Detection of bisphenol A using a novel surface plasmon resonance biosensor

We present a compact surface plasmon resonance (SPR) biosensor for the detection of bisphenol A (BpA), an endocrine-disrupting chemical. The biosensor is based on an SPR sensor platform (SPRCD) and the binding inhibition detection format. The detection of BpA in PBS and wastewater was performed at concentrations ranging from 0.05 to 1,000 ng/ml. The limit of detection for BpA in PBS and wastewater was estimated to be 0.08 and 0.14 ng/ml, respectively. It was also demonstrated that the biosensor can be regenerated for repeated use. Results achieved with the SPR biosensor are compared with those obtained using ELISA and HPLC methods.     

Electrochemical impedance spectroscopy and surface plasmon resonance studies of DNA hybridization on gold/SiOx interfaces

The use of Au/SiO(x) interfaces for the investigation of DNA hybridization using electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) simultaneously is demonstrated. Standard glass chemistry was used to link single-stranded DNA (ss-DNA) on aldehyde-terminated Au/SiO(x) interfaces. The layer thickness and amount of grafted oligonucleotides (ODNs) were calculated from SPR on the basis of a multilayer system of glass/Ti/Au/SiO(x)/grafted molecule. Capacitance and resistance values of the modified interface before and after hybridization were calculated from EIS data using an equivalent circuit and allowed the affinity rate constant, K(A) = 4.07 x 10(5) M(-1), to be determined. The EIS results were comparable to those obtained by SPR hybridization kinetics recorded in parallel.     

Thin chitosan films as a platform for SPR sensing of ferric ions

Homogeneous chitosan films of various thicknesses (10-65 nm) were deposited on thin gold films through spin coating. The resulting interfaces were characterized using surface plasmon resonance (SPR), AFM, profilometry and cyclic voltammetry. The strong chelating properties of chitosan films to Fe(3+) were investigated using cyclic voltammetry. Through SPR measurements, an affinity constant between chitosan and Fe(3+) of 9.49 x 10(5) M(-1) was determined with a detection limit as low as 250 ppb.     

The effect of low pH on the glycitein-BSA conjugate interaction with specific antiserum: competitive inhibition study using surface plasmon resonance technique

Competitive inhibition serological assay for detection of the phytoestrogen glycitein (Glyc) was developed using surface plasmon resonance (SPR) technique with protein conjugates and polyclonal antibodies initially designed for the enzyme-linked immunosorbent assays (ELISA). The efficiency of the approach to the quantification of the soy isoflavone glycitein in water was investigated using the competitive reaction of analyte (free Glyc)and immobilized Glyc-BSA-conjugate with polyclonal antibodies. It was shown that the efficiency to detect Glyc drastically depends on the pH level of the probe solution. With the decrease in pH from 7.4 to 4.0, (i) the affinity of the specific reaction increases and (ii) the level of unspecific sorption becomes saturated. Non-specific adsorption to a SPR sensor surface obscures the specific component and shaded specific response at higher pH (6.0-7.4) when used serum for the quantification of specific analytes. The standard curves obtained in acidic solutions (pH 4-5) indicate that the linear part of the dependence completely covers the range between detection limit (0.1 μg/ml) and Glyc solubility in water (0.9 μg/ml). The difference in SPR- and ELISA-based analytical protocols as well as the requirements for increasing the efficiency in quantitative SPR analysis using purified antibodies is discussed.     

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.     

Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin

A dual ligand (DL) system that combines high affinity streptavidin-biotin binding with lower affinity fibronectin-integrin ligand binding was developed to augment endothelial cell adhesion to polymers. In this study, we examined the utility of biotinylated fibronectin (bFN) as an enhancement to the previously developed DL approach. The goal was to make the system more amenable to clinical studies by eliminating xenogenic bovine serum albumin (bBSA). Fibronectin (FN) biotinylation was achieved with Sulfo-NHS-LC-Biotin. The affinity of conjugated biotin for wild-type streptavidin (WT-SA) and a mutant strain streptavidin (RGD-SA) was measured using surface plasmon resonance (SPR) spectroscopy. Enzyme-Linked ImmunoSorbent Assay (ELISA) absorbance values confirmed the accessibility of the cell binding domain on mildly biotinylated bFN when compared to unmodified native protein. SPR binding analysis confirmed similar binding behavior to bFN with WT-SA and RGD-SA. Kinetic analysis, however, showed no increase in affinity due to increased biotins per FN, an indication of the absence of positive cooperativity in the system. We verified the essential utility of bFN in affinity binding by SPR and confirmed the potential for integrin-FN linkages by ELISA. Finally, Vinculin immunostaining was used to determine focal adhesion formation using bFN in the DL system. Significantly greater focal adhesion density was achieved with the bFN in the DL system than with FN alone.     

Rational design of affinity peptide ligand by flexible docking simulation

Rational design of affinity peptide ligands of proteins by flexible docking simulation is performed using the SYBYL program package. This approach involves the use of experimental data to verify a scoring function that can be used to assess the affinity of a peptide for its target protein. The enzyme-linked immunosorbent assay (ELISA) data of several peptides displayed on phage surfaces for insulin and lysozyme, respectively, reported in literature are used for the purpose. It is found that the absolute values of the Dscore calculated from the docking correspond well to the ELISA data that relate to the affinity between the peptides and the target molecule. So, the Dscore function is used to assess the affinity of docked peptides in a pentapeptide library designed on the basis of protein (alpha-amylase) structure. As a result, a pentapeptide with a high Dscore value is selected and a hexapeptide (FHENWS) is built by linking serine to its C-terminal to lengthen the peptide. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions (including hydrogen bonds) and Van der Waals forces contribute to the affinity of the hexapeptide for alpha-amylase. Chromatographic experiments with the immobilized peptide have given further evidence for this observation. Adsorption isotherm described by the Langmuir equation indicates that the apparent binding constant of alpha-amylase to the immobilized hexapeptide was 2.5x10(5)L/mol. Finally, high affinity and specificity of the affinity adsorbent is exemplified by the purification of alpha-amylase from crude fermentation broth of Bacillus subtilis.     

Preparation of Anti—Cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

IntroductionIn fast and slow skeletal and cardiac muscles,troponin I,the inhibitory protein of the troponin-tropomyosin complex,exists in three isotype formsencoded by three separated genes.The amino acidsequences of the two skeletal and one cardiac Tn Iforms( s Tn I and c Tn I,respectively) exhibit40 %dissimilarity[1] .Moreover,human cardiac Tn I has31 additional residues on the N - terminal end,which do not exist in skeletal forms,thus it pro-vides a high potential for obtaining cardiac-…     

An on-membrane quantitative analysis system for glycyrrhizin in licorice roots and traditional Chinese medicines

An on-membrane quantitative analysis system has been developed for determining glycyrrhizin (GC) in licorice roots and traditional Chinese medicines. A GC standard and the extracts of licorice roots and traditional Chinese medicines were applied to a polyethersulfone (PES) membrane and were developed by acetonitrile/water/formic acid (45:55:2, by volume), then treated with a NaIO₄ solution followed by bovine serum albumin (BSA), resulting in a GC-BSA conjugate on a PES membrane. Anti-GC monoclonal antibody was bound and then a second antibody labeled with peroxidase directed against the first antibody. Finally a substrate reacted with the enzyme and gave staining. The stained membrane was scanned and coloring spots were analyzed quantitatively using graphic analysis by NIH Image software, indicating at least 0.5 μg of GC was clearly detectable. GC can be analyzed quantitatively between 1.0 and 8.0 μg.     

Arginine-modified DNA aptamers that show enantioselective recognition of the dicarboxylic acid moiety of glutamic acid

We have screened glutamic acid-binding aptamers from a modified DNA pool containing arginine residues using the method of systematic evolution of ligands by exponential enrichment (SELEX). Thirty-one modified DNA molecules were obtained from the enriched pool after the 17th round of selection, and their binding affinities for the target were evaluated by binding assays using affinity gels. Three modified DNA molecules having higher affinity were sequenced and we determined their affinity and specificity for the target by surface plasmon resonance (SPR) measurements. The SPR studies indicated that two of these three aptamers distinguished the dicarboxylic acid moiety of the D-isomer from that of the L-isomer; however, the third aptamer did not show enantioselectivity.     

Determination of stability constants of complexes of neutral analytes with charged cyclodextrins by affinity capillary electrophoresis

A novel procedure for the determination of stability constants in systems with neutral analytes and charged complexation agents by affinity capillary electrophoresis was established. This procedure involves all necessary corrections to achieve precise and reliable data. Temperature, ionic strength, and viscosity corrections were applied. Based on the conductivity measurements, the average temperature of the background electrolyte in the capillary was kept at the constant value of 25°C by decreasing the temperature of the cooling medium. The viscosity correction was performed using the viscosity ratio determined by an external viscosimeter. The electrophoretical measurements were performed, at first, at constant ionic strength. In this case, the increase of ionic strength caused by increasing complexation agent concentration was compensated by changing of the running buffer concentration. Subsequently the dependence of the analyte effective mobility on the complexation agent concentration was measured without the ionic strength compensation (at variable ionic strength). The new procedure for determination of the stability constants even from such data was established. These stability constants are in a very good agreement with those obtained at the constant ionic strength. The established procedure was applied for determination of the thermodynamic stability constants of (R, R)-(+)- and (S, S)-(-)-hydrobenzoin and R- and S-(3-bromo-2-methylpropan-1-ol) complexing with 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin hydrochloride.     

Characterisation of antibodies and analytes by surface plasmon resonance for the optimisation of a competitive immunoassay based on energy transfer

The determination of binding constants using surface plasmon resonance (SPR) was introduced to optimise a competitive homogeneous fluorescence energy-transfer immunoassay (ETIA) before labelling. Steroids were chosen as model for the detection of three analytes estrone, estradiol and ethinylestradiol--by taking three polyclonal antibodies (anti estrone-, anti estradiol- and anti estrogen-antibodies) and the corresponding analyte derivatives used for the immunisation. The active concentration of the antibodies was determined before and after labelling. Inhibition curves were recorded using SPR for all possible combinations of analyte, antibody, and analyte derivatives. The experiments revealed that the active antibody concentration can be reduced to 30% whereas the antibody affinity is not affected by the labelling process. Limits of the use of SPR for determination of affinity constants in solution are discussed. All possible ETIA calibration for the quantification of estrone and estradiol was performed. The lower limits of detection for estrone (0.06 microg L(-1)) and estradiol (0.17 microg L(-1)) were reached with the anti-estrogen IgG and its derivative     

Analysis of immunoarrays using a gold grating-based dual mode surface plasmon-coupled emission (SPCE) sensor chip

We have developed a novel dual mode immunoassay platform that combines the advantages of real-time, label free measurement of surface plasmon resonance (SPR) and the highly directional surface plasmon-coupled emission (SPCE) using a gold grating-based sensor chip. Since only fluorophore-labeled analyte molecules that are close to the metal surface of the sensor chip will couple to the surface plasmon, SPCE detection is highly surface-specific leading to background suppression and increased sensitivity. Theoretical calculations were done to find SPR and SPCE angles for a sensor chip optimized for Alexa Fluor 647. We have confirmed the SPR and SPCE responses on the dual mode sensor chip using Alexa Fluor 647 labeled anti-mouse IgG. Signal fluctuation of the dual mode sensor chip reader was below 1.2% and 0.8% for SPR and SPCE, respectively. The SPR response in this configuration showed a minimum detection level of 1 μg ml(-1), and the SPCE response showed a minimum detection level of 1 ng ml(-1) for the same sample. A range of human IgG concentrations in human serum was also analyzed with the dual mode sensor chip. The SPCE measurement is more sensitive than the SPR real-time measurement, and substantially extends the dynamic range of the assay platform, as well as enabling independent measurements of co-localized analytes on the same sensor chip region of interest. Since this assay platform is capable of measuring more than 1000 spatially encoded regions of interest on a 1 cm(2) sensor chip, it has the potential for high-content analyses of biological samples with both research and clinical applications.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法,表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息.同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境;二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光,使其构象发生变化.荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息,揭示KA的存在使HSA的流动性和微粘度发生变化.定量求得不同温度下(298、308和318 K)考拉维酸与HSA作用的键合参数和热力学参数.分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内,并与赖氨酸Lys195和天冬氨酸Asp451形成三个氢键,与HSA的键合模式主要是疏水作用;位点竞争实验证明考拉维酸在HSA亚结构域的位点II位发生作用.另外,获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制.结果表明,HSA对考拉维酸有较强的结合能力,提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

A new electrochemical enzyme-linked immunosorbent assay for the screening of macrolide antibiotic residues in bovine meat.

A new sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed, using the mouse monoclonal antibodies anti-erythromycin and anti-tylosin. The competitive indirect assay was performed using an erythromycin (or tylosin)-BSA conjugate as a coating molecule; after competition between free and coated analytes for the antibodies, the activity of the horseradish peroxidase-labelled antiglobulins was measured electrochemically using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. The detection limit of the assay was 0.4 ng ml(-1) for erythromycin and 4.0 ng ml(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.4 ng ml(-1) for erythromycin and 13.0 ng ml(-1) for tylosin. The specificity of the assay was assessed by studying the cross-reactivity of various macrolides other than erythromycin and tylosin. The results indicate that the monoclonal antibodies anti-erythromycin and anti-tylosin can readily distinguish the target compound from other macrolides, with the exception of roxithromycin, a semisynthetic macrolide antibiotic derived from erythromycin. Fortified and real samples were analysed by the developed ELISA method and results confirmed by micro-LC-MS-MS using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest. The ELISA assay showed precision (RSD) values ranging from 6.3 to 11.4% for erythromycin and from 7.5 to 12.6% for tylosin; the accuracy (relative error, RE) ranged from -16.0 to -9.8% and from -9.5 to 8.0% for erythromycin and tylosin, respectively. All results obtained demonstrate that the electrochemical ELISA is a suitable method for a sensitive, simple, rapid and reliable screening of the two macrolides in animal tissues.     

Determination of Fucose Concentration in a Lectin-Based Displacement Microfluidic Assay

We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.

     

Intricate Recognition of Glycolipid-Like Compounds by HIV-1 Envelope Proteins Evaluated with Surface Plasmon Resonance Imaging

Fusion of human immunodeficiency virus (HIV) to the cell membrane occurs by the specific binding of an envelope protein of HIV-1 (gp120 and gp160) and a glycosphingolipid of the cell membrane. In this study, quantitative and array-based affinity evaluation of gp120 and gp160 was performed by surface plasmon resonance (SPR) and the SPR imaging technique using a substrate immobilized with glycolipid-like compounds (Gb3, GM3, and Lac). Quantitative affinity evaluation showed that gp160 specifically bound to Gb3 and Lac compared with GM3, whereas gp120 showed lower binding affinity and specificity. Array-based evaluation showed that gp160 binds to Gb3 more favorably than Lac and GM3.     

Comparison of Electrochemical and Surface Plasmon Resonance Immunosensor Responses on Single Thin Film

This paper reports results obtained when comparing an electrochemical enzyme immunosensor and a surface plasmon resonance (SPR) based immunosensor on the same gold surface installed in an electrochemical SPR flow cell. Simultaneous electrochemical and SPR measurements were performed on a gold surface modified with multilayers of poly‐L ‐lysine and poly‐styrenesulfonate assembled with the layer‐by‐layer method. First, we obtained the SPR response induced by the formation of an immunocomplex from the shift in the SPR angle by injecting an anti tumor necrosis factor‐α antibody solution labeled with alkaline phosphatase into the flow cell containing the multilayer modified with tumor necrosis factor‐α. Then we compared this SPR result with that obtained for the electrochemical oxidation current of p‐aminophenol catalyzed by alkaline phosphatase from p‐aminophenolphosphate on the same gold film. We compared the two immunosensor responses obtained using the different measurement principles and found that there was a high correlation efficient of 0.973 between them. This was because we were able to immobilize the immunoreagents with good stability and without losing the transport of the enzyme product in the multilayer whose thickness we easily controlled with nanometer scale accuracy. We also report that the detection limit of our electrochemical immunosensor after optimization was around 100 pg/mL (0.4 pM), which is one of the lowest values yet reported for an electrochemical immunosensor.     

Evaluation of a commercial immunoassay for the detection of chlorfenapyr in agricultural samples by comparison with gas chromatography and mass spectrometric detection

A commercially available enzyme-linked immunosorbent assay (ELISA) kit with a high affinity monoclonal antibody was applied to residual analysis of insecticide chlorfenapyr in agricultural samples, and drawn a parallel between the ELISA and gas chromatography (GC) with mass spectrometry (MS). For standards prepared in water containing 5% (v/v) methanol, the sensitivity (I50 value), the dynamic range, and the limit of detection of the ELISA kit were 2.3, 1 - 10, and 0.1 ng/g, respectively. The used monoclonal antibody in the ELISA kit had a high selectivity. The ELISA kit was applied to the determination of chlorfenapyr in two kinds of fruits (apple and peach). The examination of the influence of these matrices on the reliability of the assay performance indicated that the ELISA could determine it in these samples near the regulation values in Japan simply by diluting the methanolic extract or by concentrating it, without any clean-up procedures. Recovery and precision of the proposed ELISA method were assessed by fortifying fruit samples with chlorfenapyr ranging from 0.05 to 1.5 microg/g. Mean recoveries were 94.2 and 90.3% for apple and peach, and coefficients of variation were below 16% in most cases. The results obtained from the proposed ELISA method correlate well the reference GC/MS method for both fruit samples (r > 0.98). These considerations make the ELISA kit very useful analytical tool for monitoring and regulatory programs, without the need of complex and expensive instrumentation.     

Integration of polyaniline/poly(acrylic acid) films and redox enzymes on electrode supports: an in situ electrochemical/surface plasmon resonance study of the bioelectrocatalyzed oxidation of glucose or lactate in the integrated bioelectrocatalytic systems

Electropolymerization of aniline in the presence of poly(acrylic acid) on Au electrodes yields a polyaniline/poly(acrylic acid) composite film, exhibiting reversible redox functions in aqueous solutions at pH = 7.0. In situ electrochemical-SPR measurements are used to identify the dynamics of swelling and shrinking of the polymer film upon the oxidation of the polyaniline (PAn) to its oxidized state (PAn(2+)) and the reduction of the oxidized polymer (PAn(2+)) back to its reduced state (PAn), respectively. Covalent attachment of N(6)-(2-aminoethyl)-flavin adenin dinucleotide (amino-FAD, 1) to the carboxylic groups of the composite polyaniline/poly(acrylic acid) film followed by the reconstitution of apoglucose oxidase on the functional polymer yields an electrically contacted glucose oxidase of unprecedented electrical communication efficiency with the electrode: electron-transfer turnover rate approximately 1000 s(-1) at 30 degrees C. In situ electrochemical-SPR analyses are used to characterize the bioelectrocatalytic functions of the biomaterial-polymer interface. The current responses of the bioelectrocatalytic system increase as the glucose concentrations are elevated. Similarly, the SPR spectra of the system are controlled by the concentration of glucose. The glucose concentration controls the steady-state concentration ratio of PAn/PAn(2+) in the film composition. Therefore, the SPR spectrum of the film measured upon its electrochemical oxidation is shifted from the spectrum typical for the oxidized PAn(2+) at low glucose concentration to the spectrum characteristic of the reduced PAn at high glucose concentration. Similarly, the polyaniline/poly(acrylic acid) film acts as an electrocatalyst for the oxidation of NADH. Accordingly, an integrated bioelectrocatalytic assembly was constructed on the electrode by the covalent attachment of N(6)-(2-aminoethyl)-beta-nicotinamide adenine dinucleotide (amino-NAD(+), 2) to the polymer film, and the two-dimensional cross-linking of an affinity complex formed between lactate dehydrogenase and the NAD(+)-cofactor units associated with the polymer using glutaric dialdehyde as a cross-linker. In situ electrochemical-SPR measurements are used to characterize the bioelectrocatalytic functions of the system. The amperometric responses of the system increase as the concentrations of lactate are elevated, and an electron-transfer turnover rate of 350 s(-1) between the biocatalyst and the electrode is estimated. As the PAn(2+) oxidizes the NADH units generated by the biocatalyzed oxidation of lactate, the PAn/PAn(2+) steady-state ratio in the film is controlled by the concentration of lactate. Accordingly, the SPR spectrum measured upon electrochemical oxidation of the film is similar to the spectrum of PAn(2+) at low lactate concentration, whereas the SPR spectrum resembles that of PAn at high concentrations of lactate.