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.     

Enhanced optical immunosensor based on surface plasmon resonance for determination of transferrin

Wavelength modulation surface plasmon resonance biosensors (SPR) using colloidal Au nanoparticles and double-linker sensing membrane enhancement are reported for determination of transferrin. The 2-mercaptoethylamine (MEA) was immobilized on the biosensor surface with traditional amine coupling method. The interaction between colloidal Au nanoparticles and MEA was investigated. The anti-transferrin was immobilized on the biosensor surface prepared with staphylococcal protein A (SPA). The interaction of the antibody and antigen was monitored in real time. The good response was obtained in the concentration range 1-20, 0.1-20 and 0.05-20 μg/mL for directly immune assay, double-linker assay and colloidal Au-amplified assay. The result clearly demonstrates that these methods may obtain significantly enhancement of sensitivity for the wavelength modulation SPR biosensor.     

表面等离子体子共振生物传感器用于乙肝表面抗原的测定

运用自行研制的表面等离子体子共振（SPR）生物传感器，采用自组装成膜技 术并以戊二醛作偶联剂，在传感片表面修饰HBsAg单克隆抗体，将其用于乙肝表面 抗原（HBsAg)的检测。实验结果表明SPR生物传感器对HBsAg的检出限为0.06ng/mL 。与传统的酶联免疫吸附试验（ELISA）相比，SPR生物传感器的检出灵敏度明显高 于ELISA法。用该SPR生物传感器对HBsAg质控血清与纯化的HBsAg溶液进行比较检测 ，结果表明该SPR生物传感器对HBsAg具有好的特异选择性。     

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.     

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin–streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K _D≈4.01×10⁻⁷. All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.     

Surface plasmon resonance sensing of nucleic acids: A review

Biosensors based on surface plasmon resonance (SPR) have become a central tool for the investigation and quantification of biomolecules and their interactions. Nucleic acids (NAs) play a vital role in numerous biological processes and therefore have been one of the major groups of biomolecules targeted by the SPR biosensors. This paper discusses the advances of NA SPR biosensor technology and reviews its applications both in the research of molecular interactions involving NAs (NA–NA, NA–protein, NA–small molecule), as well as for the field of bioanalytics in the areas of food safety, medical diagnosis and environmental monitoring.     

Development of surface chemistry for surface plasmon resonance based sensors for the detection of proteins and DNA molecules

The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundecanoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.     

Biofunctionalized gold nanoparticles for SPR-biosensor-based detection of CEA in blood plasma

We report on the use of new biofunctionalized gold nanoparticles (bio-AuNPs) that enable a surface plasmon resonance (SPR) biosensor to detect low levels of carcinoembryonic antigen (CEA) in human blood plasma. Bio-AuNPs consist of gold nanoparticles functionalized both with (1) streptavidin, to provide high affinity for the biotinylated secondary antibody used in the second step of the CEA sandwich assay, and with (2) bovine serum albumin, to minimize the nonspecific interaction of the bio-AuNPs with complex samples (blood plasma). We demonstrate that this approach makes it possible for the SPR biosensor to detect CEA in blood plasma at concentrations as low as 0.1?ng/mL, well below normal physiological levels (approximately nanograms per milliliter). Moreover, the limit of detection achieved using this approach is better by a factor of more than 1,000 than limits of detection reported so far for CEA in blood plasma using SPR biosensors.     

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.     

A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome

A surface plasmon resonance (SPR)-based biosensor was developed for simple diagnosis of severe acute respiratory syndrome (SARS) using a protein created by genetically fusing gold binding polypeptides (GBPs) to a SARS coronaviral surface antigen (SCVme). The GBP domain of the fusion protein serves as an anchoring component onto the gold surface, exploiting the gold binding affinity of the domain, whereas the SCVme domain is a recognition element for anti-SCVme antibody, the target analyte in this study. SPR analysis indicated the fusion protein simply and strongly self-immobilized onto the gold surface, through GBP, without surface chemical modification, offering a stable and specific sensing platform for anti-SCVme detection. AFM and SPR imaging analyses demonstrated that anti-SCVme specifically bound to the fusion protein immobilized onto the gold-micropatterned chip, implying that appropriate orientation of bound fusion protein by GBP resulted in optimal exposure of the SCVme domain to the assay solution, resulting in efficient capture of anti-SCVme antibody. The best packing density of the fusion protein onto the SPR chip was achieved at the concentration of 10 μg mL⁻¹; this density showed the highest detection response (906 RU) for anti-SCVme. The fusion protein-coated SPR chip at the best packing density had a lower limit of detection of 200 ng mL⁻¹ anti-SCVme within 10 min and also allowed selective detection of anti-SCVme with significantly low responses for non-specific mouse IgG at all tested concentrations. The fusion protein provides a simple and effective method for construction of SPR sensing platforms permitting sensitive and selective detection of anti-SCVme antibody.     

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 μg L⁻¹. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (1) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future.     

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.     

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.     

Real-time immunoassay of ferritin using surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used for the first time to determine the concentration of ferritin in both HBS-EP buffer and serum. The monoclonal antibody was immobilized on the carboxymethyl dextran-modified gold surface by an amine coupling method. The interaction of antibody with antigen was monitored in real-time. The signal was enhanced by sandwich amplification strategy to improve the sensitivity and specificity of the immunoassay, especially in serum. The linear range of the assay in serum is over 30-200 ng ml⁻¹ with the detection limit of 28 ng ml⁻¹. The sensitivity, specificity, and reproducibility of the assay are satisfactory. The analyte and enhancement antibody-binding surface could be regenerated by pH 2.0 glycine-HCl buffer and the same antibody-immobilized surface could be used for more than 50 cycles of ferritin binding and regeneration.     

A surface plasmon resonance immunosensor for detecting a dioxin precursor using a gold binding polypeptide

A surface plasmon resonance (SPR) based biosensor was developed for monitoring 2,4-dichlorophenol, a known dioxin precursor, using an indirect competitive immunoassay. The SPR sensor was fabricated by immobilizing a gold-thin layer on the surface of an SPR sensor chip with an anti-(2,4-dichlorophenol) antibody using a gold binding polypeptide (GBP) and protein G. The SPR response based on the antigen-antibody reaction in a flow system was measured by injecting a 2,4-dichlorophenol sample solution into the flow system in which the SPR sensor was located. In a direct immunoassay system using the modified sensor chip, no significant SPR angle shift less than 0.001° was observed when a 25 ppm of 2,4-dichlorophenol solution was injected. In order to improve the sensitivity of the SPR sensor, an indirect competitive immunoassay method was used in conjunction with the SPR sensor system using 2,4-dichlorophenol conjugated with bovine serum albumin (BSA). In the competitive assay, a 350 ppm 2,4-dichlorophenol-BSA conjugate solution containing 2,4-dichlorophenol at various concentrations (10-250 ppb) were injected into the SPR sensor system. The sensitivity of this indirect immunoassay was found to be extremely sensitive, compared to the direct one, and a detection limit of 20 ppb was estimated. Verification that the use of GBP for immobilizing the antibody on the sensor chip enhanced the sensitivity to 2,4-dichlorophenol was obtained by comparing the procedure with another modification, in which BSA was used instead of GBP for immobilizing the antibody on the sensor chip. The affinity constant of 2,4-dichlorophenol and its conjugate to the antibody were estimated form the SPR response.     

Label-free detection of DNA mutations by SPR: application to the early detection of inherited breast cancer

A screening analysis of DNA hybridization and the presence of DNA mutations using an surface plasmon resonance (SPR) biosensor is shown. The influence of lateral and vertical spacers, as well as several hybridization conditions, was studied to optimize the differentiation between fully complementary and mismatched DNA strands. Our results demonstrated that SPR biosensors were able to detect mismatch sequences related to inherited breast cancer, with high specificity and sensitivity. Using PCR synthetic sequences as targets, mutant sequences were clearly discriminated from fully complementary ones, and detection limits below 50 nM were achieved.     

Imaging Array SPR Biosensor Immunoassays for Sulfamethoxazole and Sulfamethazine

A homemade array surface plasmon resonance (SPR)-based imaging biosensor was used to develop sensitive and fast immunoassays to determine sulfamethoxazole (SMOZ) and sulfamethazine (SMT) in buffer. Two conjugations of sulfonamide-bovine serum albumin (BSA) were separately immobilized on two different rows of the array chip with one row as reference. The immobilization was carried out in the instrument to monitor the quantity of the conjugations immobilized. The antibody mixed with the sulfonamide in the buffer was injected over the surface of the chip to get a relative response which was inversely proportional to the concentration of the sulfonamide in the PBS buffer. Two calibration curves were constructed and the limit of detection for sufamethoxazole in buffer was 3.5 ng/mL and for sulfamethazine 0.6 ng/mL. The stability and specificity of the antibody were also studied. The monoclonal antibody did not bind with BSA.     

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.     

A Au nanoparticle and polydopamine co-modified biosensor: A strategy for in situ and label-free surface plasmon resonance immunoassays

This article reports a surface plasmon resonance (SPR) strategy capable of label-free yet amplified in situ immunoassays for sensitive and specific detection of human IgG (hIgG), a serum marker that is important for the diagnosis of certain diseases. Primarily, a wavelength-modulated Kretschman configuration SPR analyzer was constructed, and Au film SPR biosensor chips were fabricated. Specifically, based on Au nanoparticles (AuNPs) adsorbed on the surface of the Au film, the AuNP/Au film was coated with polydopamine (PDA) to fix streptavidin (SA), and then the biotinylated antibodies were connected to the surface of the biosensor chip. The SPR analyzer was utilized for in situ real-time monitoring of hIgG. Due to the immunological recognition between the receptor and target, the surface plasmon waves produced by the attenuated total reflection were affected by the changes in the surface of the biosensor chip. The resonance wavelength (λ_R) of the output spectra gradually redshifted, and the redshift degrees were directly related to the target concentration. The biosensor can realize the in situ detection of hIgG, displaying satisfactory sensitivity, excellent specificity and stability. Briefly, by monitoring the shift in λ_R after specific binding, a new SPR immunoassay can be customized for label-free, in situ and amplified hIgG detection. The operating principle of this research could be extended as a common protocol for many other targets of interest.     

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。