Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.     

Laser exposure of gold nanorods can induce intracellular calcium transients

Uncoated and poly(styrene sulphonate) (PSS)‐coated gold nanorods were taken up by NG108‐15 neuronal cells. Exposure to 780 nm laser light at the plasmon resonance wavelength of the gold nanorods was found to induce intracellular Ca²⁺ transients. The higher Ca²⁺ peaks were observed at lower laser doses, with the highest levels obtained at a radiant exposure of 0.33 J/cm². In contrast, the cells without nanoparticles showed a consistently small response, independent of the laser dose. These initial results open up new opportunities for peripheral nerve regeneration treatments and for more efficient optical stimulation techniques. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Characterization of a new maleimido functionalization of gold for surface plasmon resonance spectroscopy

Para‐maleimidophenyl (p‐MP) modified gold surfaces have been prepared by one‐step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N‐terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p‐MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read‐out for a broad variety of biomolecular interactions on the same chip. Copyright © 2014 John Wiley & Sons, Ltd.     

Cancer cell‐specific protein delivery by optoporation with laser‐irradiated gold nanorods

The delivery of macromolecules into living cells is challenging since in most cases molecules are endocytosed and remain in the endo‐lysosomal pathway where they are degraded before reaching their target. Here, a method is presented to selectively improve cell membrane permeability by nanosecond laser irradiation of gold nanorods (GNRs) with visible or near‐infrared irradiation in order to deliver proteins across the plasma membrane, avoiding the endo lysosomal pathway. GNRs were labeled with the anti‐EGFR (epidermal growth factor receptor) antibody Erbitux to target human ovarian carcinoma cells OVCAR‐3. Irradiation with nanosecond laser pulses at wavelengths of 532 nm or 730 nm is used for transient permeabilization of the cell membranes. As a result of the irradiation, the uptake of an anti‐Ki‐67 antibody was observed in about 50 % of the cells. The results of fluorescence lifetime imaging show that the GNR detached from the membrane after irradiation.     

表面等离子体共振技术在药物研究中的应用

表面等离子体共振(surface plasmon resonance,SPR)技术作为一种新型的免标记、实时在线研究生物分子间相互作用的高灵敏传感技术,已经在生命科学领域中得到了大量应用。该文简要介绍了SPR生物传感器的基本原理,重点评述了其在新药筛选和药物作用机制方面的研究进展,并对其前景进行了展望。     

Localized surface plasmon resonance based gold nanobiosensor: Determination of thyroid stimulating hormone

An immunoassay method based on the peak shift of the localized surface plasmon resonance (LSPR) absorption maxima has been developed for the determination of the thyroid stimulating hormone (TSH) in human blood serum. The anti-TSH antibody was adsorbed on the synthesized gold nanoparticles by electrostatic forces. The efficiency of the nanobiosensor was improved by optimizing the factors affecting the probe construction such as the pH and the antibody to gold nanoparticles ratio. Dynamic light scattering was applied for the characterization of the constructed probe. The amount of peak shift of the LSPR absorption maxima was selected as the basis for determination of TSH antigen. The linear dynamic range of 0.4–12.5 mIU L⁻¹ and the calibration sensitivity of 1.71 L mIU⁻¹ were obtained. The human control serum sample was analyzed for TSH by constructed nanobiosensor and the acceptable results were obtained.     

Insulin receptor‐insulin interaction kinetics using multiplex surface plasmon resonance

Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor‐insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real‐time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR‐A). We used a scaffold approach in which anti‐insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR‐A capture. The multiplex SPR system (ProteOn XPR36?, Bio‐Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two‐site binding pattern, consistent with the literature, with a high‐affinity site (dissociation constant K_D1 = 38.1 ± 0.9 nM) and a low‐affinity site (K_D2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K_D1 = 73.2 ± 1.8 nM and K_D2 = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high‐affinity site was converted to the low‐affinity site. The new SPR assay enables insulin‐eIR‐A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Antibody-based surface plasmon resonance detection of intact viral pathogen

Surface plasmon resonance (SPR) technique was used to directly detect an intact form of insect pathogen: the baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). An SPR sensor chip with three bio-functional layers was used to detect the intact AcMNPV: amine-reactive crosslinker with a disulfide bond that chemisorbs to gold film, Protein A, and a mouse IgG monoclonal antibody raised against a surface protein of the target viral pathogen. A two-channel (reference & test) micro-fluidic SPR system is used for reliable measurement. Bio-specific response to the AcMNPV is compared with the response for tobacco mosaic virus (TMV) as control. Successive exposure of the sensor chip to both viruses verifies a specific response to AcMNPV. This serves as a prerequisite to the development of a new type of viral pathogen detection sensors.     

Coupling of chemical extraction and expanded-bed adsorption for simplified inclusion-body processing: optimization using surface plasmon resonance

Integration of the chemical extraction of recombinant inclusion-body protein from Escherichia coli, and its recovery by metal-affinity expanded-bed adsorption (IMAC-EBA) under denaturing conditions, was investigated. The viral coat protein L1 with a hexa-histidine tag was expressed in Escherichia coli HMS174(DE3) as a model protein. Interference of released host DNA with adsorbent fluidization in the EBA step was solved by selective precipitation using spermine and low-speed centrifugation. However, the capacity and selectivity of the adsorbent for L1 remained lower than anticipated. The binding of L1 to immobilized Ni(2+) was therefore studied in detail using surface plasmon resonance (SPR). The Tris buffer and ethylene-diamine tetraacetic acid (EDTA) used in the extraction mixture were found to interfere significantly with the L1-Ni(2+) interaction. The SPR studies suggest that L1 binding could be improved by replacing the Tris buffer with HEPES and by adding CaCl(2) to inactivate the EDTA. The modified chemical extraction conditions resulted in effective L1 extraction from cytoplasmic inclusion bodies, at high cell density (OD(600 )= 80) and without the use of reducing agent, into a medium optimized for subsequent IMAC recovery. The modified buffer conditions resulted in an improved binding capacity and a good L1 purification factor (12.7) and recovery yield (71%). This work demonstrates that it is possible to reduce the complexity and hence the cost associated with traditional processes used to prepare purified denatured protein, ready for refolding, from cytoplasmic inclusion bodies.     

Development of a localized surface plasmon resonance-based gold nanobiosensor for the determination of prolactin hormone in human serum

A localized surface plasmon resonance immunoassay has been developed to determine prolactin hormone in human serum samples. Gold nanoparticles were synthesized, and the probe was prepared by electrostatic adsorption of antibody on the surfaces of gold nanoparticles. The pH and the antibody-to-gold nanoparticle ratio, as the factors affecting the probe functions, were optimized. The constructed nanobiosensor was characterized by dynamic light scattering. The sensor was applied for the determination of prolactin antigen concentration based on the amount of localized surface plasmon resonance peak shift. A linear dynamic range of 1–40 ng ml⁻¹, a detection limit of 0.8 ng ml⁻¹, and sensitivity of 10 pg ml⁻¹ were obtained. Finally, the nanobiosensor was applied for the determination of prolactin in human control serum sample.     

Surface plasmon resonance imaging analysis of hexahistidine-tagged protein on the gold thin film coated with a calix crown derivative

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His₆-Ub-hPTHF(1–34)) expressed inEscherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinker^TM B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrices. The soluble and insoluble fractions of anE. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His₆-Ub-hPTHF (1–34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.     

Identification of carbohydrates binding to lectins by using surface plasmon resonance in combination with HPLC profiling

A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of ligand-fishing. Labeling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNase B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing blood group H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose could selectively be picked from the analyte solution as demonstrated by HPLC profiling.     

Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance

It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE® 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.     

Compensation for loss of ligand activity in surface plasmon resonance experiments

The determination of equilibrium binding constants is an important aspect of the analysis of protein-protein interactions. In recent years surface plasmon resonance experiments (e.g., with a BIAcore instrument) have provided a valuable experimental approach to determining such constants. The standard method is based on measuring amounts of analyte bound at equilibrium for different analyte concentrations. During the course of a typical surface plasmon resonance experiment the measured equilibrium levels for a given analyte concentration often decrease. This appears to be due to a loss of activity of the protein coupled to the sensor chip or other phenomena. The loss in signal can lead to an erroneous determination of the equilibrium constant. A data analysis approach is introduced that aims to compensate for the loss of activity so that its influence on the results of the experiments is reduced.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.     

Comparison of a carboxylated terthiophene surface with carboxymethylated dextran layer for surface plasmon resonance detection of progesterone

Functionalization of a gold surface is usually accomplished by covalent binding via self-assembled monolayers (SAMs) on the gold surface, followed by attachment of flexible polymeric linker layers such as dextran hydrogels. However, these techniques require multiple steps and also have nonspecific interactions and steric problems. In this study, a self-assembled carboxylated terthiophene monolayer was formed onto a gold surface to create a sensitive and stable surface plasmon resonance (SPR) biosensing system. Compared with a commercial carboxymethyl dextran chip (CM5), the terthiophene SAM surface provided more than six times more antibody-binding signals and nearly three times the SPR assay sensitivity for progesterone (P₄).