Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor

Acetylcholinesterase (AChE) inhibitors are potentially lethal but also have applications as therapeutic drugs for neurodegenerative diseases such as Alzheimer’s. Enzyme inhibitor binding are difficult to be detected directly by surface plasmon resonance (SPR) due to their small molecular weight. In this article, we describe the detection of AChE inhibitor binding by SPR without the use of competitive binding or antibodies. AChE was immobilized on the gold surface of an SPR sensor through covalent attachment to a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol. The activity of the immobilized protein and the surface density were determined by using a standard photometric assay. Binding of two reversible inhibitors, which are used as therapeutic drugs, was detectable by SPR without the need to further modify the surface or the use of other reagents. The binding affinities (K_A) obtained from the fits were 3.8 × 10³ M⁻¹ for neostigmine and 1.7 × 10³ M⁻¹ for eserine, showing a higher affinity of the sensor for neostigmine. We believe that the SPR sensor’s ability to detect these inhibitors is due to conformational changes of the enzyme structure on inhibitor binding.     

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.     

Development of surface plasmon resonance immunosensor through metal ion affinity and mixed self-assembled monolayer

An immunosensor based on surface plasmon resonance (SPR) with enhanced performance was developed through a mixed self-assembled monolayer. A mixture of 16- mercaptohexadecanic acid (16-MHA) and 1-undecanethiol with various molar ratios was self-assembled on gold (Au) surface and the carboxylic acid groups of 16-MHA were then coordinated to Zn ions by exposing the substrate to an ethanolic solution of Zn(NO(3))(2)d6H2O. The antibody was immobilized on the SPR surface by exposing the functionalized substrate to the desired solution of antibody in phosphatebuffered saline (PBS) molecules. The film formation in series was confirmed by SPR and atomic force microscopy (AFM). The functionalized surface was applied to develop an SPR immunosensor for detecting human serum albumin (HSA) and the estimated detection limit (DL) was 4.27 nM. The limit value concentration can be well measured between ill and healthy conditions.     

Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance

This paper describes how the technique of surface plasmon resonance (SPR) can be utilized to follow (in real time) the attachment of Pseudomonas aeruginosa bacteria on bare gold and gold modified with a self-assembled monolayer (SAM) of mercaptounadecanoic acid. We show that SPR is able to discriminate between the adsorption of live versus dead (thermally shocked) bacteria. Moreover, the SPR distinguishes between the adsorption of wild-type versus mutant bacteria (single gene knockouts), the concentration of the bacterial suspension, and between bacteria adsorbing on SAM-modified and bare gold. SPR is able to measure bacterial adsorption within seconds of the bacterial suspension being introduced. Finally, a qualitative correlation between results from SPR with a crystal violet staining assay for different mutant bacteria was observed.     

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays.     

Comparison of surface plasmon resonance and capacitive immunosensors for cancer antigen 125 detection in human serum samples

This paper presents a comparison between surface plasmon resonance (SPR) and capacitive immunosensors for a flow injection label-free detection of cancer antigen 125 (CA 125) in human serum. Anti-CA 125 was immobilized on gold surface through a self-assembled monolayer. Parameters affecting the responses of each system were optimized. Under optimal conditions, SPR provided a detection limit of 0.1 U ml⁻¹ while 0.05 U ml⁻¹ was obtained for the capacitive system. Linearity for SPR was between 0.1 and 40 U ml⁻¹ and 0.05–40 U ml⁻¹ for capacitive system. These immunosensors were applied to analyze CA 125 concentrations in human serum samples and compared with conventional enzyme linked fluorescent assay (ELFA). Both systems showed good agreement with ELFA (P < 0.05). Moreover, these immunosensors were very stable and provided good reproducible responses after regeneration, up to 32 times for SPR and 48 times for capacitive system with relative standard deviation lower than 4%. The SPR immunosensor provided advantages in term of fast response and real-time monitoring while capacitive immunosensor offered a sensitive and cost-effective method for CA 125 detection.     

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.     

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.     

Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance

A direct human ferritin immunosensor was developed using anti-human ferritin monoclonal antibodies (MAbs) immobilized on the gold surface of a self-assembled surface plasmon resonance (SPR) apparatus. A kind of self-assembled monolayer (SAM) prepared by cystamine-glutaraldehyde method was applied to immobilize the MAbs. The reusability of the sensor chip adopting the SAM was found to be better than the other immobilization methods including adsorption, protein A, concanavalin A method. Ten cycles of measurements could be performed on the same chip regenerated with a 0.1M HCl solution. A linear relationship existed between the angle shifts (millidegrees) and the log values of ferritin concentrations in the range from 0.2 to 200 ng/ml in buffer and human serum. When used for 15 days, the angle shifts were all >95% of those on the response at the first day. A 10 M NaOH solution was used for clearing nonspecific binding in human serum. Correlation coefficient was 0.991 between this SPR method and chemiluminescent immunoassay for determination of ferritin in clinical human serum samples. The SPR sensor offers advantages of simplicity of immobilization, high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.     

Poly-(3-hexylthiophene) self-assembled monolayer based cholesterol biosensor using surface plasmon resonance technique

Cholesterol oxidase (ChOx) has been covalently immobilized onto 1-fluoro-2-nitro-4-azidobenzene (FNAB) modified poly-(3-hexylthiophene) (P3HT) self-assembled monolayer (SAM) onto gold coated glass plates. These ChOx/FNAB/P3HT/Au bio-electrodes have been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical impedance technique, cyclic voltammetric technique and atomic force microscopic (AFM) technique, respectively. The ChOx/FNAB/P3HT/Au bio-electrodes were utilized for the estimation of cholesterol concentration in standard solutions using surface plasmon resonance (SPR) technique. It is shown that this SPR biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with detection limit of 50 mg/dl, sensitivity of 1.0 4 m degrees /(mg dl), reusability of around 15 times and a shelf-life of about 10 weeks when stored at 4 degrees C.     

Comparison of different protein immobilization methods on quartz crystal microbalance surface in flow injection immunoassay.

In this study, a quartz crystal microbalance (QCM) system operated repetitively in flow injection analysis (FIA) mode, is reported. Four immobilization approaches of seven different methods include: (i) physical adsorption; (ii) two thioamine thiolation methods, using cysteamine and cystamine for gold chemisorption and further coupling; (iii) two oxidized dextran spacer methods, coupling of cysteamine and cystamine thiolated QCM surface with periodate-oxidized dextran for further Schiff acid-base reaction; and (iv) two thiol-gold chemisorption-based self-assembled monolayer (SAM), applying short-chain, C(3), and long-chain, C(11), mercapto fatty acids to insolubilize human serum albumin (HSA) on QCM surface. Effects of these protein immobilization methods on FIA immunoassay of anti-HSA were compared. At the 0.01 mg/ml anti-HSA level, the lowest analyte concentration tested, the SAM using 11-mercaptoundecanoic acid as QCM surface activating agent generated a larger frequency shift than the other immobilization methods. This implied that the use of thiolated long-chain fatty acid constructed as self-assembled monolayer may thereby potentially be a useful protein immobilization method in QCM-FIA application.     

A fusion protein expression analysis using surface plasmon resonance imaging

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.     

An optical biosensor for monitoring recombinant proteins in process media.

This paper describes the construction of a sensor for the direct monitoring of a recombinant protein, the human insulin analogue (MI3). The surface plasmon resonance (SPR) sensor incorporates an immobilised, sterilisable affinity-ligand that has been designed to bind to MI3. In practice, gold SPR devices were fabricated with; a 2D assembly of ethanethiol-modified ligand, a 2D mixed-assembly of ethanethiol-modified ligand and mercaptoethanol, a 3D coating of ligand-modified terminal-thiolated poly(vinyl)alcohol (PVA) or a 3D hydrogel of dextran coupled to a self-assembled monolayer (SAM) of mercaptohexaneundecanl-ol. Routine measurement of the concentration MI3 in the concentration range 1-100 mg/l in pilot-scale samples of crude fermentation broth have been achieved with high sensitivity levels and a high signal-to-noise ratio. Analysis can be achieved within < 10 min with the active surface being regenerable for at least 60 cycles over a 6 month period. The coupling of a robust, sterilisable and highly-selective sensor-coating with suitable transducer technologies promises to deliver sensors that are capable of direct in situ monitoring of biopharmaceuticals in industrial bioprocesses.     

Sub-attomole oligonucleotide and p53 cDNA determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification

Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10(-20)mol, corresponding to 1.38 fM in a 15 microl solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyses conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values <16%) and to possess high sequence specificity. It is also demonstrated to be viable for sequence-specific p53 cDNA analysis. The successful elimination of nonspecific adsorption of, and the signal amplification by, ODN-capped Au-NPs renders the SPR attractive for cases where the DNA concentration is extremely low and the sample availability is severely limited.     

Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface

Cell surface display was used as a strategy to display the gold-binding polypeptide (GBP) fusion protein on the surface of Escherichia coli , and consequently to immobilize the cells on the gold surface. The DNA encoding the GBP was fused to the truncated fadL gene and was expressed by the tac promoter. For the display of the core streptavidin (cSA) of Streptomyces avidinii , the cSA gene was fused to the truncated oprF gene. After the dual display of FadL–GBP and OprF–cSA on the surface of E. coli , binding of cells on the gold surface and the interaction of OprF–cSA with the biotin–horseradish peroxidase (HRP) were studied by surface plasmon resonance (SPR) analysis. Cells displaying the FadL–GBP fusion protein could be immobilized on the SPR sensor chip as shown by the SPR angle shift of 0.5°, which was stably bound at least for 60 h with a washing solution. When the FadL–GBP and OprF–cSA fusion proteins were displayed on the same cell surface, the former was used to immobilize the cells on the gold surface and the latter was used for the interaction studies with the biotin–HRP, which demonstrates that the strategy should be useful for developing whole-cell biosensor chips.     

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.     

Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping.

Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.     

Surface plamon resonance imaging of DNA based biosensors for potential applications in food analysis

The adsorption processes of oligonucleotides immobilised onto suitable photolithographic patterned gold substrates have been investigated in aqueous buffer solution by using a home made surface plasmon resonance (SPR) imaging equipment. A rapid self-assembled method for the construction of DNA chips to be used in SPR imaging experiments have been followed. The immobilised DNA molecules (probes) adopted in our SPR experiments anchored to a gold surface via thiol group were 5'thiol-modified containing a (CH(2))(15) tail. The hybridisation processes taking place with its complementary sequence have been observed and characterized by monitoring phenomena by a SPR imaging system. The two analysed oligonucleotides (probes and target) are of interest in plant gene biotechnological application and differing for the presence at the 5'-end of a poly T16 spacer. Dynamic investigation of smallest changes in SPR imaging pictures performed in liquid phase in the presence of DNA complementary probes have been performed. Quantitative information in terms of threshold of sensitivity has been extracted by using a specific images treatment.     

Grating-based surface plasmon resonance detection of core-shell nanoparticle mediated DNA hybridization

In this report, we have investigated enhanced surface plasmon resonance (SPR) detection of DNA hybridization using gold core - silica shell nanoparticles in localized plasmonic fields. The plasmonic fields were localized by periodic linear gratings. Experimental results measured for hybridization of 24-mer single-stranded DNA oligomers suggest that core-shell nanoparticles (CSNPs) on gratings of 400 nm period provide enhanced optical signatures by 36 times over conventional thin film-based SPR detection. CSNP-mediated DNA hybridization produced 3 times larger angular shift compared to gold nanoparticles of the same core size. We have also analyzed the effect of structural variation. The enhancement using CSNPs was associated with increased surface area and index contrast that is combined by improved plasmon coupling with localized fields on gratings. The combined approach for conjugated measurement of a biomolecular interaction on grating structures is expected to lower the limit of detection to the order of a few tens of fg/mm(2).     

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.