Comparative study of random and oriented antibody immobilization as measured by dual polarization interferometry and surface plasmon resonance spectroscopy

Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL.     

A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome

A surface plasmon resonance (SPR)-based biosensor was developed for simple diagnosis of severe acute respiratory syndrome (SARS) using a protein created by genetically fusing gold binding polypeptides (GBPs) to a SARS coronaviral surface antigen (SCVme). The GBP domain of the fusion protein serves as an anchoring component onto the gold surface, exploiting the gold binding affinity of the domain, whereas the SCVme domain is a recognition element for anti-SCVme antibody, the target analyte in this study. SPR analysis indicated the fusion protein simply and strongly self-immobilized onto the gold surface, through GBP, without surface chemical modification, offering a stable and specific sensing platform for anti-SCVme detection. AFM and SPR imaging analyses demonstrated that anti-SCVme specifically bound to the fusion protein immobilized onto the gold-micropatterned chip, implying that appropriate orientation of bound fusion protein by GBP resulted in optimal exposure of the SCVme domain to the assay solution, resulting in efficient capture of anti-SCVme antibody. The best packing density of the fusion protein onto the SPR chip was achieved at the concentration of 10 μg mL⁻¹; this density showed the highest detection response (906 RU) for anti-SCVme. The fusion protein-coated SPR chip at the best packing density had a lower limit of detection of 200 ng mL⁻¹ anti-SCVme within 10 min and also allowed selective detection of anti-SCVme with significantly low responses for non-specific mouse IgG at all tested concentrations. The fusion protein provides a simple and effective method for construction of SPR sensing platforms permitting sensitive and selective detection of anti-SCVme antibody.     

Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance

Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand – diethylaminoethyl (DEAE) – onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations.     

Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles

Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21 pM, providing a simple, sensitive and selective protocol for small target molecules detection.     

A surface plasmon resonance study on the optical properties of gold nanoparticles on thin gold films

We report on an investigation of the optical properties of gold nanoparticles assembled as thin films of different thickness. The nanoparticles were linked to the surface of a gold chip by dithiol reagents and studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. There is good correlation between the experimental findings and theoretical simulation, and the respective data reveal the presence of ordered nanostructures in the assemblies. The shift in the SPR angle is linearly dependent on the particle size and the ratio of the different particles. SPR spectroscopy also reveals important information in terms of the optical constants of such films. This shall be further applied to in-situ quality control in the fabrication of optoelectronic, solar cell and semiconductor devices.

Site-Directed Antibody Immobilization by Resorc[4]arene-Based Immunosensors

One of the main problems in the development of immunosensors is to overcome the complexity of binding antibodies to the sensor surface. Most immobilizing methods lead to a random orientation of antibodies with a lower binding site density and immunoaffinity. In order to control the orientation of antibody immobilization, several resorc[4]arene derivatives were designed and synthesized. After the spectroscopic characterization of resorc[4]arene self-assembled monolayers (SAMs) onto gold films, the surface coverage and the orientation of insulin antibody (Ab-Ins) were assessed by a surface plasmon resonance (SPR) technique and compared with a random immobilization method. Experimental results combined with theoretical studies confirmed the dipole–dipole interaction as an important factor in antibody orientation and demonstrated the importance of the upper rim functionalization of resorcarenes. Accordingly, resorcarene 5 showed a major binding force towards Ab-Ins thanks to the H-bond interactions with the amine protein groups. Based on these findings, the resorcarene-based immunosensor is a powerful system with improved sensitivity providing new insight into sensor development.     

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin–streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K _D≈4.01×10⁻⁷. All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.     

Surface plasmon resonance-enabled mass spectrometry arrays

Biosensors that utilize surface plasmon resonance (SPR) as a method of detection of protein interactions can be used for selective separation of proteins prior to MS analysis. The combination of SPR and MS results in a unique multiplexed detection technology capable of both quantitative and qualitative protein analysis. To further the development of a high-throughput SPR-MS approach, the possibility of arraying binding ligands on SPR chips for affinity capture of proteins and their MS analysis was explored. Antibodies to beta-2-microglobulin, cystatin C, transferrin, and insulin-like growth factors I and II were arrayed on a large number of SPR chips. Human plasma samples were injected over the antibody array chips in an SPR Biosensor, after which on-chip MS analysis was performed to detect the bound proteins. Signals from the targeted proteins were observed for each antibody-derivatized chip, indicating successful antibody immobilization and protein capture. The SPR-MS arrays are robust, highly reproducible, and are capable of high-throughput analysis.     

A surface plasmon resonance immunosensor for detecting a dioxin precursor using a gold binding polypeptide

A surface plasmon resonance (SPR) based biosensor was developed for monitoring 2,4-dichlorophenol, a known dioxin precursor, using an indirect competitive immunoassay. The SPR sensor was fabricated by immobilizing a gold-thin layer on the surface of an SPR sensor chip with an anti-(2,4-dichlorophenol) antibody using a gold binding polypeptide (GBP) and protein G. The SPR response based on the antigen-antibody reaction in a flow system was measured by injecting a 2,4-dichlorophenol sample solution into the flow system in which the SPR sensor was located. In a direct immunoassay system using the modified sensor chip, no significant SPR angle shift less than 0.001° was observed when a 25 ppm of 2,4-dichlorophenol solution was injected. In order to improve the sensitivity of the SPR sensor, an indirect competitive immunoassay method was used in conjunction with the SPR sensor system using 2,4-dichlorophenol conjugated with bovine serum albumin (BSA). In the competitive assay, a 350 ppm 2,4-dichlorophenol-BSA conjugate solution containing 2,4-dichlorophenol at various concentrations (10-250 ppb) were injected into the SPR sensor system. The sensitivity of this indirect immunoassay was found to be extremely sensitive, compared to the direct one, and a detection limit of 20 ppb was estimated. Verification that the use of GBP for immobilizing the antibody on the sensor chip enhanced the sensitivity to 2,4-dichlorophenol was obtained by comparing the procedure with another modification, in which BSA was used instead of GBP for immobilizing the antibody on the sensor chip. The affinity constant of 2,4-dichlorophenol and its conjugate to the antibody were estimated form the SPR response.     

Continuous Immunoassay for Sulfamethazine by Surface Plasmon Resonance-Based Biosensor

Regeneration of the sensor chip surface is difficult in many surface plasmon resonance (SPR) biosensor assays. Improper regeneration will reduce life span of the sensor chip and decrease the quality of the data. Considering the advantages of reducing the regeneration frequency, a theoretically feasible continuous SPR biosensor immunoassay for sulfamethazine (SMT) was developed. In the continuous inhibitive immunoassay, the sensor chip surface is regenerated only once after a definite number of tests instead of every test. The SMT-bovine serum albumin (BSA) conjugate was covalently immobilized to a carboxymethyldextran modified gold film. The immobilization conditions of the antigen were studied and the working dilution of the antibody was optimized. The antibody was mixed with SMT of different concentrations prepared with PBS buffer to construct the calibration curve. The limit of detection was 0.5 ng mL^?1. The continuous SPR biosensor assay was proved to be simpler and more practical than a normal one.     

Enhanced surface plasmon resonance by Au nanoparticles immobilized on a dielectric SiO2 layer on a gold surface

This paper introduces strategies for enhancement of a surface plasmon resonance (SPR) signal by adopting colloidal gold nanoparticles (AuNPs) and a SiO₂ layer on a gold surface. AuNPs on SiO₂ on a gold surface were compared with an unmodified gold surface and a SiO₂ layer on a gold surface with no AuNPs attached. The modified surfaces showed significant changes in SPR signal when biomolecules were attached to the surface as compared with an unmodified gold surface. The detection limit of AuNPs immobilized on a SPR chip was 0.1 ng mL⁻¹ for the prostate-specific antigen (PSA), a cancer marker, as measured with a spectrophotometer. Considering that the conventional ELISA method can detect ∼10 ng mL⁻¹ of PSA, the strategy described here is much more sensitive (∼100 fold). The enhanced shift of the absorption curve resulted from the coupling of the surface and particle plasmons by the SiO₂ layer and the AuNPs on the gold surface.     

Analysis of immunoarrays using a gold grating-based dual mode surface plasmon-coupled emission (SPCE) sensor chip

We have developed a novel dual mode immunoassay platform that combines the advantages of real-time, label free measurement of surface plasmon resonance (SPR) and the highly directional surface plasmon-coupled emission (SPCE) using a gold grating-based sensor chip. Since only fluorophore-labeled analyte molecules that are close to the metal surface of the sensor chip will couple to the surface plasmon, SPCE detection is highly surface-specific leading to background suppression and increased sensitivity. Theoretical calculations were done to find SPR and SPCE angles for a sensor chip optimized for Alexa Fluor 647. We have confirmed the SPR and SPCE responses on the dual mode sensor chip using Alexa Fluor 647 labeled anti-mouse IgG. Signal fluctuation of the dual mode sensor chip reader was below 1.2% and 0.8% for SPR and SPCE, respectively. The SPR response in this configuration showed a minimum detection level of 1 μg ml(-1), and the SPCE response showed a minimum detection level of 1 ng ml(-1) for the same sample. A range of human IgG concentrations in human serum was also analyzed with the dual mode sensor chip. The SPCE measurement is more sensitive than the SPR real-time measurement, and substantially extends the dynamic range of the assay platform, as well as enabling independent measurements of co-localized analytes on the same sensor chip region of interest. Since this assay platform is capable of measuring more than 1000 spatially encoded regions of interest on a 1 cm(2) sensor chip, it has the potential for high-content analyses of biological samples with both research and clinical applications.     

A novel detection of single-stranded DNA binding protein based on ss-DNA modified chip using surface plasmon resonance microscopy

An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide,and used for thedetermination of single-stranded binding protein(SSB)by surface plasmon resonance microscopy(SPR).The experiment resultsshowed that SSB binds ss-DNA with high specificity,and relative signal of SPR response is proportional to the concentration of SSBin the range of 0.1-100 ng/mL with a detection limit(S/N=3)of 0.07 ng/mL.     

Dendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface

We report here a multistep route for the immobilization of DNA and proteins on chemically modified gold substrates using fourth-generation NH(2)-terminated poly(amidoamine) dendrimers supported by an underlying amino undecanethiol (AUT) self-assembled monolayer (SAM). Bioactive ultrathin organic films were prepared via layer-by-layer self-assembly methods and characterized by fluorescence microscopy, variable angle spectroscopic ellipsometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). The thickness of the AUT SAM base layer on the gold substrates was determined to be 1.3 nm from ellipsometry. Fluorescence microscopy and AFM measurements, in combination with analyses of the XPS/ATR-FTIR spectra, confirmed the presence of the dendrimer/biopolymer molecules on the multilayer sensor surfaces. Model proteins, including streptavidin and rabbit immunoglobulin proteins, were covalently attached to the dendrimer layer using linear cross-linking reagents. Through surface plasmon resonance measurements, we found that sensor surfaces containing a dendrimer layer displayed an increased protein immobilization capacity, compared to AUT SAM sensor surfaces without dendrimer molecules. Other SPR studies also revealed that the dendrimer-based surfaces are useful for the sensitive and specific detection of DNA-DNA interactions. Significantly, the multicomponent films displayed a high level of stability during repeated regeneration and hybridization cycles, and the kinetics of the DNA-DNA hybridization process did not appear to be influenced by surface mass transport limiting effects.     

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.     

Environmentally friendly synthesis of highly monodisperse biocompatible gold nanoparticles with urchin-like shape

We report a facile and environmentally friendly strategy for high-yield synthesis of highly monodisperse gold nanoparticles with urchin-like shape. A simple protein, gelatin, was first used for the control over shape and orientation of the gold nanoparticles. These nanoparticles, ready to use for biological systems, are promising in the optical imaging-based disease diagnostics and therapy because of their tunable surface plasmon resonance (SPR) and excellent surface-enhanced Raman scattering (SERS) activity.     

A novel polymerization of ultrathin sensitive imprinted film on surface plasmon resonance sensor

A new surface-initiated polymerization based on pasting the initiator on a sensor chip surface was applied to prepare a malachite green (MG) imprinted ultrathin film on a surface plasmon resonance (SPR) sensor. First, the initiator (2,2-azoisobutyronitrile) was pasted on the gold surface using polyvinyl chloride (PVC). The initiator-covered gold chip was then soaked in a pre-polymerization solution prepared by dissolving methacrylic acid (functional monomer), ethylene glycol dimethacrylate (cross-linker), and MG (template) in DMSO in a weighing bottle. Finally, the weighing bottle was placed in a vacuum oven and thermal-initiated polymerization was conducted at 60 °C for 16 h. This method was simple and time-saving compared with the commonly used surface-initiated polymerization. More importantly, the molecularly imprinted polymer (MIP) film prepared using this method was much thicker than that of commonly used methods; the adsorption quantity was also much larger. The MIP modified SPR sensor showed high sensitivity and selectivity as well as good stability in detecting MG. The results suggest that the ultrathin MIP film prepared using the new method in this study is suitable to serve as the recognition element of the SPR sensor.     

Immobilization of light-harvesting chlorophyll a/b complex (LHCIIb) studied by surface plasmon field-enhanced fluorescence spectroscopy

The major light-harvesting chlorophyll a/ b complex (LHCIIb) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that engage in rapid excitation energy transfer. This property makes LHCIIb potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Such applications would require the immobilization of LHCIIb (or similar dye-protein complexes) on a solid surface. In this work, the immobilization of recombinant LHCIIb is tested and optimized on functionalized gold surfaces via a histidine 6 tag (His tag) in the protein moiety. Immobilization efficiency and kinetics are analyzed by using surface plasmon resonance (SPR) and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The latter was also used to assess the integrity of immobilized LHCIIb by recording Chl b-sensitized Chl a emission spectra. Since His tags have been included in a substantial number of recombinant proteins, the immobilization technique developed here for LHCIIb presumably can be extended to a large range of other membrane and water-soluble proteins.     

Spin-coated methacrylic acid copolymer thin films for covalent immobilization of small molecules on surface plasmon resonance substrates

Hydrophilic copolymers of methacrylic acid and ethylene glycol dimethacrylate are simply and rapidly prepared as thin films by spin-coating on gold-coated glass slides with a concurrent photo-crosslinking step. Coating techniques were optimized for use on gold surfaces both separately and as part of surface plasmon resonance (SPR) sensor chips. The population of carboxylic acid functional groups as binding sites in the polymer matrix, as reflected in the corresponding hydrophilicity, could be easily adjusted through changes to the stoichiometric ratio of the monomers, allowing for good control of immobilization capacity. The polymers used adhered to the gold surfaces both with and without use of thiol moieties. Coating thickness was measured by ellipsometry and coatings of 30–40 nm thickness were routinely achieved on gold-coated slides. This dimension is dependent on the spin speed and the viscosity of the polymerization mixture applied. The polymers were further characterized by contact angle measurements and infrared spectroscopy before being applied to immobilization of the steroid cortisol in a BIAcore SPR instrument, where binding to a monoclonal antibody was studied and the surface coatings optimized for maximum specific binding capacity. Optimized surfaces could be regenerated and re-used, and have potential applications as immobilization matrices in plasmonic biosensors with a very rapid coating technique.     

Enzyme-encapsulated silica monolayers for rapid functionalization of a gold surface

We report a simple and rapid method for the deposition of amorphous silica onto a gold surface. The method is based on the ability of lysozyme to mediate the formation of silica nanoparticles. A monolayer of lysozyme is deposited via non-specific binding to gold. The lysozyme then mediates the self-assembled formation of a silica monolayer. The silica formation described herein occurs on a surface plasmon resonance (SPR) gold surface and is characterized by SPR spectroscopy. The silica layer significantly increases the surface area compared to the gold substrate and is directly compatible with a detection system. The maximum surface concentration of lysozyme was found to be a monolayer of 2.6 ng/mm(2) which allowed the deposition of a silica layer of a further 2 ng/mm(2). For additional surface functionalization, the silica was also demonstrated to be a suitable matrix for immobilization of biomolecules. The encapsulation of organophosphate hydrolase (OPH) was demonstrated as a model system. The silica forms at ambient conditions in a reaction that allows the encapsulation of enzymes directly during silica formation. OPH was successfully encapsulated within the silica particles and a detection limit for the substrate, paraoxon, using the surface-encapsulated enzyme was found to be 20 microM.