Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance

Biosurface fabrication using the Fab′ fragment of immunoglobulin (IgG) was carried out by self-assembly (SA) technique. The pepsin-digested monoclonal antibody (Mab) against bovine insulin containing the F(ab′)₂ fragment and residual proteins was separated using affinity chromatography and dialysis. To prevent the nonspecific binding of F(ab′)₂ onto gold (Au) substrate, the native disulfide bridge was reduced using dithiothreitol (DTT) to convert F(ab′)₂ into Fab′, which made the immobilization to be carried out via the native thiol (–SH) group. The fabricated biosurface using SA technique showed the formation of stable thin film through AFM topography. Through the concentration change of DTT and Fab′, the absorption characteristics against the Au surface were investigated using surface plasmon resonance (SPR) with the flow cell. The amount of immobilized antibody fragment and the antigen binding capacity were regulated with respect to the reduction state and concentration of F(ab′)₂. Based on the biosurface of the fabricated Fab′, the insulin-detection was carried out by the measurement of SPR. The proposed antibody surface could successfully detect the bovine insulin at the concentration from 100 ng/mL to 10 μg/mL.     

Surface plasmon resonance biosensor for enrofloxacin based on deoxyribonucleic acid

A DNA-based surface plasmon resonance biosensor for enrofloxacin was developed. Heating denatured DNA immobilized on the gold-coated glass surface was exploited. The immobilization was performed by a layer-by-layer co-deposition with a cationic polymer. The sensor performance was tested with real biological probes. Direct and simple determination of enrofloxacin in milk samples was demonstrated. The sensor response obeys Langmuir binding isotherm being almost linear until about 20 μg mL⁻¹. The detection limit in milk samples was estimated to be 3 μg mL⁻¹.     

Microfluidic device for immunoassays based on surface plasmon resonance imaging

We have designed and fabricated a polydimethylsiloxane (PDMS) microfluidic device containing an array of gold spots onto which antigens or antibodies of interest can be attached. We use surface plasmon resonance (SPR) imaging to monitor the antibody-antigen recognition and binding events. This combination offers two significant advantages: (1) the microfluidic device dramatically reduces reaction time and sample consumption; and (2) the SPR imaging yields real-time detection of the immunocomplex formation. Thus, an immunoreaction may be detected and quantitatively characterized in about 10 min. The sensitivity of this method is at the subnanomolar level. When gold nanoparticles are selectively coupled to the immunocomplex to cause signal amplification, the sensitivity reaches the ten to one hundred picomolar level but the time required increases to about 60 min.     

Surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety

This review describes the exploitation of exclusively optical surface plasmon resonance (SPR) biosensors for the direct and indirect detection of pathogenic microorganisms in food chains and the environment. Direct detection is, in most cases, facilitated by the use of defined monoclonal or polyclonal antibodies raised against (a part of) the target pathogenic microorganisms. The antibodies were immobilized to a solid phase of the sensor to capture the microbe from the sample. Alternatively, antibodies were used in an inhibition-like assay involving incubation with the target organism prior to analysis of nonbound antibodies. The free immunoglobins were screened on a sensor surface coated with either purified antigens or with Fc or Fab binding antibodies. Discussed examples of these approaches are the determination of Escherichia coli O1 57:H7, Salmonella spp., and Listeria monocytogenes. Another direct detection strategy involved SPR analysis of polymerase chain reaction products of Shiga toxin-2 genes reporting the presence of E. coli O157:H7 in human stool. Metabolic products have been exploited as biomarkers for the presence of a microbial agent, such as enterotoxin B and a virulence factor for the occurrence of Staphylococcus aureus and Streptococcus suis, respectively. Indirect detection, on the other hand, is performed by analysis of a humoral immune response of the infected animal or human. By immobilization of specific antigenic structures, infections with Herpes simplex and human immunodeficiency viruses, Salmonella and Treponema pallidum bacteria, and Schistosoma spp. parasites were revealed using human, avian, and porcine sera and avian eggs. Bound antibodies were easily isotyped using an SPR biosensor to reveal the infection history of the individual. Discussed studies show the recent recognition of the suitability of this type of instrument for (rapid) detection of health-threatening microbes to food and environmental microbial safety.     

Surface plasmon resonance immunosensor for histamine based on an indirect competitive immunoreaction

The use of a surface plasmon resonance immunosensor for the analysis of histamine (β-imidazole ethylamine) is described. The method is based on an indirect competitive reaction of an anti-histamine antibody in a sample solution with histamine immobilized on a sensor chip and with histamine in the sample solution. A sensor chip immobilized with histamine was prepared using a self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA) as an anchor membrane, followed by an amino-coupling reaction with histamine after activation of the 11-MUA layer on the sensor chip by treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide. The sensor chip can be reused, after regeneration with a 10 mM HCl solution, which dissociates the anti-histamine antibody complex from histamine on the sensor chip. The affinity constants for the immunocomplex of the anti-histamine antibody with histamine in the solution and for that of the anti-histamine antibody with histamine immobilized on the sensor chip were calculated to be 1.5 × 10⁷ and 7.2 × 10⁵ M⁻¹, respectively, by assuming a Langmuir-type adsorption of the anti-histamine antibody to histamine immobilized on the sensor chip. The detection limit of the method was determined to be 3 ppb.     

Surface plasmon resonance sensor for lysozyme based on molecularly imprinted thin films

Molecularly imprinted polymers (MIPs) selective for lysozyme were prepared on SPR sensor chips by radical co-polymerization with acrylic acid and N,N′-methylenebisacrylamide. Gold-coated SPR sensor chips were modified with N,N′-bis(acryloyl)cystamine, on which MIP thin films were covalently conjugated. The presence of NaCl during the polymerization and the re-binding tests affected the selectivity and the optimization of NaCl concentration in the pre-polymerization mixture and the re-binding buffer could enhance the selectivity in the target protein sensing. When the lysozyme-imprinted polymer thin films were prepared in the presence of 40 mM NaCl, the selectivity factor (target protein bound/reference protein bound) of MIP in the re-binding buffer containing 20 mM NaCl was 9.8, meanwhile, that of MIP in the re-binding buffer without NaCl was 1.2. A combination of SPR sensing technology with protein-imprinted thin films is a promising tool for the construction of selective protein sensors.     

Kappa-casein based electrochemical and surface plasmon resonance biosensors for the assessment of the clotting activity of rennet

We report for the first time the development of kappa-casein (κ-CN)-based electrochemical and surface plasmon resonance (SPR) biosensors for the assessment of the clotting activity of rennet. Electrochemical biosensors were developed over gold electrodes modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate, while SPR measurements were performed on regenerated carboxymethylated dextran gold surfaces. In both types of biosensor, κ-CN molecules were immobilized onto modified gold surfaces through covalent bonding. In electrochemical biosensors, interactions between the immobilized κ-CN molecules and chymosin (the active component of rennet) were studied by performing cyclic voltammetry, differential pulsed voltammetry, and electrochemical impedance spectroscopy (EIS) measurements, using hexacyanoferrate(II)/(III) couple as a redox probe. κ-CN is cleaved by rennet at the Phe105–Met106 bond, producing a soluble glycomacropeptide, which is released to the electrolyte, and the positively charged insoluble para-κ-casein molecule, which remains attached to the surface of the electrode. This induced reduction of the net negative charge of the sensing surface, along with the partial degradation of the sensing layer, results in an increase of the flux of the redox probe, which exists in the solution, and consequently, to signal variations, which are associated with the increased electrocatalysis of the hexacyanoferrate(II)/(III) couple on the gold surface. SPR experiments were performed in the absence of the redox probe and the observed SPR angle alterations were solely attributed to the cleavage of the immobilized κ-CN molecules. Various experimental variables were investigated and under the selected conditions the proposed biosensors were successfully tried to real samples. The ratios of the clotting power units in various commercial solid or liquid samples, as they are calculated by the EIS-based data, were almost identical to those obtained with a reference method. In addition, EIS measurements showed an excellent reproducibility, lower than 5%.     

3R,4R-dihydroxy-L-proline: a potent and specific β-D-glucuronidase inhibitor

The title compound, a naturally-occurring amino acid found in virotoxins, competitively inhibits bovine β-D-glucuronidase but does not affect other glycosidases.     

Molecular design of potent inhibitor specific for cathepsin B based on the tertiary structure prediction.

To design a potent inhibitor specific for cathepsin B (rat liver), the tertiary structure was predicted based on the crystal structure of the papain complexed with (+)-(2S,3S)-3-(1-[N-(3-methylbutyl)amino]leucylcarbonyl)oxirane-2- carbolylic acid (E-64-c), a thiol protease inhibitor. Taking advantage of the structural characteristics of the predicted active site, seventeen inhibitors were chemically synthesized by molecular modeling, and one of them, N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-p rol ine (CA-074) was shown to be the first potent inhibitor specific for cathepsin B. The relationship between the structure and inhibitory activity is discussed based on the model structure of the cathepsin B-inhibitor complex.     

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin–strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10⁻¹¹ M to 1 × 10⁻⁶ M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.     

Surface plasmon resonance and Raman scattering effects studied for layers deposited on Spreeta sensors

The possibility to detect and prospectively to characterize deposited organic layers directly on Spreeta sensors by Fourier transform Raman spectroscopy was studied. A special holder of sensors that enabled measurement of FT Raman spectra was developed. The effects of various angles of incidence of the laser beam on the sensor surface were studied with respect to the intensity of Raman spectra and to the risks of artifacts. No effect of measurement of FT Raman spectra on SPR functionality of sensors was proven. The key role of the surface morphology of the sensing gold layer on repeatability of SPR curves and the possibility to check the surface by optical microscopy was demonstrated.     

Design and application of a flow cell for carbon-film based electrochemical enzyme biosensors

A flow cell has been designed for use with an electrochemical enzyme biosensor, based on low-cost carbon-film electrodes. Three types of mediators were used: cobalt and copper hexacyanoferrates and poly(neutral red) (PNR), covered with glucose oxidase (GOx) immobilised by cross-linking with glutaraldehyde in the presence of bovine serum albumin or inside a oxysilane sol–gel network. Mixtures of sol–gel precursors were made from 3-aminopropyl-triethoxysilane (APTOS) together with methyltrimethoxysilane (MTMOS), methyltriethoxysilane (MTEOS), tetraethyloxysilane (TEOS) or 3-glycidoxypropyl-trimethoxysilane (GOPMOS), and the best chosen for encapsulation. Optimisation in batch mode, using amperometric detection at fixed potential, showed the PNR-GOx modified carbon-film electrodes to be best for flow analysis for both glutaraldehyde and sol–gel enzyme immobilisation. Both types of enzyme electrode were tested under flow conditions and the reproducibility and stability of the biosensors were evaluated. The biosensors were used for fermentation monitoring of glucose in grape must and interference studies were also performed.     

Surface plasmon resonance as a tool for the estimation of adsorbed polymeric layer characteristics: Theoretical considerations and experiment

The potential application of Surface Plasmon Resonance (SPR) spectroscopy in evaluating the thickness and volume fraction of adsorbed macromolecular layers is discussed in this work. The sensitivity of SPR spectroscopy to different layer concentration and to the layer extension normal to the surface (thickness) is theoretically illustrated. A new approach for the interpretation of SPR data is presented, which is applicable whenever the functional form of the density profile is known. The use of the proposed procedure for the fitting of experimental results from PS‐PEO brush self‐assembly on alumina surface has allowed the determination of the layer parameters, which have been found to be in accordance with theoretical mean‐field and scaling predictions, being also in good agreement with previous results from neutron reflectivity experiments. Furthermore, it has been confirmed that the dependence of the brush layer thickness d on the molecular weight M_w obeys the scaling law d ～ M_w^0.63. Since surface plasmon measurements can be acquired quite fast, it is suggested that under the present analysis scheme, the technique may be implemented to probe the average conformational properties of adsorbed macromolecular layers during their formation or under external stimuli. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 2060–2070, 2007     

A highly specific immunoassay for microcystin-LR detection based on a monoclonal antibody

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive and fast means to detect these toxins, but cross-reactivity (CR) characteristic of different antibodies was variable. Here, we have produced and characterized a monoclonal antibody (Clone MC8C10) with highly specificity against the most frequent and most toxic variant of microcystins, MC-LR. MC8C10 is more specific against MC-LR among the reported antibodies before. The immunogen was synthesized from the modified MC-LR and bovine serum albumin (BSA). An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with MC8C10 was established to detect the MCs in waters, which showed highly specificity with MC-LR, and have a detection limit for MC-LR 0.1 μg L⁻¹, the 50% inhibition concentration (IC₅₀) for MC-LR was 1.8 ± 0.1 μg L⁻¹ and the quantitative detection range was from 0.3 to 10 μg L⁻¹. The [4-arginine] microcystins and the nodularin-R showed lower cross-reactivates (CR < 10%), and other MCs such as MC-LF and MC-LW are not recognized (CR < 10⁻⁴). The analysis results of real water samples with ic-ELISA showed that all the coefficients of variation were less than 15%, and the recovery was (100.3 ± 5.9)%. So the highly specific ic-ELISA will commendably suit for sensitive analysis for MC-LR in surface water as well as drinking water.     

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-β-alanine-ovalbumin (TNP-β-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-β-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 × 10⁻¹¹ to 3 × 10⁻⁷ g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 × 10⁻¹¹ g/ml.     

A coumarinylaldoxime as a specific sensor for Cu and its biological application

In this Letter we present a new probe, (E)-7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde oxime (JB), which can detect Cu²⁺ ions in HEPES buffer under physiological conditions. Benesi–Hildebrand and Job plots demonstrate that the stoichiometry of the Cu²⁺ complex formed is 2:1. Possible interference with other analytes was examined, and the decrease of the fluorescence of JB at 510 nm when it reacts with Cu²⁺ was shown to be highly selective. This probe accumulates in the plasmalemma of human neuroblastoma SH-SY5Y cells. Molecular dynamics (MD) simulations revealed that JB interacts with the lipid bilayer at the level of the glycerol moieties.     

Surface plasmon resonance as a time-resolved probe of structural changes in molecular films: considerations for correlating resonance shifts with adsorbate layer parameters

Surface plasmon resonance (SPR) spectroscopy is an efficient probe of transient structural changes in molecular films. To analyze kinetic SPR data for such systems, generally it is necessary to adapt an adequate theoretical framework that would allow one to express the measured optical quantities (time-dependent shifts of the resonance angle or wavelength) in terms of the structural parameters (layer thickness, mass density, or surface coverage) of the sample molecules. We present here theoretical calculations and illustrative experimental results to address certain essential elements of this type of data analysis for transient SPR systems. The phenomenological framework we consider here is based on multilayer reflectivity calculations, and can be applied to a broad class of systems involving ordered molecular layers on supporting gold films. A typical application of these calculations is demonstrated through the analysis of specific SPR experiments designed to probe the kinetics of pH-induced structural changes in a molecular film of 11-mercaptoundecanoic acid (MUA) on a thin gold film.     

Total synthesis and biological mode of action of largazole: a potent class I histone deacetylase inhibitor

The efficient total synthesis of the recently described natural substance largazole (1) and its active metabolite largazole thiol (2) is described. The synthesis required eight linear steps and proceeded in 37% overall yield. It is demonstrated that largazole is a pro-drug that is activated by removal of the octanoyl residue from the 3-hydroxy-7-mercaptohept-4-enoic acid moiety to generate the active metabolite 2, which is an extraordinarily potent Class I histone deacetylase inhibitor. Synthetic largazole and 2 have been evaluated side-by-side with FK228 and SAHA for inhibition of HDACs 1, 2, 3, and 6. Largazole and largazole thiol were further assayed for cytotoxic activity against a panel of chemoresistant melanoma cell lines, and it was found that largazole is substantially more cytotoxic than largazole thiol; this difference is attributed to differences in the cell permeability of the two substances.     

Surface characterization and efficiency of a matrix-free and flat carboxylated gold sensor chip for surface plasmon resonance (SPR)

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM–MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen–antibody interaction. For both interactions, the well characterized densely functionalized SAM–MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.     

Surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin B

An immunosensor based on surface plasmon resonance (SPR) with a mixed self-assembled monolayer (SAM) was developed to determine staphylococcal enterotoxin B (SEB). The SAM on a gold surface was fabricated by adsorbing a mixture of 16-mercapto-1-hexadecanoic acid (16-MHA) and hexanethiol at various molar ratios. Initially, full-length anti-SEB was randomly immobilized onto the SAM to form the immunosensing surface. Through optimization of surface functionalization and anti-SEB immobilization, the SPR sensors can be applied to the determination of SEB in a linear range of 0.01?~?1.0 μg.mL^?1. Furthermore, a smaller antibody fragment (F(ab)’) was generated and immobilized randomly (via amino groups) or in an oriented manner (via ?SH groups) to form the immunosensing surface. The oriented immobilization of F(ab)’ led to a 50% increase in the antigen binding efficiency compared to randomly immobilized covalent F(ab’) fragments. The resulting calibration curve showed higher sensitivity. In addition, the specificity and applicability of the proposed immunosensor to milk samples were also demonstrated. Furthermore, the sensor can be regenerated using 0.1 M HCl, and 70% of the initial response was maintained over 3 cycles.