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.     

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。     

Determination of sulfamethazine in milk by biosensor immunoassay

A biosensor based on surface plasmon resonance (SPR) measurement was developed for use in an immunoassay for detection of sulfamethazine (SMZ) in milk. The biospecific surface was a carboxymethyl dextran-modified gold-surface sensor chip to which SMZ was covalently bound. The assay was based on inhibition of the binding of polyclonal antibodies to immobilized SMZ by SMZ in the sample. The SPR response changed inversely in relation to the antibiotic concentration in the sample. Calibration curves were constructed for SMZ in buffer and in milk at a concentration which included the maximum residue limit (0 to 200 micrograms/kg). The analysis time per sample varied from 8 to 30 min. Different flow rates and antibodies were modified alternatively during the study to assess their influence on the performance of the assay. The active antibody concentration was calculated at approximately 1880 and 180 nM for the antibody anti-SMZ 1 and the antibody anti-SMZ 2, respectively. No cross-reactivity of antibodies with other antibiotics was found. Under optimal conditions, the detection limits in milk for SMZ were 8 and 1.7 micrograms/kg, respectively, for antibody 1 and antibody 2, at a flow rate of 20 microL/min.     

Comparative study of random and oriented antibody immobilization as measured by dual polarization interferometry and surface plasmon resonance spectroscopy

Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL.     

Surface plasmon resonance sensor chips for the recognition of bovine serum albumin via electropolymerized molecularly imprinted polymers

In this paper,a surface plasmon resonance(SPR)sensor chip for detection of bovine serum album(BSA)was prepared by electropolymerization of 3-aminophenylboronic acid(3-APBA)based on molecularly imprinted polymer(MIP)technique.The surface morphology of MIP and non-imprinted(NIP)flms were characterized by scanning electroscopy(SEM).SEM images exhibited nanoscale cavities formed on the MIP films surface homogeneously due to the removal of BSA templates.The effects of pH,ion strength of rebinding BSA,the specific binding and selective recognition were studied for MIP films.Results indicated that the BSA-imprinted films exhibited a good adsorption of template protein(0.02–0.8 mg/mL)in0.05 mol/L sodium phosphate buffer at pH 5.0 with the limit of detection(LOD)of 0.02 mg/mL.     

Use of polyclonal antibodies to ochratoxin A with a quartz–crystal microbalance for developing real-time mycotoxin piezoelectric immunosensors

A piezoelectric immunosensor was tested for ochratoxin A (OTA) mycotoxin detection through the immobilization of OTA–bovine serum albumin (OTA–BSA) conjugate on gold-coated quartz crystals (AT-cut/5 MHz). Immunoassays were performed in a flow-injection system through frequency decreases in a quartz–crystal microbalance (QCM) because of a mass increasing during immunoreaction with anti-OTA antibodies. Three immobilization procedures for OTA–BSA (direct adsorption and covalent attachment to two alkane thiol self-assembled monolayers) were characterized with QCM in real time. Covalent attachment of the OTA–BSA conjugates through gold nanoparticles was also tested for amplifying the signal. Binding of the excess of antibodies to the immobilized OTA in an indirect competitive analysis decreased linearly the resonant frequency in the range of the OTA concentration from 10 to 128 ng/mL, with a detection limit of 8 ng/mL (signal/noise ratio of 3). A pepsin 2 mg/mL (pH = 2.1) solution was used to release antigen–antibody complexes, regenerating the biorecognition surface.     

Amperometric Immunosensor Based on L-Cysteine/Gold Colloidal Nanoparticles for Carbofuran Detection

In this study, anti-carbofuran monoclonal antibodies (Ab) were immobilized onto a gold electrode surface modified with multilayers of L-cysteine and gold colloidal nanoparticles (GNPs). Furthermore, horseradish peroxidase (HRP) as enzyme membrane was used for blocking unspecific sites and amplifying signal. The conformational properties of the immunosensor were characterized using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The concentration of antibody solution, pH of working buffer and incubation time were studied in detail for optimization of analytical performance. Under optimal conditions, the variation of current response was proportional to the concentration of carbofuran which ranged from 0.01 ng/mL to 50 ng/mL with a correlation coefficient of 0.9912. The detection limit was 0.01 ng/mL (S/N = 3). The proposed immunosensor exhibited good reproducibility and stability and it can be used for the rapid detection of carbofuran pesticide.     

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.     

Biosensor assay of sulfadiazine and sulfamethazine residues in pork

Biosensor-based immunochemical screening assays for the detection of sulfadiazine (SDZ) and sulfamethazine (SMT) in muscle extract from pigs were developed. Samples were extracted with aqueous buffer and then centrifuged. This simple and straightforward preparation allowed up to 40 samples to be processed and analysed in 1 d. The limits of detection for the assays were found to be 5.6 ng g-1 for SDZ and 7.4 ng g-1 for SMT. These figures were well below the European and US legal limits for sulfonamides (100 ng g-1). The precision (RSD) between runs was < 8% and the recovery was between 91 and 98%. The validation proved the assays to be accurate and the analysis of routine field samples showed good correlation with an established TLC screening procedure. No false negative or positive results were obtained with blank and spiked samples. The influence of cross-reacting metabolites on immunoassays was demonstrated by testing incurred tissue samples, collected from sulfonamide treated pigs after only a short withdrawal period. The quantitative results obtained by biosensor analysis were a combination of parent sulfonamide plus N4-acetyl metabolite while the HPLC method used for confirmatory analysis detected only the parent sulfonamide. This gave rise to some false positive results and highlighted the need to use real samples in evaluating any assay thoroughly. False negative results were not obtained.     

Determination of sulfonamides in bovine milk with column-switching high performance liquid chromatography using surface imprinted silica with hydrophilic external layer as restricted access and selective extraction material

A novel restricted access-molecularly imprinted material (RA-MIP) with selectivity for sulfonamides was synthesized using initiator-transfer agent-terminator (iniferter) method, a "living"/controlled radical polymerization technique. The material was prepared by grafting two layers with different functions on the silica support. To perform a "grafting from" polymerization, iniferter was immobilized on the surface of silica. The internal sulfamethazine imprinted polymer and the external poly(glycidyl methacrylate) [poly(GMA)] were then grafted successively. The hydrophilic structures were formed on the external layer of the material by the hydrolysis of the linear poly(GMA) for protein removal. The result has shown that this restricted access-MIP grafted silica (RA-MIP-SG) not only has the selectivity for the template and its analog, but also has the ability of exclusion for bovine serum albumin (BSA). It indicated that the material possesses both properties of molecularly imprinted polymer (MIP) and restricted access material (RAM). Using RA-MIP-SG as pre-column, a column-switching HPLC method was established for the determination of sulfonamides in bovine milk. Direct sample injection was performed in the analysis, which provides a convenient analytical procedure. Good linearity in the range of 2-400 ng mL(-1) (R>0.998) has been obtained for seven sulfonamides in the study. The recoveries of all the analytes at three concentration levels are between 93% and 107% with the RSD<8.0%. The limits of quantification and limits of detection are less than or equal to 2.7 ng mL(-1) and 0.8 ng mL(-1) respectively. It demonstrated this RA-MIP-SG can be applied in sample extraction and clean-up for the determination of sulfonamides in bovine milk by direct injection and column-switching HPLC with good efficiency and reliability.     

固相萃取–高效液相色谱法测定地表水中4种磺胺类抗生素

建立固相萃取–高效液相色谱法测定地表水中磺胺嘧啶、磺胺二甲嘧啶、磺胺氯哒嗪、醋磺胺甲恶唑4种磺胺类抗生素。样品采用HLB柱进行萃取富集,流动相为甲醇–水(体积比为20∶80),流量为0.5 m L/min,用SPD检测器检测,检测波长为270 nm;采用外标法定量。4种磺胺类抗生素质量浓度在4~160 ng/L范围内与色谱峰面积的线性关系良好,相关系数不低于0.998 2,方法检出限为1.0~1.7 ng/L,样品加标回收率为75.2%~97.4%,测定结果的相对标准偏差为2.8%~6.1%(n=7)。该方法操作简便,灵敏度高,可用于地表水中磺胺类抗生素的检测。     

Amplified Immunoassay of Human IgG Using Real—time Biomolecular Interaction ANalysis （BIA） Technology

An automated biomolecular interaction analysis instrument(BIAcore)based on surface plasmon resonance(SPR)has been used to determine human immunoglobulin G(IgG) in real time.Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran-modified gold film surface.The samples of human IgG prepared in HBS buffer were poured over the immpbilized surface.The signal amplification antibody was applied to amplify the response signal.After each measurement,the surface was regenerated with 0.1mol/L H3PO4.The assay was rapid,requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding,antibody amplification and regeneration.The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L.The same antibody immobilized surface could be used for more than 110 cycles of binding,amplification and regeneration.The results demonstrate that the sensitivity,specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.     

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.     

基于表面等离子体共振技术用鸡蛋黄抗体 IgY测定人血清中转铁蛋白

采用表面等离子体共振(SPR)方法, 用鸡蛋黄抗体(IgY)取代传统免疫检测中哺乳动物抗体IgG作为识别分子偶联于CM5传感芯片上, 对人血清中的转铁蛋白进行了检测. 考察了IgY在传感芯片上的偶联条件及芯片的再生条件. 结果表明, 在pH=4.0, IgY浓度为100 μg/mL, 流速为5 μL/min的最佳偶联条件下, SPR响应信号和转铁蛋白浓度在50~500 ng/mL范围内具有良好的线性关系, 检出限为39.56 ng/mL, 对人血清样品检测的日间变异系数<8%, 日内变异系数<5%, 平均回收率为86.22% ~94.51%.     

improvement of the Specificity of Surface Plasmon Resonance with BSA-modified Chip

Recently, surface plasmon resonance (SPR) become more and more popular without the need of the label technology1-3. However, sometimes, a number of experimental artifacts complicate the final biosensor analysis4-7. The utilization of a reference surface c…     

Surface Plasmon Resonance Immunoassay for Ochratoxin A Based on Nanogold Hollow Balls with Dendritic Surface

Abstract

A surface plasmon resonance (SPR)‐immunosensor based on nano‐size gold hollow ball (GHB) with dendritic surface has been developed for detection of Ochratoxin A (OTA). A thionine thin film was initially electropolymerized onto the SPR‐probe surface, and then anti‐OTA monoclonal antibody (anti‐OTA) was immobilized onto the SPR‐probe surface by means of GHB conjugation. The binding of target molecules onto the immobilized antibodies causes an increase in the resonant angle of the sensor chip, and the resonant angle shift was proportional to the OTA concentration in the range of 0.05–7.5 ng/ml with a detection limit of 0.01 ng/ml at a signal/noise ration of 3. A glycine‐HCl solution (pH 2.8) was used to release antigen‐antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of OTA into three milk samples was assayed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable. Compared with the conventional enzyme‐linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Sensitive electrochemical immunosensor array for the simultaneous detection of multiple tumor markers

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.     

Determination of sulfonamide residues in eggs by liquid chromatography

A method was developed for determining residual sulfonamide antibacterials such as sulfamethazine (SMZ), sulfamonomethoxine (SMM), sulfadimethoxine (SDM), and sulfaquinoxaline (SQ) in eggs using liquid chromatography with a photodiode array detector. The spiked and blank samples were cleaned up by using an Ultrafree-MC/PL centrifugal ultrafiltration unit. A Mightysil RP-4 GP column and a mobile phase of 28% (v/v) ethanol-H2O with a photodiode array detector were used for the determination. Average recoveries from eggs spiked with each drug at 0.1, 0.2, 0.4, and 1.0 ppm were > or = 80.9%, with relative standard deviations between 1.3 and 4.7%. The limits of quantitation were 0.060 ppm for SMZ, 0.045 for SMM, 0.044 for SDM, and 0.093 for SQ. The analysis of one sample required < 30 min and < 5 mL ethanol as solvent.     

Surface plasmon resonance immunosensor for IgE analysis using two types of anti-IgE antibodies with different active recognition sites.

A simple and novel method for the determination of an IgE antibody based on a surface plasmon resonance immunosensor for the diagnosis of an allergy is described. The method involves the use of an anti-IgE(D) antibody and an anti-IgE(H) antibody, which reacts with the Ce2 domain and the Ce3 domain of the IgE antibody. The anti-IgE(D) antibody was immobilized on the gold surface of a sensor chip by physical adsorption. An IgE antibody sample was incubated by adding it to an anti-IgE(H) antibody solution to form an anti-IgE(H) immunocomplex through a reaction of the Ce3 domain of the IgE antibody. The incubated solution was introduced onto the sensor chip and the immunocomplex of the IgE-anti-IgE(H) then reacted with the anti-IgE(D) antibody immobilized on the sensor chip through the Ce2 domain of the IgE antibody part of the IgE-anti-IgE(H) immunocomplex. The detection limit of the present method for the determination of the IgE antibody was about 10 ppb. The affinity constants for the anti-IgE(H) antibody immunocomplex with the IgE antibody in solution and that of the anti-IgE(H) antibody immunocomplex with the IgE antibody immobilized on the sensor chip by a biotin-streptavidin interaction were estimated to be 4.1 x 10(7) M(-1) and 5.8 x 10(6) M(-1), respectively. The affinity constant for the immunocomplex of the anti-IgE(H) antibody with the IgE antibody with the anti-IgE(D) immobilized on the sensor chip was estimated to be 4.9 x 10(7) M(-1), 20-times larger than the affinity constant for the IgE antibody immunocomplex with the anti-IgE(D) antibody immobilized on the sensor chip, based on a direct immunoassay method of the IgE antibody under the same experimental conditions.     

Development and molecular recognition of Calixcrownchip as an electrochemical ALT immunosensor

The developments of immunosensors with a variety of formats are increasingly finding applications in clinical diagnostics and biological researches. A strategy for the immunoassay and preparation of Calixcrownchips is proposed. This strategy is based on the immobilization of antigens or antibodies on the surface of Calixcrown and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody or antigen, with its activity determined by using tetramethylbenzidine (TMB) as an electrochemical substrate. The present study includes general considerations of the competitive immunoreaction protocols. Alanine aminotransferase (ALT) monoclonal antibody (anti-ALT-mAb) was successfully immobilized on thiol derivative of Calixcrown fixed to a gold surface. ALT antigen was detected by competitive immunoreactions based on microarrays of anti-ALT-mAb or antigen immobilized on the surface of the Calixcrownchip. For the anti-ALT-mAb immobilized microarray the dynamic range is 0.05 ng/mL–10 μg/mL, the detection limit is 0.05 ng/mL and the sensitivity is 10 nA/(ng/mL) respectively. The Calixcrownchip immunosensor microarray provided much better technical performance than a comparable enzyme sensor with immobilized-anti-ALT-mAb. To investigate the complexation site, the structures of the complexes formed between the crown-5-ether moiety of Calix[4]arene and protonated Arginine and Lysine were determined by minimizing the complex formation energies. The complex stability depends on the number of amine groups in the alkyl chain of the amino acid and also the number of methylene groups between the amine groups of the amino acid.