNA传感器相关研究提供参考。     

Development of surface plasmon resonance immunosensor through metal ion affinity and mixed self-assembled monolayer

An immunosensor based on surface plasmon resonance (SPR) with enhanced performance was developed through a mixed self-assembled monolayer. A mixture of 16- mercaptohexadecanic acid (16-MHA) and 1-undecanethiol with various molar ratios was self-assembled on gold (Au) surface and the carboxylic acid groups of 16-MHA were then coordinated to Zn ions by exposing the substrate to an ethanolic solution of Zn(NO(3))(2)d6H2O. The antibody was immobilized on the SPR surface by exposing the functionalized substrate to the desired solution of antibody in phosphatebuffered saline (PBS) molecules. The film formation in series was confirmed by SPR and atomic force microscopy (AFM). The functionalized surface was applied to develop an SPR immunosensor for detecting human serum albumin (HSA) and the estimated detection limit (DL) was 4.27 nM. The limit value concentration can be well measured between ill and healthy conditions.     

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction. The immunosensor chips were constructed by various concentrations of protein A. To determine the surface characteristics of different self-assembly monolayers (SAMs), several quantitative and kinetic measurements were carried out. The extent of immobilization of anti-CEA and the immune response of anti-CEA antibody against CEA were measured using the SPR instrument and CV device. The terminal functional groups of protein A have different effects on the adsorption and covalent binding of immunoprotein depending on the steric hindrance. Through the parallel measurements, we demonstrate that SPR and CV are sensitive to measure the antigen–antibody binding capacity.     

Comparative study of surface plasmon resonance, electrochemical and electroassisted chemiluminescence methods based immunosensor for the determination of antibodies against human growth hormone

An indirect immunoassay format with human growth hormone (hGH) immobilized on the self-assembled monolayer (SAM) modified surface plasmon resonance (SPR) chip has been shown to detect specific anti-hGH antibodies using the combination of three different physical phenomena in the same channel of the SPR analyzer. For the enhancement of analytical signal and sensitivity of the immunosensor horseradish peroxidase (HRP) labeled secondary antibodies, specifically interacting with the formed immune complexes, were used. The electroassisted chemiluminescence (ECL) protocol offered the limit of detection (LOD) as low as 0.061 nM and this result was very similar to that obtained by SPR, which was 0.051 nM. In the case of anti-hGH detection using pulsed amperometry (PA) with 3,3',5,5'-tetramethylbenzidine (TMB) and H(2)O(2) in the electrochemical system the LOD was the lowest - 0.027 nm. Lower reproducibility of the analytical signal and higher limit of detection was observed using cyclic voltammetry (CV) where LOD was 0.056 nM. PA detection shows 1.89, 2.07 and 2.26 times higher sensitivity if compared with SPR, CV and ECL, respectively. This work demonstrates successful simultaneous exploitation of several techniques to detect the specific anti-hGH antibodies using indirect immunoassay format on the same area of the SPR-chip.     

Immunosensor for the detection of Vibrio cholerae O1 using surface plasmon resonance

An immunosensor for the detection of Vibrio cholerae O1 was developed on the basis of surface plasmon resonance (SPR). A protein G layer was fabricated by means of the chemical coupling between the free amine (-NH₂) groups of protein G and the activated carboxyl groups present on a self-assembled monolayer (SAM) consisting of a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2). A monoclonal antibody, which was confirmed to be specific to V. cholera O1 by the Western blotting technique, was immobilized on the protein G layer. The formation of the SAM, the protein G layer and the sequential binding of the antibody against V. cholera O1 were investigated with SPR spectroscopy. As the number of fabricated layers increased, the minimum angle of plasmon resonance was increased accordingly. The target bacteria, V. cholera O1, was measured with the fabricated immunosensor, whose detection range was between 10⁵ and 10⁹ cells/mL.     

Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance

Three types of imaging, namely layer structure, electrochemical reaction, and enzyme sensor response, were achieved by applying surface plasmon resonance (SPR) measurement to an electrochemical biosensor. We constructed glucose oxidase based mediator type sensors on a gold electrode by spotting the mediator that contained horseradish peroxidase and spin coating the glucose oxidase film. The layer structure of the sensor was imaged by means of angle scanning SPR measurement. The single sensor spot (about 1 mm in diameter) consisted of about 100 x 100 pixels and its spatial structure was imaged. The multilayer structure of the enzyme sensor had a complex reflectance-incident angle curve and this required us to choose a suitable incident angle for mapping the redox state. We chose an incident angle that provided the most significant reflection intensity difference by using data obtained from two angle scanning SPR measurements at different electrode potentials. At this incident angle, we controlled the electrochemical states of the spotted mediator in cyclic voltammetry and imaged the degree to which the charged site density changed. Finally, we mapped the enzymatic activity around the mediator spot by the enzymatic reoxidation of pre-reduced mediator in the presence of glucose.     

Nanoscale fabrication of protein A on self-assembled monolayer and its application to surface plasmon resonance immunosensor

The fabrication of protein A film on self-assembled monolayer was done for the construction of immunosensor using surface plasmon resonance (SPR) measurement. The layer of heterobifunctional linker, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was self-assembled on the gold (Au) surface. Due to the succinimidyl functional group in SPDP to be reacted with amine (NH₂) group of protein A, the covalent immobilization of protein A was subsequently induced toward Au surface. The characteristics of film formation were investigated using SPR with respect to the various concentrations of SPDP and protein A. The optimal concentration for the film formation was found to be 0.1 mg/mL of SPDP and 0.1 mg/mL of protein A, respectively. The surface topography of protein A layer using atomic force microscopy showed that the heteromolecular layer was formed successfully. The antibody, anti-bovine serum albumin (BSA), was immobilized onto protein A layer, and the fabricated antibody layer was applied for the detection of BSA. The extent of BSA–antibody binding was measured using SPR and its lower detection limit of BSA was 100 pM.     

Analysis of carbohydrates using liquid chromatography--surface plasmon resonance immunosensing systems

An immunosensing system based on surface plasmon resonance (SPR) was used for on-line detection and characterization of carbohydrate molecules separated by high-performance liquid chromatography. These analytes, with or without serum, were continuously separated and analyzed in the combined liquid chromatography-surface plasmon resonance (LC-SPR) system. By using weak and readily reversible monoclonal antibodies, the SPR system allowed specific on-line monitoring of the substances. To increase the specificity of the immunosensor, nonrelevant antibodies were used as reference in a serial flow cell. The sensitivity of the LC-SPR system was dependent on molecular weight of the carbohydrate, affinity of binding, and design of the sensor.     

Electrodissolution of inorganic ions/DNA multilayer film for tunable DNA release

A layer-by-layer film composed of DNA and inorganic zirconium ion (Zr(4+)) was fabricated on the surface of gold thin film, and an electric field triggered disintegration of the multilayer film was studied by using electrochemical surface plasmon resonance (EC-SPR). EC-SPR results demonstrated that the film was disassembled upon the application of an electric field and the disassembly rate varied with the applied potential, leading to the controlled release of DNA. The electrodissolution could be switched off by removing the electric potential and reactivated by reapplying the potential. By incorporating plasmid DNA (pDNA) in to this controlled release system, the multilayer film could sustain the consecutive release of pDNA electrochemically. The released pDNA retained its integrity and transfection activity, and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The electrochemical systems, with advantages of miniaturization, surface-tailoring, safety, simplicity, convenience, automation, low-cost, and free of immune reactions, made the electrical route a very attractive gene-delivery alternative.     

Fabrication of self-assembled protein A monolayer and its application as an immunosensor

The self-assembled layer of modified protein A was fabricated. In order to modify protein A, the surface group of protein A was substituted with thiol (-SH) functionality by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and dithiothreitol (DTT). The formation of a self-assembled protein A layer on a Au substrate and its increased binding capacity to antibody were confirmed by surface plasmon resonance (SPR) spectroscopy. The surface structure of self-assembled protein A layer, and the binding status of anti-bovine serum albumin (anti-BSA) and BSA were determined by atomic force microscopy (AFM). Treatment on the self-assembled protein A layer with a detergent, such as Tween 20, increased the binding capacity of anti-BSA, because protein A aggregation was reduced significantly by the detergent; this was confirmed by SPR spectroscopy. The self-assembled layer of chemically modified protein A with enhanced binding capacity can be used for immunosensor fabrication.     

Label-free electrochemical immunosensors based on surface-initiated atom radical polymerization

A novel label-free immunosensing strategy for sensitive detection of tumor necrosis factor-alpha antigen (TNF-α) via surface-initiated atom transfer radical polymerization (SI-ATRP) was proposed. In this strategy, the Au electrode was first modified by consecutive SI-ATRP of ferrocenylmethyl methacrylate (FMMA) and glycidyl methacrylate (GMA), and TNF-α antibody was coupled to the copolymer segment of GMA (PGMA) by aqueous carbodiimide coupling reaction. Subsequently, the target TNF-α antigen was captured onto the Au electrode surface through immunoreaction. The whole process was confirmed by scanning electron microscopy (SEM) and surface plasmon resonance (SPR) measurements. With introduction of redox polymer segment of FMMA (PFMMA) as electron-transfer mediator, the antigen-coupled Au electrode exhibited well electrochemical behavior, as revealed by cyclic voltammetry measurement. This provided a sensing platform for sensitive detection of TNF-α with a low detection limit of 3.9pgmL(-1). Furthermore, the "living" characteristics of the ATRP process can not only be readily controlled but also allow further surface functionalization of the electrodes, thus the proposed method presented a way for label-free and flexible detection of biomolecules.     

Volatile organic compound specific detection by electrochemical signals using a cell-based sensor

A cell-based in vitro exposure system was developed to determine whether oxidative stress plays a role in the cytotoxic effects of volatile organic compounds (VOCs) such as benzene, toluene, xylene, and chlorobenzene, using human epithelial HeLa cells. Thin films based on cysteine-terminated synthetic oligopeptides were fabricated for immobilization of the HeLa cells on a gold (Au) substrate. In addition, an immobilized cell-based sensor was applied to the electrochemical detection of the VOCs. Layer formation and immobilization of the cells were investigated with surface plasmon resonance (SPR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The adhered living cells were exposed to VOCs; this caused a change in the SPR angle and the VOC-specific electrochemical signal. In addition, VOC toxicity was found to correlate with the degree of nitric oxide (NO) generation and EIS. The primary reason for the marked increase in impedance was the change of aqueous electrolyte composition as a result of cell responses. The p53 and NF-kappaB downregulation were closely related to the magnitude of growth inhibition associated with increasing concentrations of each VOC. Therefore, the proposed cell immobilization method, using a self-assembly technique and VOC-specific electrochemical signals, can be applied to construct a cell microarray for onsite VOC monitoring.     

表面等离子体共振技术在分子生物学中的应用

表面等离子体共振(SPR)技术可以实时、原位地测定生物分子间的相互作用而无需任何标记,可以连续监测吸附和解离过程,并可以进行多组分复合物的相互作用的研究。SPR技术在DNA的复制和转录、DNA的修复、核酸与药物的作用以及肽库和抗体库的筛选等分子生物学领域的应用研究取得了令人瞩目的进展,显示了常规技术无法比拟的优越性。     

Direct surface plasmon resonance immunosensor for in situ detection of benzoylecgonine, the major cocaine metabolite

In this paper the development of the first direct surface plasmon resonance (SPR) immunoassay for the detection of benzoylecgonine (BZE) is described. Immunosensor chips consisting of a high affinity monoclonal anti-BZE-antibody (anti-BZE-Ab) immobilized at high density to a sensor chip were prepared. First, BZE detection in Hepes buffer was achieved by direct, real time monitoring of the binding between BZE in solution and the surface bound antibody. The detection protocol was based on calibration curves obtained from reaction rate data and end point data analysis of sensorgrams registered after injection of a series of known BZE concentrations over the chips. Moreover, immunosensor accuracy, reproducibility, stability and robustness were tested to demonstrate their good performance as reusable devices. The immunosensor was used for BZE detection in oral fluid (OF) showing that, within 180 s, our immunoassay detects BZE concentrations as low as 4 μg/L in filtered OF-buffer (1:4) samples. This value is remarkably lower than current cut off levels established by the Substance Abuse and Mental Health Services Administration. These results manifest the potential use of this direct SPR immunoassay for the in situ sensitive detection of recent cocaine abuse, of utility in roadside drug OF testing. Moreover, it exemplifies the high potential of direct SPR immunoassays for the rapid, sensitive detection of small molecules in contrast with the more established indirect methods.     

Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex

Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy–immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content.     

Metallic Nanowire Array–Polymer Hybrid Film for Surface Plasmon Resonance Sensitivity Enhancement and Spectral Range Enlargement

In this paper, the coupling interaction is investigated between a metallic nanowire array and a metal film under the Kretschmann condition. The plasmonic multilayer is composed of a metallic nanowire array embedded in a polymer layer positioned above a metal film, exploiting the classical surface plasmon resonance (SPR) configuration. We analyze the influence of various structural parameters of the metallic nanowire array on the SPR spectrum of thin metal film. The results show that the coupling interactions of nanowires with the metal film can greatly affect SPR resonance wavelength and increase SPR sensitivity. The coupling strength of metallic nanowire array and metal film also impacts resonance wavelength, which can be used to adjust SPR range but have little effect on its sensitivity. The results are confirmed using a dipole coupling resonance model of metallic nanowire. We demonstrated that this nanostructured hybrid structure can be used for high sensitivity SPR monitoring in a large spectral range, which is important for advanced SPR measurement including fiber-optic SPR sensing technology.     

Determination of monoenzyme- and bienzyme-stimulated precipitation by a cuvette-based surface plasmon resonance instrument.

This paper describes the use of a cuvette-based surface plasmon resonance (SPR) instrument to measure biocatalyzed precipitation reactions. Enzyme-modified SPR sensor disk forms the base of a cuvette, in which the substrate solution is added with stirring. The determination of the substrate concentration relies on the measurement of SPR angle shift (Deltatheta(SPR)) induced by the deposition of the insoluble products without involving in any electrochemical reactions. As examples, horseradish peroxidase (HRP)-modified monoenzyme SPR sensor and HRP-glucose oxidase bienzyme-layered sensor are created to determine hydrogen peroxide and glucose via the catalyzed oxidation of 4-chloro-1-naphthol (4-CN). The deposition of the oxidized 4-CN-insoluble products leads to SPR angle shifts, which are linear to H(2)O(2) and glucose in the concentration ranges of 0.067-7.24 x 10(-5) and 0.7-8.3 x 10(-4) mM, respectively. The SPR sensitivities are greater than those of nonelectrochemical quartz crystal microbalance (QCM) (the parallel results in this study) and compare favorable with those of electrochemical QCM and electrochemical SPR methods. This study opens the field for enhanced SPR measurements by using biocatalyzed precipitation as a signal amplification method.     

Interaction of low-molecular-weight chitosan with mimic membrane studied by electrochemical methods and surface plasmon resonance

Chitosan has shown its potential as a non-viral gene carrier and an adsorption enhancer for subsequent drug delivery to cells. These results showed that chitosan acted as a membrane perturbant. However, there is currently a lack of direct experimental evidence of this membrane perturbance effect, especially for chitosans with low molecular weight (LMW). In this report, the interaction between a lipid (didodecyl dimethylammonium bromide; DDAB) bilayer and chitosan with molecular weight (MW) of 4200 Da was studied with cyclic voltammetry (CV), electrochemical impedance spectroscopy and surface plasmon resonance (SPR). A lipid bilayer was formed by fusion of oppositely charged lipid vesicles on a mercaptopropionic acid (MPA)-modified gold surface to mimic a cell membrane. The results showed that the LMW chitosan could disrupt the lipid bilayer, and the effect seemed to be in a concentration-dependent manner.