Online optimization of surface plasmon resonance-based biosensor experiments for improved throughput and confidence

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.     

Phase-polarisation contrast for surface plasmon resonance biosensors

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.     

Estimation of analyte concentration by surface plasmon resonance-based biosensing using parameter identification techniques

Surface plasmon resonance-based biosensors have been applied to the determination of macromolecule concentration. Up to now, the proposed experimental approaches have relied either on the generation of a calibration curve that exploits only a few data points from each sensorgram or on multiple injections of the unknown sample at various flow rates. In this article, we show that prior knowledge of the kinetic parameters related to the interaction of the species with a given partner could advantageously reduce the number of injections required by both aforementioned methods, thereby reducing experimental time while maintaining a good level of confidence on the determined concentrations.     

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

A surface plasmon resonance-based solution affinity assay is described for measuring the K _d of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar K _d values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays

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.     

基于SPR生物传感器的免疫学检测

利用一种全新的生物大分子相互作用检测仪表面等离子激元共振（SPR）生物传感器,对乙肝表面抗原,抗体,破伤风类毒素,抗体等生物制品进行生物特异性相互作用分析（BIA）,并对其在免疫学检测上的特征进行了探讨。     

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.     

Phase-polarisation contrast for surface plasmon resonance biosensors

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.     

Real time analysis of the RNAI-RNAII-Rop complex by surface plasmon resonance: from a decaying surface to a standard kinetic analysis

RNA loop-loop complexes are motifs that regulate biological functions in both prokaryotic and eukaryotic organisms. In E. coli, RNAI, an antisense RNA encoded by the ColE1 plasmid, regulates the plasmid replication by recognizing through loop-loop interactions RNAII, the RNA primer that binds to the plasmidic DNA to initiate the replication. Rop, a plasmid-encoded homodimeric protein interacts with this transient RNAI-RNAII kissing complex. A surface plasmon resonance (SPR)-based biosensor was used to investigate this protein-nucleic acid ternary complex, at 5 degrees C, in experimental conditions such as the protein binds either to the loop-loop complex while it dissociates or to a saturated stable RNAI-RNAII surface. The results show that RNAI hairpin dissociates from the RNAII surface 110 times slower in the presence of Rop than in its absence. Rop binds to RNAI-RNAII with an on-rate of 3.6 x 10(6) M(-1) s(-1) and an off-rate of 0.11 s(-1), resulting in a binding equilibrium constant equal to 31 nM. A Scatchard-plot analysis of the interaction monitored by SPR confirms a 1:1 complex of Rop and RNAI-RNAII as observed for non-natural Rop-loop-loop complexes.     

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.     

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

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.     

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2–2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1–2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.     

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.     

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.     

Evaluation of alpha-D-mannopyranoside glycolipid micelles-lectin interactions by surface plasmon resonance method

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.     

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。     

Circulating microRNA breast cancer biomarker detection in patient sera with surface plasmon resonance imaging biosensor

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.     

Surface plasmon resonance based biosensor technique: A review

Optical Surface plasmon resonance (SPR) biosensors represent the most advanced and developed optical label‐free biosensor technology. Optical SPR biosensors are a powerful detection and analysis tool that has vast applications in environmental protection, biotechnology, medical diagnostics, drug screening, food safety and security. This article reviews the recent development of SPR biosensor techniques, including bulk SPR and localized SPR (LSPR) biosensors, for detecting interactions between an analyte of interest in solution and a biomolecular recognition. The concepts of bulk and localized SPs and the working principles of both sensing techniques are introduced. Major sensing advances on biorecognition elements, measurement formats, and sensing platforms are presented. Finally, the discussions on both biosensor techniques as well as comparison of both SPR sensing techniques are made. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)