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1.
A surface plasmon resonance (SPR)-immunosensor for detection of benzo[a]pyrene (BaP) is developed by using a model BaP-hapten compound, BaP-bovine serum albumin conjugate (BaP-BSA), and an anti-BaP-BSA monoclonal antibody. BaP-BSA conjugate is immobilized on a gold thin-film sensor chip by means of simple physical adsorption. The number of BaP-hapten units in BaP-BSA conjugate is estimated to be 28 from the difference in molecular weight (MW) between BaP-BSA conjugate and BSA based on the results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurement. Anti-BaP-BSA antibody on contact with the BaP-BSA conjugate immobilized sensor chip causes an increase in the incident angle of the sensor chip. Binding of anti-BaP-BSA antibody with surface-immobilized BaP-BSA conjugate is inhibited by the presence of BaP in analyte solution, because of the inhibition effect of BaP. The SPR immunosensor for BaP functioning with the indirect competitive immunoreaction of anti-BaP-BSA antibody between the analyte (BaP) in testing solution and the BaP-BSA conjugate immobilized on the sensor chip provides a rapid determination (response time: ca. 15 min) of BaP in the concentration range of 0.01-1000 ppb. The antibody anchored to the sensor chip by antigen-antibody binding is removed on treatment with a pepsin solution (pH 2.0) for few minutes. The SPR sensor chip is found to be reusable for more than 20 times with a little decrease (<7%) in the sensor response. Detection of BaP by direct competitive immunoreactions is also carried out by enzyme-linked immunosorbent assay (ELISA). The concentration of BaP could be determined as low as 0.01 ppb and 2 ppb using the SPR sensor and the ELISA method, respectively. The SPR sensor is found to detect BaP selectively in the presence of 2-hydroxybiphenyl (HBP); the incident angle shift of the SPR sensor for BaP is found to be same irrespective to the presence or the absence of a same concentration (as much as 30 ppb) of HBP together.  相似文献   

2.
In this study, a direct detection system for herbicides inhibiting photosynthetic electron transfer was developed using the photosynthetic reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The heavy-subunit-histidine-tagged RCs (HHisRCs) were immobilized on an SPR sensor chip via nickel chelation chemistry as a binder for one of the triazine herbicides, atrazine. Immediately after injection of atrazine solution on the HHisRCs-immobilized chip, the SPR responses increased and reached plateaus within 1 min. The SPR signals were proportional to the sample concentrations of atrazine in the range 1-100 microg/ml. To evaluate the binding specificity to atrazine, chlorinated aromatic herbicides, DCMU and MCPP, were investigated using the HHisRCs-immobilized chip. An RC inhibitor, DCMU, could also be detected with a higher detection limit of 20 microg/ml than atrazine (1 microg/ml). MCPP showed no signals because its inhibition mechanism against plants is different from that of atrazine and DCMU. These results indicated that the sensor chip immobilized RCs could be used for the specific detection of photosynthetic inhibitors.  相似文献   

3.
Surface plasmon resonance (SPR) is routinely applied on determining association or dissociation constant rates of antigen-antibody complexes. In a SPR system such as Biacore, the capture method is a widely accepted procedure in kinetic analysis for association or dissociation of soluble antigen analytes with antibody ligands initially captured by anti-Fc molecules immobilized on the sensor chip. Appropriate preparations of anti-immunoglobulin G (IgG)-Fc molecules on sensor chips have not been examined yet for stable kinetic analysis of antibodies with several affinities to soluble antigens. Here, we constructed murine monoclonal antibodies (MoAbs) with various affinities to hen egg lysozyme (HEL) and performed kinetic analysis of these MoAbs captured by rat MoAbs against mouse IgG-Fc immobilized on the sensor chip. When capture molecules maximally immobilized on the sensor chip, we observed no apparent dissociation of MoAbs with extremely high affinity to soluble HEL antigens. In contrast, on the limited amount (1000-2000 response units) of capture molecule immobilized on the sensor chip, we could perform stable kinetic analysis of MoAbs with highest affinities to the antigen as well as those with lower or moderate binding affinities. Thus, in some cases, accurate kinetic analysis of high-affinity antibodies can be performed by minimization of capture molecule densities on the sensor chip in SPR.  相似文献   

4.
A SPR-based immunosensor for the detection of isoproturon   总被引:1,自引:0,他引:1  
The proof of principle of a reusable surface plasmon resonance (SPR)-based immunosensor for the monitoring of isoproturon (IPU), a selective and systemic herbicide, is presented. The detecting rat monoclonal anti-isoproturon antibody (mAb IOC 7E1) was reversibly immobilized through the use of a capture mouse anti-rat (kappa-chain) monoclonal antibody (mAb TIB 172), which was covalently immobilized on the sensor chip surface. Such strategy features a controlled binding of the captured detecting antibody as well as facilitates the surface regeneration. The capture of the anti-IPU mAb by the antibody (TIB 172) coated sensor surface could be carried out up to 120 times (immobilization/regeneration cycles) without any evidence of activity loss. With a high test midpoint and a low associated SPR signal, the direct detection format was shown to be unsuitable for the routine analysis of isoproturon. However, the limit of detection (LOD) could be easily enhanced by using a strategy based on a surface competition assay, which improved all immunosensor parameters. Moreover, the sensitivity and working range of the indirect format were found to be dependent on the surface density of the anti-IPU mAb IOC 7E1. As expected for competitive formats, the lowest surface coverage (0.5 ng/mm(2)) allowed a lower detection of the herbicide isoproturon with a calculated LOD of 0.1 microg/l, an IC(50) (50% inhibition) of 5.3+/-0.6 microg/l, and a working range (20-80% inhibition) of 1.3-16.3 microg/l.  相似文献   

5.
A fractal analysis is used to analyze the influence of: (a) electrostatic interactions on binding and dissociation rate coefficients for antibodies HH8, HH10, and HH26 in solution to hen egg-white lysozyme (HEL) immobilized on a sensor chip surface [Biophys. J. 83 (2002) 2946]; and (b) the binding and dissociation of recombinant Fab in solution to random NHS-coupled Cys-HEL and oriented thiol-coupled Cys-HEL immobilized on a sensor chip surface [Methods 20 (2000) 310]. Single- and dual-fractal models were employed to fit the data. Values of the binding and the dissociation rate coefficient(s) and the fractal dimensions were obtained from a regression analysis provided by Corel Quattro Pro 8.0 (Corel Corporation Limited, Ottawa, Canada. 1997). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. It is of interest to compare the results obtained by the fractal analysis with that of the original analysis [Biophys. J. 83 (2002) 2946]. For example, as one goes from the binding of 21 nM HH10/HEL to the binding of 640 nM HH10/HEL(K97A), Sinha et al. [Biophys. J. 83 (2002) 29461 indicate that the enhancement of diffusional encounter rates may be due to 'electrostatic steering' (a long-range interaction). Our analysis indicates that there is an increase in the value of the fractal dimension, Df1 by a factor of 1.12 from a value of 2.133-2.385. This increase in the degree of heterogeneity on the surface leads to an increase in the binding rate coefficient, k1 by a factor of 1.59 from 12.92 to 20.57. The fractal analysis of binding and dissociation of recombinant Fab in solution to random NHS-coupled Cys-HEL and oriented thiol-coupled Cys-HEL immobilized on a sensor chip [Methods 20 (2000) 310] surface are consistent with the degree of heterogeneity present on the sensor chip surface for the random and the oriented case. As expected, the random case will exhibit a higher degree of heterogeneity than the oriented case, leading to subsequently a higher binding rate coefficient.  相似文献   

6.
Researchers now recognize the utility of surface plasmon resonance technology to evaluate interactions of microbial pathogens with host components. The surface adhesin and candidate vaccine antigen P1 of Streptococcus mutans, the main causative agent of dental caries, interacts with a high molecular weight glycoprotein called salivary agglutinin, or gp340, in the salivary pellicle. We optimized a BIAcore assay to measure P1-mediated Ca(2+) dependent binding of S. mutans whole cells to this physiological ligand immobilized on a Pioneer F1 sensor chip. Regeneration conditions allowed cells to be eluted from the sensor chip permitting multiple reuse of the agglutinin-coated surface. An isogenic P1-deficient S. mutans mutant did not bind to immobilized agglutinin demonstrating specificity of the detected interaction. Glutaraldehyde-fixation of bacterial cells showed the assay measured a whole cell-ligand interaction and was not an artifact of solubilized or leached proteins. Adherence inhibition assays demonstrated varying degrees of disruption of the S. mutans-agglutinin interaction by anti-P1 monoclonal antibodies recognizing different epitopes, whereas a polyclonal reagent demonstrated more complete inhibition. This report describes an improved method to assess salivary agglutinin-mediated adherence of S. mutans in vitro under physiological-like conditions and to evaluate the effectiveness of antibodies of differing specificities to inhibit binding.  相似文献   

7.
A novel sensor chip for use in surface plasmon resonance (SPR) biosensors has been developed to capture vesicles which may contain membrane-bound receptors. Sulforhodamine-containing vesicles were shown by fluorescence microscopy to be immobilized intact on the sensor chip. Binding of cholera toxin to captured vesicles containing ganglioside GM(1) was demonstrated using SPR, and the derived kinetic and affinity constants were similar to literature values. Biotinylated vesicles captured on the sensor chip were used to bind streptavidin and then biotinylated ss-DNA. The hybridization of complementary ss-DNA to the immobilized ss-DNA was then analyzed using SPR. The values obtained were similar to those obtained for an identical interaction analyzed using a commercially available streptavidin-containing sensor chip. Binding of vancomycin-group antibiotics to captured vesicles containing a bacterial cell wall mucopeptide analogue was demonstrated. No binding of the bacterial endotoxin Cry1A(c) to captured vesicles containing its cell surface receptor could be demonstrated.  相似文献   

8.
An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).  相似文献   

9.
Surface plasmon resonance (SPR) spectroscopy has been used to study DNA assembly, DNA hybridization, and protein-DNA interactions on two streptavidin (SA) sensor chips. On one chip, SA molecules are immobilized on a biotin-exposed surface, forming an ordered two-dimensional (2D) SA monolayer. The other chip, BIAcore's SA chip, contains SA molecules immobilized within a three-dimensional (3D) carboxylated dextran matrix. Compared to the 2D chip, the 3D SA matrix allows for a slower immobilization rate of biotinylated DNA due to diffusion limitation in the dextran matrix, but with twice the amount of the immobilized DNA due to the greater number of reactive sites, which in turn enables a higher sensitivity for DNA hybridization detection. Interestingly, having a greater DNA probe dispersion in the 3D matrix does not induce a higher DNA hybridization efficiency. In a study of protein binding to immobilized DNA (estrogen receptor to estrogen response elements), aiming at assessing the DNA sequence dependent protein binding behavior, the 2D and 3D chips produce different binding characteristics. On the 2D chip, the protein binding exhibits a better selectivity to the specific sequences, regardless of binding stringency (e.g. salt concentration), whereas on the 3D chip, the liquid handling system needs to be optimized in order to minimize transport limitations and to detect small affinity differences. Through this study we demonstrate that the physicochemical structure of SPR chips affects the apparent binding behaviors of biomolecules. When interpreting SPR binding curves and selecting a sensor chip, these effects should be taken into account.  相似文献   

10.
A biosensor based on mammalian metallothionein (MT) for the detection of metal ions was developed and characterized. MT was immobilized onto a carboxymethylated dextran matrix as a biosensor for the detection of metal ions by surface plasmon resonance (SPR). The optimal pH for the immobilization step was determined to be 4. The temperature for the analysis was also defined, and the highest interaction was observed at 30 degrees C. The MT sensor chip binds cadmium (Cd), zinc (Zn) or nickel (Ni), but not magnesium (Mg), manganese (Mn) and calcium (Ca). Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection is at the micromolar level. The interaction between the metal ions and the sensor chip is influenced significantly by the presence of NaCl, Tween 20 and the pH of the reaction buffer. By decreasing the NaCl in the reaction buffer to 1 mM, the MT chip effectively differentiates cadmium from zinc and nickel. Kinetic parameters of the metal-MT interactions were also determined by using this chip. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel, which is the same as that determined for MT in solution. Thus, the MT chip can be an effective biosensor for the detection and measurement of several metal ions.  相似文献   

11.
A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg white apoA-I rHDL immobilized on a biosensor chip surface (), native, mildly oxidized, and strongly oxidized LDL in solution to a heparin-modified Au-surface of a surface plasmon resonance (SPR) biosensor (), and TRITC-labeled HDL in solution to a bare optical fiber surface (). Single-and dual-fractal models were used to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.  相似文献   

12.
As populations age, osteoporosis is becoming an important public health care problem. Urinary level of the cross-linked N-telopeptide of type I collagen has been reported to be a sensitive marker of bone resorption. Recently, we synthesized and characterized 10 overlapping peptides covering the N-telopeptide of alpha-2 type I collagen and reported their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibodies determined by a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). In this study, we design an assay based on the surface plasmon resonance (SPR) technology to detect binding interaction of each peptide fragment of NTX with the anti-NTX monoclonal antibodies. Anti-NTX monoclonal antibodies were immobilized on the surface of sensor chip by amine-coupling procedure. Serial dilutions of each peptide were prepared and injected separately onto the antibodies-immobilized sensor chip. The real-time association and dissociation interactions of each peptide were detected and reported as sensorgrams. Binding response of each peptide to the monoclonal antibodies was determined, and the SPR results were compared with the ELISA results. We demonstrate that the trends of binding potency of peptide fragments detected by SPR are in good correlation to the results obtained by ELISA, indicating that our developed SPR-based method can be further applied to detect the NTX fragments in urine and to monitor the bone loss in humans. The potent peptide fragments identified by both assays are promising for further preparation of specific monoclonal antibodies in order to develop bioassays for bone loss in humans.  相似文献   

13.
The sugar binding specificity of the mushroom Pleurotus ostreatus lectin (POL) was analyzed by surface plasmon resonance. The lectin was immobilized to a sensor chip, and asialo-bovine submaxillary mucin (asialo-BSM), one of the most potent inhibitors in the hemagglutination inhibition assay, tightly bound to the lectin. The binding specificity of various mono- or oligosaccharides to the lectin was evaluated by the coinjection method. The dissociation of asialo-BSM was promoted by injection of some haptenic saccharides. For the most part, the order of acceleration ability of the sugars to the dissociation in the coinjection experiment agreed with that of the inhibitory potency of each sugar evaluated by the hemagglutination inhibition assay. In conclusion, POL recognized a galactosyl residue, and the specificity was increased by substitution at the C-2 position of the galactosyl residue with a fucosyl or acetylamino group. This method using the coinjection method proved useful in analysis of carbohydrate-lectin binding specificity.  相似文献   

14.
We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC50) (or 80% inhibition concentration, IC80) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG.  相似文献   

15.
A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg white apoA-I rHDL immobilized on a biosensor chip surface (1), native, mildly oxidized, and strongly oxidized LDL in solution to a heparin-modified Au-surface of a surface plasmon resonance (SPR) biosensor (2), and TRITC-labeled HDL in solution to a bare optical fiber surface (3). Single-and dual-fractal models were used to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (4). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.  相似文献   

16.
The growth factor receptor-binding protein 2-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, therefore, peptidic inhibitors of the Grb2-SH2 domain has been chosen as our target for the development of antiproliferative agents. The inhibitory effects of peptide analogs on the Grb2-SH2 domain have been determined by using surface plasmon resonance (SPR) technology developed with the BIACORE biosensor. Recently, we reported the analysis of interactions between peptides and the GST-Grb2-SH2 that was immobilized on the surface of sensor chip by using BIACORE biosensor (the protein-immobilized method). Herein, we analyze interactions of peptides with the GST-Grb2-SH2 that was captured by the anti-GST antibodies immobilized on the surface of sensor chip (the protein-captured method). Results obtained by both methods are in good correlation, indicating the immobilization of GST-Grb2-SH2 on the sensor chip did not significantly affect the binding of Grb2-SH2 with peptides. Both SPR-based assays are very sensitive bioanalytical methods and can be applied in screening inhibitors of target proteins or purifying GST-fusion proteins, however, considering the efficiency and the cost, the GST-Grb2-SH2-immobilized method is suggested for routinely determining the binding potency of inhibitors of Grb2-SH2.  相似文献   

17.
In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.  相似文献   

18.
A fractal analysis is presented for the binding and dissociation of different cancer markers on biosensor surfaces. The data analyzed include putrescine in solution to PDDA/APTES/MWCNT/Puo-modified GCE (glassy carbon electrode) (8) and vascular endothelial growth factor (VEGF) in solution to the soluble form of the VEGF receptor (SFlt-1 or VEGF-1) immobilized on a sensor chip (1). Single- and dual-fractal models were used to fit the data. Values of the binding and dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (13). The binding rate coefficients and the affinity values are sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the heterogeneity present on the biosensor chip surface. The analysis presented provides physical insights into these cancer biomarker-receptor reactions occurring on the different biosensor surfaces.  相似文献   

19.
In this study, a direct detection system for triazine derivative herbicides was developed using the photosynthetic reaction center (RC) from the purple bacterium,Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The histidine-tagged RCs were immobilized on an SPR gold chip using nickel-nitrilotriacetic acid groups as a binder for one of the triazine herbicide, atrazine. The SPR responses were proportional to the sample concentrations of atrazine in the range 0.1–1 μg/mL. The sensitivity of the direct detection of atrazine using the RC-assembled sensor chip was higher than that using the antibody-immobilized chip. The other types of herbicides, DCMU or MCPP, were not detected with such high sensitivity. The results indicated the high binding selectivity of the RC complex.  相似文献   

20.
A fractal analysis is presented for the binding and dissociation of different cancer markers on biosensor surfaces. The data analyzed include putrescine in solution to PDDA/APTES/MWCNT/Puo-modified GCE (glassy carbon electrode) () and vascular endothelial growth factor (VEGF) in solution to the soluble form of the VEGF receptor (SFlt-1 or VEGF-1) immobilized on a sensor chip (). Single- and dual-fractal models were used to fit the data. Values of the binding and dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (). The binding rate coefficients and the affinity values are sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the heterogeneity present on the biosensor chip surface. The analysis presented provides physical insights into these cancer biomarker-receptor reactions occurring on the different biosensor surfaces.  相似文献   

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