A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST–GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2°. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays. 相似文献
Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml. 相似文献
A technique of phase-polarisation contrast (PPC) for the enhancement of the contrast of a surface plasmon resonance (SPR) intensity profile is proposed and experimentally realised. The technique exploits the peculiarities of light phase and polarisation behaviour under SPR. It applies to non-optimum SPR coupling conditions and enables one to lower the resonant minimum of reflected intensity nearly to zero, and hence to increase substantially the ratio of the intensity from the resonance to that at the minimum. We observed the contrast enhancement by more than one order of magnitude when we applied the PPC scheme. The PPC can be efficiently employed in commercial SPR sensors, as it significantly reduces restrictions on allowable parameters of SPR-supporting metal films and biomolecular layers immobilised on them, facilitates SPR observation, and increases the accuracy of SPR shift measurements. 相似文献
Although it has been revealed that astrocytes, generally known as star‐shaped glial cells, play critical roles in the functions of central nervous system, there have been few efforts to directly modulate their activities and responses. In this study, an optical stimulation strategy for producing intracellular Ca2+ transients of astrocytes is demonstrated using near‐infrared (NIR) light and localized surface plasmon resonance. It is presented that NIR stimulation of micro‐second duration combined with gold nanorods (GNRs) efficiently produces stronger Ca2+ transients of astrocytes, which seems to be associated with a local heat generation by photothermal effects of GNRs. Since the proposed scheme can directly activate astrocytes with a high reliability, it is expected that GNR‐mediated NIR stimulation could be utilized to facilitate minimally invasive physiological studies on the astrocyte functions.
Photos of intracellular Ca2+ transient of astrocytes with membrane‐bound GNRs after optical stimulation at 30 s. 相似文献
We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody–carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM–CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For the amplification of the SPR signal, the CNT was conjugated with a polyclonal antibody, and then the conjugates were reacted with antibodies coupled with the target proteins. This amplification strategy increases the dynamic range of the immunoassays and enhances the detection sensitivity. The SPR immunoassays, combined with the CNT-assisted signal amplification method, provided a wide dynamic range over four orders of magnitude for both EPO and GM–CSF (0.1–1000 ng/ml). The CNT amplification method is expected to realize the detection of picogram levels and a wide dynamic detection range of multiple proteins, enabling it to offer a robust analysis tool for the development of biopharmaceutical production. 相似文献
In this article, we report for the first time, the detection of circulating miRNA as a breast cancer biomarker in patient sera using surface plasmon resonance imaging biosensor. The advantage of this approach lies in the rapid, label-free and sensitive detection. The sensor excites plasmonic resonance on the gold sensor surface and specific DNA-miRNA molecular bindings elucidate responses in the plasmonic resonance image. Experiments of detecting synthetic miRNA molecules (miR-1249) were performed and the sensor resolution was found to be 63.5 nM. The sensor was further applied to screen 17 patient serum samples from National Cancer Centre Singapore and Tan Tock Seng Hospital. Sensor intensity response was found to differ by 20% between malignant and benign cases and thus forms, a potential and an important metric in distinguishing benignity and malignancy. 相似文献
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. 相似文献
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. 相似文献
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 ProLinkerTM 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. 相似文献
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). 相似文献
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. 相似文献
The emergence of surface plasmon resonance-based optical biosensors has facilitated the identification of kinetic parameters for various macromolecular interactions. Normally, these parameters are determined from experiments with arbitrarily chosen periods of macromolecule and buffer injections, and varying macromolecule concentrations. Since the choice of these variables is arbitrary, such experiments may not provide the required confidence in identified kinetic parameters expressed in terms of standard errors. In this work, an iterative optimization approach is used to determine the above-mentioned variables so as to reduce the experimentation time, while treating the required standard errors as constraints. It is shown using multiple experimental and simulated data that the desired confidence can be reached with much shorter experiments than those generally performed by biosensor users. 相似文献
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. 相似文献
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. 相似文献
It is established that achieving higher binding affinities in carbohydrate-protein interactions requires multivalent presentations of the sugar ligands at the receptor binding site. Several inhibition, calorimetric, mass balance, and other studies have reiterated the beneficial effects of molecular level clustering of the sugar ligands for tight binding to the receptors. We have undertaken an effort to study the multivalent effects involving larger assemblies, represented by micelles, and their lectin interactions. The micelles were constituted with monomer bearing one- or two-sugar moieties at the monomolecular level and with varying the distances between the sugar moieties. Micellar aggregation studies and dynamic light scattering (DLS) studies afforded details of the aggregation numbers and the hydrodynamic diameters of various glycolipid (GL) micelles. The GL micelles were used as analytes of surface plasmon resonance (SPR) experiments on a lectin concanavalin A (Con A)-immobilized surface. SPR studies of the micelle-lectin interactions demonstrate that the ligand-receptor binding can be fit into the bivalent analyte model of interaction. Furthermore, micelles formed from two-sugar containing GLs are able to elicit favorable kinetic association rate constants in comparison to the micelles constituted with one-sugar containing GLs. The kinetic rate constants across the micelles and the effect of the sugar valencies in the GLs are discussed. 相似文献