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.     

New immobilization method for immunoaffinity biosensors by using thiolated proteins

A new immobilization method for immunoaffinity (IA) biosensors that ensures the high surface density and the stability of the IA layer was developed. For the immobilization of biomolecules, the molecular recognition protein was first thiolated by covalent conjugation of mercaptopropionic acid, and then the thiolated protein was attached on the gold surface of the transducer. In this work, horseradish peroxidase (HRP) and its antibody were used as a model antigen-antibody, and the following properties of the IA layer prepared by thiolated protein were estimated: (i) biological integrity of HRP after the immobilization process by using activity assay, (ii) charge transfer resistance by immobilization, (iii) mass loading by the surface plasmon resonance (SPR) biosensor, (iv) number of binding sites, and (v) feasibility test for the measurement of capacitive change by the antigen-antibody interaction. Based on these parameters, the immobilization method by using thiolated protein was determined to be feasible for application to IA biosensors.     

Self-assembled monolayer-assisted silicon nanowire biosensor for detection of protein-DNA interactions in nuclear extracts from breast cancer cell

The large number of estrogen receptor (ER) binding sites of various sequence patterns requires a sensitive detection to differentiate between subtle differences in ER-DNA binding affinities. A self-assembled monolayer (SAM)-assisted silicon nanowire (SiNW) biosensor for specific and highly sensitive detection of protein-DNA interactions, remarkably in nuclear extracts prepared from breast cancer cells, is presented. As a typical model, estrogen receptor element (ERE, dsDNA) and estrogen receptor alpha (ERα, protein) binding was adopted in the work. The SiNW surface was coated with a vinyl-terminated SAM, and the termination of the surface was changed to carboxylic acid via oxidation. DNA modified with amine group was subsequently immobilized on the SiNW surface. Protein-DNA binding was finally investigated by the functionalized SiNW biosensor. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were employed to characterize the stepwise functionalization of the SAM and DNA on bare silicon surface, and to visualize protein-DNA binding on the SiNW surface, respectively. We observed that ERα had high sequence specificity to the SiNW biosensor which was functionalized with three different EREs including wild-type, mutant and scrambled DNA sequences. We also demonstrate that the specific DNA-functionalized SiNW biosensor was capable of detecting ERα as low as 10 fM. Impressively, the developed SiNW biosensor was able to detect ERα-DNA interactions in nuclear extracts from breast cancer cells. The SAM-assisted SiNW biosensor, as a label-free and highly sensitive tool, shows a potential in studying protein-DNA interactions.     

Immunosensor for detection of Legionella pneumophila using surface plasmon resonance

Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila. A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine (-NH(2)) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10(5) cells/ml.     

Antibody-antigenic peptide interactions monitored by SPR and QCM-D. A model for SPR detection of IA-2 autoantibodies in human serum

This work reports on a complementary use of surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) technologies to study interactions between a peptide antigen and polyclonal antibodies, in an experimental format suitable for diagnostic assays of autoimmune diseases. In the chosen model, a synthetic peptide from the juxtamembrane region of IA-2 (a type 1 diabetes associated antigen) was immobilized by an optimized chemical protocol applicable to both BIACORE and QCM-D sensors. A thorough study of the peptide immobilization was performed to optimize the signal-to-noise ratio using mixed self-assembled monolayers (SAM) on a gold surface. Introduction of polyethylene glycol (EG₆) chains into mixed SAM layers and addition of an anionic surfactant to the human serum reduced non-specific binding without modifying the viscoelasticity properties of the layer. Under our conditions, the antibody SPR detection limit was determined to be 0.2 nM in diluted human serum. This value is in agreement with the reported rank distribution of IA-2 antibodies in diabetic patient sera. Label-free and real-time technologies such as SPR and/or QCM-D could be precious tools in future diagnostic assays.     

Conducting polyamic acid membranes for sensing and site-directed immobilization of proteins

A biosensor platform based on polyamic acid (PAA) is reported for oriented immobilization of biomolecules. PAA, a functionalized conducting polymer substrate that provides electrochemical detection and control of biospecific binding, was used to covalently attach biomolecules, resulting in a significant improvement in the detection sensitivity. The biosensor sensing elements comprise a layer of PAA antibody (or antigen) composite self-assembled onto gold (Au) electrode via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linking. The modified PAA was characterized by Fourier transform infrared (FTIR), (1)H nuclear magnetic resonance (NMR), and electrochemical techniques. Cyclic voltammetry and impedance spectroscopy experiments conducted on electrodeposited PAA on Au electrode using ferricyanide produced a measurable decrease in the diffusion coefficient compared with the bare electrode, indicating some retardation of electron transfer within the bulk material of the PAA. Thereafter, the modified PAA surface was used to immobilize antibodies and then to detect inducible nitric oxide synthase and mouse immunoglobulin G (IgG) using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and amperometric techniques. ELISA results indicated a significant amplified signal by the modified PAA, whereas the SPR and amperometric biosensors produced significant responses as the concentration of the antigen was increased. Detection limits of 3.1×10(-3)ng/ml and 2.7×10(-1)ng/ml were obtained for SPR and amperometric biosensors, respectively.     

Nano-scale probe fabrication using self-assembly technique and application to detection of<Emphasis Type="Italic">Escherichia coli</Emphasis> O 157∶H7

A self-assembled monolayer of protein G was fabricated to develop an immunosensor based on surface plasmon resonance (SPR), thereby improving the performance of the antibody-based biosensor through immobilizing the antibody molecules (IgG). As such, 11-mercaptoundecanoic acid (11-MUA) was adsorbed on a gold (Au) support, while the non-reactive hydrophilic surface was changed through substituting the carboxylic acid group (-COOH) in the 11-MUA molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholide (EDAC). The formation of the self-assembled protein G layer on the Au substrate and binding of the antibody and antigen were investigated using SPR spectroscopy, while the surface topographies of the fabricated thin films were analyzed using atomic force microscopy (AFM). A fabricated monoclonal antibody (Mab) layer was applied for detectingE. coli O157∶H7. As a result, a linear relationship was achieved between the pathogen concentration and the SPR angle shift, plus the detection limit was enhanced up to 10² CFU/mL.     

Immunosensor for the detection of Vibrio cholerae O1 using surface plasmon resonance

An immunosensor for the detection of Vibrio cholerae O1 was developed on the basis of surface plasmon resonance (SPR). A protein G layer was fabricated by means of the chemical coupling between the free amine (-NH₂) groups of protein G and the activated carboxyl groups present on a self-assembled monolayer (SAM) consisting of a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2). A monoclonal antibody, which was confirmed to be specific to V. cholera O1 by the Western blotting technique, was immobilized on the protein G layer. The formation of the SAM, the protein G layer and the sequential binding of the antibody against V. cholera O1 were investigated with SPR spectroscopy. As the number of fabricated layers increased, the minimum angle of plasmon resonance was increased accordingly. The target bacteria, V. cholera O1, was measured with the fabricated immunosensor, whose detection range was between 10⁵ and 10⁹ cells/mL.     

Detection of CD133-marked cancer stem cells by surface plasmon resonance: Its application in leukemia patients

Here, we reported the development of a label-free and real-time surface plasmon resonance (SPR) based biosensor for cancer stem cells (CSCs) detection using cell surface biomarker; CD133. The fabricated biosensor was used for detection of this marker in some acute myeloid leukemia (AML) patients and the results were compared with those obtained from flow cytometry (FC) method. CD133 antibody was immobilized on the gold chip surface via EDC/NHS coupling method and binding of the candidate cells to the modified gold sensor surface was monitored after isolation of mononuclear cells from bone marrow of the patients. The method was validated in terms of various parameters such as CD133- antibody concentration and cell density. The CD133-marked cells were investigated in seven AML patients. All SPR results were compared with those obtained from FC method. A very good correlation (R² = 0.96) was obtained between SPR and FC responses related to CD133-marked cells densities. In conclusion, in this study, a label-free and real-time SPR cytometry method was developed to detect CD133 and it was successfully applied to follow this cancer stem cell biomarker in AML patients.     

Development of instrumentation to allow the detection of microorganisms using light scattering in combination with surface plasmon resonance

This paper describes work carried out to develop a biosensor which allows two separate detection principles to operate simultaneously at the same surface. A prototype device was constructed that provided Kretschmann-configuration surface plasmon resonance (SPR) measurement of refractive index (RI) changes using an 820 nm LED light source, whilst a 635 nm diode laser was used to produce light scattering signals from bacterial spores. Both effects occurred at a gold-coated surface. The RI changes were measured conventionally from the side of the gold layer nearer to the light sources. The scattered light was imaged from the opposite face which was in contact with the aqueous sample. Specific detection of bacterial spores through the light scattering mode using antibody capture was investigated. The flow dynamics and interactions with the surface of individual spores were observed. A comparison with SPR for detection using the same antibody/antigen pair was made. Spore suspensions that were readily detectable by light scattering at 10(7) ml(-1) did not provide significant responses by SPR. The potential for future developments is discussed.     

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction. The immunosensor chips were constructed by various concentrations of protein A. To determine the surface characteristics of different self-assembly monolayers (SAMs), several quantitative and kinetic measurements were carried out. The extent of immobilization of anti-CEA and the immune response of anti-CEA antibody against CEA were measured using the SPR instrument and CV device. The terminal functional groups of protein A have different effects on the adsorption and covalent binding of immunoprotein depending on the steric hindrance. Through the parallel measurements, we demonstrate that SPR and CV are sensitive to measure the antigen–antibody binding capacity.     

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.     

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: a femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG3-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and FT-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG6-COOH and HS-OEG3-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.     

Characterization of a self-assembled monolayer of thiol on a gold surface and the fabrication of a biosensor chip based on surface plasmon resonance for detecting anti-GAD antibody

A biosensor chip utilizing surface plasmon resonance (SPR) was fabricated for detecting anti-glutamic acid decarboxylase (GAD) antibody, which is an indicator of the presence of type I diabetes mellitus. The sensor surfaces were constructed from various thiol mixtures of different molar ratios of 3-mercaptopropionic acid (3-MPA) to 11-mercaptoundecanoic acid (11-MUA). To determine the surface characteristics of the different alkanethiol monolayers, several quantitative and kinetic measurements were carried out. The extent of immobilization of streptavidin (SA) and biotin-GAD (the anti-GAD receptor) and the immune response of anti-GAD antibody against GAD were measured using the SPR biosensor. The terminal functional group of a thiol has different effects on the adsorption and covalent binding of protein depending on the steric hindrance. The protein chip described herein permits simple, rapid detection of anti-GAD antibody.     

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.     

Nanoscale fabrication of protein A on self-assembled monolayer and its application to surface plasmon resonance immunosensor

The fabrication of protein A film on self-assembled monolayer was done for the construction of immunosensor using surface plasmon resonance (SPR) measurement. The layer of heterobifunctional linker, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was self-assembled on the gold (Au) surface. Due to the succinimidyl functional group in SPDP to be reacted with amine (NH₂) group of protein A, the covalent immobilization of protein A was subsequently induced toward Au surface. The characteristics of film formation were investigated using SPR with respect to the various concentrations of SPDP and protein A. The optimal concentration for the film formation was found to be 0.1 mg/mL of SPDP and 0.1 mg/mL of protein A, respectively. The surface topography of protein A layer using atomic force microscopy showed that the heteromolecular layer was formed successfully. The antibody, anti-bovine serum albumin (BSA), was immobilized onto protein A layer, and the fabricated antibody layer was applied for the detection of BSA. The extent of BSA–antibody binding was measured using SPR and its lower detection limit of BSA was 100 pM.     

Human GM-CSF interaction with the alpha-chain of its receptor studied using surface plasmon resonance.

A surface plasmon resonance (SPR) based biosensor has been used for studying the interaction of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) with genetically engineered alpha-chain subunits of its specific receptor (GM-Ralpha). Western blot analysis of GM-Ralpha confirmed the correct size (80 kDa) and reactivity of these proteins against anti-GM-Ralpha polyclonal or monoclonal antibodies. GM-CSF was immobilized, using standard amine coupling methods, to the dextran-modified gold biosensor surface in order to capture GM-Ralpha subsequently injected over the sensing layer. GM-Ralpha were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of GM-Ralpha with dilutions of soluble GM-CSF before injection of the mixture over the GM-CSF matrix, prevented ligand binding in a dose dependent manner. In contrast, unrelated soluble cytokines or serum proteins (e.g. G-CSF, albumin, etc.) were found to exert no inhibition. Complexes formation blockage by pre-incubation of constant amounts of GM-Ralpha with dilutions of neutralizing anti-GM-Ralpha antibodies was concentration dependent, further assessing the specificity of the interaction. To investigate the possibility of relating the effect on binding affinity of critical conformational changes at the contact site, experiments of multisite binding were performed, flowing a set of neutralizing monoclonal antibodies reacting to different epitopes on GM-CSF over the GM-CSF matrix, before injecting GM-Ralpha. The results indicated that antibody interaction with helix D and helix A of GM-CSF markedly inhibited GM-CSF binding to GM-Ralpha. Comparable results were obtained using the biosensor technology and enzyme-linked immunoassays, in representative experiments performed with the same reagents. These experiments demonstrate that SPR can be successfully used for studying complementary interactions between GM-CSF and its receptor alpha-chains in solution without using labels or secondary tracers and, compared with conventional immunoanalysis methods, significantly saving time.     

Quantitative assay of hepatitis B surface antigen by using surface plasmon resonance biosensor

We performed a basic experiment for the rapid, on-line, real-time measurement of hepatitis B surface antigen using a surface plasmon resonance biosensor. We immobilized anti-HBsAg (hepatitis B surface antigen) polyclonal antibody, as a ligand, to the dextran layer on a CM5 chip surface that had previously been activated byN-hydroxysuccinimide. A sample solution containing HBsAg was fed through a microfluidic channel, and the reflecting angle change due to the mass increase from the binding was detected. The binding characteristics between HBsAg and its polyclonal antibody followed the typical monolayer adsorption isotherm. When the entire immobilized antibody had interacted, no additional, non-specific binding occurred, suggesting the immunoreaction was very specific. The bound antigen per unit mass of the antibody was independent of the immobilized ligand density. No significant steric hindrance was observed at an immobilization density of approximately 17.6 ng/mm². The relationship between the HBsAg concentration in the sample solution and the antigen bound to the ligand was linear up to ca. 40 μg/mL. This linearity was much higher than that of the ELISA method. It appeared the antigen-antibody binding increased as the immobilized ligand density increased. In summary, this study showed the potential of this SPR biosensor-based method as a rapid, simple and multi-sample on-line assay. Once properly validated, it may serve as a more efficient method for HBsAg quantification for replacing the ELISA.     

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.     

Surface plasmon resonance immunosensor for detection of<Emphasis Type="Italic">Legionella pneumophila</Emphasis>

An immunosensor based on surface plasmon resonance (SPR) onto a protein G layer by self-assembly technique was developed for detection ofLegionella pneumophila. The protein G layer by self-assembly technique was fabricated on a gold (Au) surface by adsorbing the 11-mercaptoundecanoic acid (MUA) and an activation process for the chemical binding of the free amine (-NH₂) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of the protein G layer by self-assembly technique on the Au substrate and the binding of the antibody and antigen in series were confirmed by SPR spectroscopy. The surface topographies of the fabricated thin films on an Au substrate were also analyzed by using an atomic force microscope (AFM). Consequently, an immunosensor for the detection ofL. pneumophila using SPR was developed with a detection limit of up to 10² CFU per mL.