Synthesis of hydrophobic nanoparticles for real‐time lysozyme detection using surface plasmon resonance sensor

Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR‐based sensor is developed to detect lysozyme with hydrophobic poly (N‐methacryloyl‐(L)‐phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection‐Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM⁻¹, and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real‐time, label‐free, and a low‐detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.     

A surface plasmon resonance probe with a novel integrated reference sensor surface

A surface plasmon resonance (SPR) sensor probe with integrated reference surface is described. In order to fabricate the integrated reference surface, two dielectric layers with different thickness were deposited on the single gold SPR sensor surface via plasma polymerization of hexamethyldisiloxane. The working sensor surface was a 34 nm dielectric layer with immobilized bovine serum albumin (BSA) antigen and an adjacent thin 1 nm dielectric layer without BSA provided reference surface. A specific immunoreaction of anti-BSA antibody was detected after immersion of the SPR probe into sample solution. Simultaneous observation of reference and working surface response enabled determination of the immunoreaction without the need for the baseline measurement. Moreover, compensation of nonspecific adsorption could be confirmed using anti-human serum albumin antibody.     

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.     

Detection of porphyrin using a short peptide immobilized on a surface plasmon resonance sensor chip

In this paper the development and feasibility of a novel detection system for a low molecular weight chemical, in which a peptide was utilized as a binding molecule, are described. Surface plasmon resonance (SPR) apparatus was used as a transducer. The porphyrin binding peptide, PSP2, was used as a model peptide ligand, while a porphyrin derivative, H₂TMpyP, was used as a model low-molecular-weight chemical. PSP2 was covalently immobilized onto the SPR sensor chip and SPR measurement using the PSP2-immobilized chip for various concentrations of porphyrin was carried out. H₂TMpyP was detectable in the range from 100 ng ml⁻¹ to 10 μg ml⁻¹ with a linear correlation and good precision and the PSP2-immobilized chip could be regenerated within 1 min after measurement in this system. From comparison of the detection manners of three porphyrin derivatives, the ability of a short peptide to discriminate between differences in molecular structure was demonstrated. Moreover, the self-assembled monolayer (SAM) of PSP2 was successfully prepared on the gold substrate and H₂TMpyP could be detected using the PSP2-SAM chip.     

Surface plasmon resonance imaging on a microchip for detection of DNA-modified gold nanoparticles deposited onto the surface in a non-cross-linking configuration

We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a product of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10nm). The height of the detected volume size is 40-70nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.     

Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.     

Surface plasmon resonance sensor using molecularly imprinted polymer for detection of sialic acid

A surface plasmon resonance (SPR) sensor using a molecularly imprinted polymer-coated sensor chip for the detection of sialic acid was developed. The thinly coated polymer was prepared by co-polymerizing N,N,N-trimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate and ethyleneglycol dimethacrylate in the presence of p-vinylbenzeneboronic acid ester with sialic acid. The sensor showed a selective response to ganglioside of which sialic acid is located at the non-reducing end and gave a linear relationship from 0.1 to 1.0 mg of ganglioside.     

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor–coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K_d) by 8–11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3,7-dihydroxy-5β-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.     

Spectral surface plasmon resonance imaging for the detection of clenbuterol via three-dimensional immobilization of bioprobes

A method of immobilizing clenbuterol (CLEN) on the sensor chip for spectral surface plasmon resonance imaging (SPRi) was experimentally investigated. The bioprobes on the sensor chip were prepared by immobilizing bovine serum albumin (BSA) protein and conjugating CLEN molecules to BSA, which provides more active points and free orientations for specific binding. The calibration curve showed that the wavelength resonance shift decreased as the concentration of CLEN analyte increased, consistent with the inhibition principle. The limit of detection (LOD) was estimated to be 6.32 μg/ml. This method proved to be highly specific, high throughput, label free, and operationally convenient.     

Binding properties of antimicrobial agents to dipeptide terminal of lipid II using surface plasmon resonance

We developed a surface plasmon resonance (SPR) assay to estimate the interactions of antimicrobial agents with the dipeptide terminal of lipid II (d-alanyl-d-alanine) and its analogous dipeptides (l-alanyl-l-alanine and d-alanyl-d-lactate) as ligands. The established SPR method showed the reproducible immobilization of ligands on sensor chip and analysis of binding kinetics of antimicrobial agents to ligands. The ligand-immobilized chip could be used repeatedly for at least 200 times for the binding assay of antimicrobial agents, indicating that the ligand-immobilized chip is sufficiently robust for the analysis of binding kinetics. In this SPR system, the selective and specific binding characteristics of vancomycin and its analogs to the ligands were estimated and the kinetic parameters were calculated. The kinetic parameters revealed that one of the remarkable binding characteristics was the specific interaction of vancomycin to only the d-alanyl-d-alanine ligand. In addition, the kinetic binding data of SPR showed close correlation with the antimicrobial activity. The SPR data of other antimicrobial agents (e.g., teicoplanin) to the ligands showed correlation with the antimicrobial activity on the basis of the therapeutic mechanism. Our SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to the dipeptide terminal of lipid II.     

Label-free immunosensing for alpha-fetoprotein in human plasma using surface plasmon resonance

In this study, we attempted to develop a surface plasmon resonance (SPR)-based immunoassay sensor to detect alpha-fetoprotein (AFP) in human plasma at the nanogram level, as is required for clinical diagnosis of hepatocellular tumors. A self-assembled monolayer (SAM) surface of tri(ethylene glycol) (TEG) and carboxyl group-terminated hexa(ethylene glycol) (HEG) was employed to suppress the nonspecific adsorption of plasma components onto the sensor surface. AFP was detected by a sandwich-type immunoassay using two kinds of antibodies, primary and secondary, in this system. The SPR signal shift was further enhanced by applying an antibody (polyclonal) against the second antibody. With this method, the SPR signals were highly intensified, and so nanogram levels (ng/ml) of AFP could be easily detected with a high signal/noise ratio, as is necessary for clinical diagnosis. It is expected that our SPR-based immunoassay method can also be applicable to the detection of several other tumor markers that are present in low concentrations in human blood.     

Selection of RNA aptamers against human influenza virus hemagglutinin using surface plasmon resonance

Aptamers are functional nucleic acids possessing high affinity and specificity to their cognate ligands and are isolated from a library of nucleic acids by iterative rounds of selection and amplification. In the current study, we used surface plasmon resonance (Biacore) as an efficient methodology for selecting aptamers that bind to hemagglutinin (HA) of human influenza virus. This procedure allowed us to monitor and select the target-bound aptamers specifically and simultaneously. These studies not only yielded an aptamer that binds to the HA of influenza virus with high affinity but also revealed the consensus sequence, 5'-GUCGNCNU(N)(2-3)GUA-3, for HA recognition.     

Real-time analysis of antibody interactions with whole enveloped human cytomegalovirus using surface plasmon resonance

Human cytomegalovirus (CMV) is a large enveloped virus that encodes multiple glycoproteins required for virus-cell binding and fusion. To assess the binding properties of antibodies with target glycoprotein in a natural context of infection, we investigated the feasibility of using the surface plasmon resonance (SPR) technique for studying the direct binding of antibodies with CMV virions. Direct immobilization of whole virions to sensor surface and a surface regeneration procedure allowed for quantitative and reproducible measurements of binding affinity and binding kinetics of antibody–whole virion interactions. The conformational and functional integrity of viral particles was not compromised by the regeneration condition as evaluated with antibodies recognizing conformational epitopes and by electron microscopy. Binding of an irrelevant antibody was not observed, indicating the high specificity of the method. A panel of anti-gB antibodies was measured and the binding affinities correlated fairly well with those determined by ELISA. These data demonstrated that the interaction of anti-gB antibody with whole virion of large enveloped CMV can be quantitatively studied using SPR. This method has been successfully applied for screening and selection of anti-CMV antibodies and can be potentially extended to study antibody–glycoprotein interactions of other related herpesviruses.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

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.     

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.     

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.     

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.     

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.