Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

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

Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode

We have developed an affinity biosensor system based on avidin-biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 x 10(-12) mol/cm(2) on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.     

Antibody linking to atomic force microscope tips via disulfide bond formation

Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

A MEMS based amperometric detector for E. coli bacteria using self-assembled monolayers.

We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA–rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.     

Directed capture of enzymes and bacteria on bioplastic films

The development of smart coatings for a variety of uses depends on the ability of the coating material to perform specific functions. We have used water dispersible polyurethane preparations for the immobilization of binding proteins under mild conditions. In these experiments, antibodies against the enzyme beta-galactosidase or the bacterium Escherichia coli were immobilized in polyurethane coatings and then used to effectively capture their cognate antigen. Further, a second, more general, capture protocol was developed which involves the incorporation of the protein avidin in the plastics. This system efficiently captures biotinylated beta-galactosidase. Biotinylated anti-E. coli antibody captured by avidin bioplastics resulted in a nearly 5-fold increase in the number of bound bacteria when compared to blank polyurethane. The use of avidin in a bioplastic allows any biotinylated antibody to be applied to all or part of the surface resulting in a patterning of capture agents on a preformed surface.     

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K(a): approximately 10(15) M(-1)) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.     

AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium-tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157:H7

The microscopic surface molecular structures and macroscopic electrochemical impedance properties of the epoxysilane monolayer and anti-Escherichia coli antibody layer on an indium-tin oxide (ITO) electrode surface were studied in this paper. Characterization of stepwise changes in microscopic features of the surfaces and electrochemical properties upon the formation of each layer were carried out using both atomic force microscopy (AFM) and electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-) as a redox couple. AFM images of the self-assembled monolayer (SAM) evidenced the dense, complete, and homogeneous morphology of the epoxysilane monolayer on the ITO surface. The uniformity of the epoxysilane SAM allowed antibodies to attach to the epoxy surface groups of the silanes in a similarly uniform fashion. The effects of epoxysilane monolayer and the antibody layer on the electrochemical properties of the electrode were quantitatively analyzed in terms of double layer capacitance, electron transfer resistance, Warburg impedance and solution resistance using Randles model as the equivalent circuit. It was demonstrated that the epoxysilane monolayer and the antibody layer act as barriers for the electron transfer between the electrode surface and the redox species in the solution, resulting in most significant increases in the electron transfer resistance compared to all the electric elements. Immunoreaction with E. coli O157:H7 cells demonstrated specific recognition of the immobilized anti-E. coli antibodies as evidenced by AFM imaging and impedance spectroscopy. It was found that the binding of E. coli cells mainly affected the electron transfer resistance and Warburg impedance.     

Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.     

A streptavidin surface on planar glass substrates for the detection of biomolecular interaction

Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a label-free, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved.     

A MEMS based amperometric detector for E. Coli bacteria using self-assembled monolayers

We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA–rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.     

SEIRA spectroscopy on a membrane receptor monolayer using lipoprotein particles as carriers

Surface-enhanced infrared absorption (SEIRA) difference spectroscopy can probe reactions in a protein monolayer tethered to a nanostructured gold surface. SEIRA studies of membrane proteins, however, remain challenging due to sample stability, effects of the metal surface on function, and the need for a membrane-mimicking environment. Here we demonstrate and characterize a model system for membrane receptor investigations using SEIRA spectroscopy. The system employs nanoscale apolipoprotein bound bilayer (NABB) particles, similar to discoidal high-density lipoprotein particles, as soluble carriers for the G-protein-coupled receptor rhodopsin. The His-tag of the engineered apolipoprotein allows for selective binding of the NABBs to a Ni-NTA modified surface, while the lipid environment of the particle ensures stability and protection of the embedded receptor. Using SEIRA spectroscopy, we followed specific binding of rhodopsin-loaded NABB particles to the surface and formation of a membrane protein monolayer. Functionality of the photoreceptor in the immobilized NABBs was probed by SEIRA difference spectroscopy confirming protein conformational changes associated with photoactivation. Orientation of the immobilized NABB particles was assessed by comparing SEIRA data with polarized attenuated total reflection-Fourier-transform infrared spectroscopy. Thus, SEIRA difference spectroscopy supported by the NABB technology provides a promising approach for further functional studies of transmembrane receptors.     

Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

Optical biosensors for real-time measurement of analytes in blood plasma

The preparation of assemblies consisting of multiple molecular layers of bovine serum albumin (BSA), monoclonal antibodies against horseradish peroxidase (anti-HRP), and monoclonal antibodies against methotrexate (anti-MTT), as well as interaction of the assemblies with human blood plasma were observed using a grating coupler and Young interferometer (YI). The assemblies could be arranged according to decreasing amounts of nonspecific deposits bound irreversibly to them from blood plasma as follows-an adsorbed antibody monolayer saturated with adsorbed BSA, antibody multilayers linked with polycations, antibodies covalently immobilized on a BSA layer densely crosslinked with glutaraldehyde (GA), slightly crosslinked BSA double layer, slightly crosslinked antibody double layers. The occurrence of human serum albumin (HSA), human fibrinogen (Fg), IgG, and IgM in the plasma deposits was studied by binding the respective antibodies. IgG, IgM, and Fg were detected in plasma deposits on the immobilized assemblies while the composition of a plasma deposit on the unmodified sensor surface reflected roughly the plasma composition containing mainly adsorbed HSA and Fg. A crosslinked anti-HRP double layer was immobilized on a waveguiding branch of YI and a similar anti-MTT double layer was immobilized on the other branch. The sensor response to blood plasma was fairly decreased owing to a compensation of the respective optical changes in the two branches, in which a similar non-specific adsorption took place. The addition of HRP or MTT to plasma induced specific responses of the corresponding branches.     

Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates

Chaperonin 10 protein from Rattus norvegicus (Rat cpn10) has been reported to bind chaperonin 60 from Escherichia coli (GroEL) in an ATP-dependent manner. Chemically synthesized Rat cpn10 was immobilized in a defined orientation to agarose-bound monomeric avidin using a reversible biotinylated affinity label ( 1 ), attached to the N^α-terminal residue. The resulting affinity chromatographic matrix was then used to isolate binding proteins from a crude cell lysate. Following affinity separation the bound ligand and ligate was released by treatment with organic base. Rat cpn10 was prepared using a highly effective synthetic protocol involving HBTU/HOBt activation and capping with N-(2-chlorobenzyloxycarbonyloxy) succinimide to terminate unreacted amino groups. The biotinylated Fmoc-based molecule ( 1 ) was introduced specifically onto the N^α-terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of side-chain protecting groups using a low-TFMSA/high-HF procedure. Crude biotinylated Rat cpn10 (Rat cpn10+ 1 ) was immobilized on monomeric avidin with a binding efficiency of approximately 75% and unlabelled truncated/capped impurities eluted off the column with buffer. The biotinylated Rat cpn10–avidin affinity matrix was then used to isolate GroEL from a crude cell lysate. The identity of the purified protein was confirmed by SDS–PAGE and binding to a specific anti-GroEL monoclonal antibody (MoAb). These results extend the applicability of the biotinylated label ( 1 ), providing a reversible non-covalent anchor for immobilization of peptide and protein ligands, thus simplifying isolation of ligates and enabling recovery of synthetic material under mild conditions. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and characterization of a photocleavable cross-linker and its application on tunable surface modification and protein photodelivery

A heterobifunctional photocleavable cross-linker based on an o-nitrobenzyl ester moiety was synthesized. The cross-linker has N-hydroxysuccinimidyl and disulfide groups attached at each end and thus can anchor a protein to a gold-coated substrate surface. Steady-state spectroscopic studies suggest that the cross-linker undergoes a clean C-O fragmentation upon irradiation with a quantum yield of 0.1. Consequently, immobilized proteins (such as avidin or antibodies) on a substrate surface can be released efficiently (>95%) under UV irradiation (lambda > 300 nm) without degrading the protein functionality. We also demonstrated protein delivery via bioconjugation of protein molecules to a gold-coated atomic-force microscope (AFM) tip. When the proteins are photoreleased from the AFM tip, they are delivered to the substrate surface as protein clusters of uniform size. This has been confirmed using both AFM and fluorescence microscopy. The application of bioconjugation in this study opens a new avenue for tunable surface modification and controllable protein delivery in studies of biological systems on the nanometer scale.     

Visualization of intracellular trafficking: use of biotinylated ligands in conjunction with avidin-gold colloids

A simple, sensitive method to visualize the binding and internalization of protein ligands by cells in culture is described. A biotinylated toxin was used as ligand, and succinoylated avidin adsorbed onto 5.2 nm gold sols was the electron-dense marker. This method affords direct localization of proteins that are on the cell surface or intracellular without need for techniques that alter membrane integrity.     

分子识别诱导的蛋白质和核酸多层膜的有序组装

通过生物大分子之间的特异性结合,采用表面等离激元共振技术监测,报导了支撑于固体表面脂单层膜上进行的亲和素、生物素标记的质粒ＤＮＡ、以及从系统性红斑狼疮患者血清中获得的抗ＤＮＡ抗体多层膜的有序组装。这种生物大分子的组装技术可以用于生物传感器以检测特定的抗原抗体。     

The effect of avidin-biotin interactions in detection systems for in situ hybridization.

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.     

分子识别诱导的蛋白质和核酸多层膜的有序组装

通过生物大分子之间的特异性结合,采用表面等离激元共振技术监测,报导了支撑于固体表面脂单层膜上进行的亲和素、生物素标记的质粒ＤＮＡ,以及从系统性红斑狼疮患者血清中获得的抗ＤＮＡ抗体多层膜的有序组装。这种生物大分子的组装技术可以用于生物传感器以检测特定的抗原抗体。