Nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor

Nanodiscs are self-assembled soluble discoidal phospholipids bilayers encirculated by an amphipathic protein that together provide a functional stabilized membrane disk for the incorporation of membrane-bound and membrane-associated molecules. The scope of the present work is to investigate how nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera toxin and its glycolipid receptor G(M1) constitute a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodiscs containing the glycolipid receptor G(M1) in lipid bilayers, enabling measurements of binding of its soluble interaction partner cholera toxin B subunit to the receptor with the sensorchip-based surface plasmon resonance (SPR) technology. The measured stoichiometric and kinetic values of the interaction are in agreement with those reported by previous studies, thus providing proof-of-principle that nanodiscs can be employed for kinetic SPR studies.     

Mass sensitivity calculation of the protein layer using love wave SAW biosensor

Love waves, a variety of surface acoustic waves (SAWs), can be used to detect very small biological surface interactions and so have a wide range of potential applications. To demonstrate the practicality of a Love wave SAW biosensor, we fabricated a 155-MHz Love wave SAW biosensor and compared it with a commercial surface Plasmon resonance (SPR) using glycerol-water solution with known densities and viscosities to calibrate the response signals of the biosensors. And the mass per unit area of anti-mouse IgG bound with protein G onto the sensitive layer of the biosensor was calculated on the basis of the calibration result. The sensitivity of the Love wave SAW biosensor was the same as or greater than that of the SPR biosensor. Furthermore, the Love wave SAW biosensor was capable of measuring a much wider range of viscosities than the SPR biosensor. Although the operating principle of the Love wave SAW biosensor is completely different from that of the SPR biosensor, the subtle changes in the viscoelastic properties of the biological layer that accompany biological binding reactions on the sensitive layer can be monitored and measured in the same ways as with the SPR biosensor.     

Direct immobilization of protein g variants with various numbers of cysteine residues on a gold surface

Protein G is an antibody binding protein, which specifically targets the Fc region of an antibody. It therefore has been widely used to immobilize different types of antibodies in numerous immunoassays. Here, we have engineered Streptococcus protein G to contain various numbers of cysteine residues at the N-terminus and therefore to form well-oriented protein G films on bare gold. SPR and SPR imaging analyses indicated that a gold surface treated with cysteine-tagged protein G possesses a superior antibody binding ability compared to one treated with tag-free protein G. AFM images indicated a higher surface coverage by antibody binding on the cysteine-tagged protein G surface than the intact protein G surface. The proper orientation of cysteine-tagged protein G on a gold surface also afforded better orientation of immobilized antibodies, resulting in enhanced antigen detection. Moreover, the protein G surfaces maintained their high antibody binding ability during multiple rounds of antibody interaction tests. The cysteine-tagged protein G constructed in this study can be a valuable link for oriented antibody immobilization in a variety of immunosensors.     

A kinetic study of analyte-receptor binding and dissociation for surface plasmon resonance biosensors applications

A fractal analysis, which takes into account the effect of surface heterogeneity brought about by ligand immobilization on the reaction kinetics in surface plasmon resonance (SPR) biosensors, is presented. The binding and dissociation of estrogen receptors (ERs), ERa and ER/spl alpha/ and ER/spl beta/, in solution to different ligands immobilized on the SPR biosensor is analyzed within the fractal framework. The heterogeneity on the biosensor surface is made quantitative by using a single number, the fractal dimension D/sub f/. The analysis provides physical insights into the binding of these receptors to different ligands and compounds, particularly the endocrine disrupting compounds (EDCs). These EDCs have deleterious effects on humans and on wildlife. Single- and dual-fractal models were employed to fit the ER-binding data obtained from the literature. Values of the binding and dissociation rate coefficient and fractal dimensions were obtained from a regression analysis provided by Corel Quattro Pro, 8.0. Values for the affinity K/sub D/(=k/sub d//k/sub a/) were also calculated. This provides us with some extra flexibility in designing biomolecular assays. The analysis should provide further information on the mode of action and interaction of EDCs with the ERs. This would help in the design of agents and modulators against these EDCs.     

Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions

The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format.     

Surface plasmon resonance investigations of human epidermal growth factor receptor 2

This investigation utilizes surface plasmon resonance (SPR) spectroscopy to detect and quantify human epidermal growth factor receptor 2 (HER-2), an oncogene product that is over-expressed in some aggressive forms of breast cancer. Specifically, the HER-2 trans-membrane protein p185 and its extra cellular fragment p105 are analytes targeted in this work by using a gold-based biosensor slide on which an anti-HER-2 antibody has been immobilized by attachment to Protein G that is fixed to the gold film. A detection limit of > or =11 ng/mL for p185 resulted when trastuzumab was used as the anti-HER-2 antibody on the biosensor slide. Experiments with semi-purified p105 revealed that it binds weakly and reversibly to trastuzumab, therefore complicating its detection and quantification. Results of studies that reacted a 13-amino-acid peptide (PP13) from the HER-2 kinase domain with its specific antibody were critically different than p185 and p105 studies. Spectral analysis of the reflectivity at constant bulk buffer refractive index revealed a progressive negative SPR shift over time. A negative shift suggests that a loss of protein mass from the anti-PP13 antibody-Protein G biosensor is occurring. Several possibilities that may explain these negative SPR shifts are discussed.     

Study on protein conformation and adsorption behaviors in nanodiamond particle-protein complexes

In the present research, the conformation of bovine serum albumin (BSA) in the nanodiamond particle (ND)-BSA complex was studied by Fourier transform infrared spectroscopy, fluorescence spectroscopy, UV-vis spectroscopy, and circular dichroism spectroscopy. The spectroscopic study revealed that most BSA structural features could be preserved in the complex though the BSA underwent conformational changes in the complex due to ND-BSA interaction. In addition, BSA adsorption isotherms and zeta-potential measurements were employed to investigate the pH dependence of the ND-BSA interaction. The changes in surface charge of the ND-BSA complex with pH variations indicated that the binding of BSA to ND might lead to not only the adsorption of BSA onto the ND surface but also the partial breakup of ND aggregates into relatively small ND-BSA aggregates because of the strong binding force between ND and BSA. The results show that ND is an excellent platform for protein immobilization with high affinity and holds great potential to be used for biosensor applications.     

Imaging technique for the screening of protein-protein interactions using scattered light under surface plasmon resonance conditions

We propose a novel technique to detect protein-protein interactions in microarray format. The technique involves measuring scattered light under surface plasmon resonance (SPR) conditions. We have shown that the maximum scattering angle correlates with the traditionally employed reflection minimum. Panoramic scanning of scattered light under SPR conditions has all the functional advantages of the SPR technique. In addition, the proposed technique simplifies device design, increases the dynamic range of analysis, and integrates data with those from surface-plasmon field-enhanced fluorescence spectroscopy. We demonstrate the technique by showing direct protein-protein interaction between protein A and either rabbit antibodies or human serum.     

Detection of synthetic RGDS(PO3H2)PA peptide adsorption using a titanium surface plasmon resonance biosensor

The purpose of this study was to measure the time-dependent chemical interaction between synthetic RGDS(PO₃H₂)PA (P-RGD) peptide and titanium surfaces using a titanium surface plasmon resonance (SPR) biosensor and to determine the degree of peptide immobilization on the surfaces. An SPR instrument for ‘single-spot’ analysis was used for nanometer-scale detection of biomolecular adsorption using a He–Ne laser light according to Knoll’s method. The oxidized titanium surface was etched when exposed to H₃PO₄ solutions with a pH of 2.0 or below. The amount of P-RGD adsorbed at pH 1.9 was approximately 3.6 times as much as that at pH 3.0 (P < 0.05). P-RGD naturally adsorbed on the oxidized titanium surface as a consequence of the bonding and dissociation mechanism of the phosphate functional group. Furthermore, the control of pH played a very important role in the interaction between P-RGD and the surface. These findings show that pH control may promote progressive binding of biomolecules with the phosphate functional group to the titanium surface.     

Self-directed and self-oriented immobilization of antibody by protein G-DNA conjugate

A versatile biolinker for efficient antibody immobilization was prepared by site-specific coupling of protein G to DNA oligonucleotide. This protein G-DNA conjugate ensures the controlled immobilization of an antibody to the intended area on the surface of bioassay chips or particles, while maintaining the activity and orientation of the bound antibody. Streptococcus protein G tagged with a cysteine residue at the N-terminus was chemically linked to amine-modified, single-stranded DNA. SPR analysis indicated that the protein G-DNA conjugates sequence-specifically bind to complementary surface-bound DNA probes. More importantly, the resulting protein G, which is hybridized onto the DNA surface, possesses a greater antibody/antigen binding ability than even properly oriented protein G linked on the chip surface by chemical bonding. Antibody targeting on glass slides could also be achieved by using this linker system without modifying or spotting antibodies. Moreover, the protein G-DNA conjugate provided a simple but effective method to label DNA-functionalized gold nanoparticles with target antibodies. The DNA-linked protein G construct introduced in this study offers a useful strategy to manage antibody immobilization in many immunoassay systems.     

Analysis of C-reactive protein on amide-linked N-hydroxysuccinimide-dextran arrays with a spectral surface plasmon resonance biosensor for serodiagnosis

A new label-free array system using amide-linked (AL) NHS-dextran and a spectral SPR biosensor are presented for the high-throughput analysis of C-reactive protein (CRP) in human sera. The AL NHS-dextran layer on the surface of gold arrays was composed of an amide linkage between NHS-modified carboxymethyl-dextran and amine-modified 11-mercaptoundecanoic acid. The topology of the AL NHS-dextran layer was analyzed by atomic force microscopy, and it was found to be superior to the previously used epoxide-linked carboxymethyl-dextran layer in its immobilization of proteins. Specific immunoreactions and a dose-dependent increase of SPR signals were demonstrated on the AL NHS-dextran layer. Then, the label-free array system was successfully applied to the rapid analysis of CRP in 120 human sera. CRP levels in human sera determined by the array-based spectral SPR biosensor showed a good correlation with those determined by the latex-enhanced turbidimetry immunoassay (n = 120, r = 0.945, p < 0.0001). Thus, the array-based spectral SPR biosensor based on the AL NHS-dextran surface is a potential system for rapid and label-free serodiagnosis of human diseases.     

Microfluidic electrochemical sensor array for characterizing protein interactions with various functionalized surfaces

We present a unique microfluidic platform to allow for quick and sensitive probing of protein adsorption to various functionalized surfaces. The ability to tailor a sensor surface for a specific analyte is crucial for the successful application of portable gas and fluid sensors and is of great interest to the drug screening community. However, choosing the correct surface chemistry to successfully passivate against nonspecific binding typically requires repeated trial and error experiments. The presented device incorporates an array of integrated electrochemical sensors for fast, sensitive, label-free detection of these binding interactions. The layout of the electrodes allows for loading various surface chemistries in one direction while sensing their interactions with particular compounds in another without any cross-contamination. Impedance data is collected for three commonly used passivation compounds (mercaptohexanol, polyethylene glycol, and bovine serum albumin) and demonstrates their interaction with three commonly studied proteins in genetic and cancer research (cAMP receptor protein, tumor necrosis factor α, and tumor necrosis factor β). The ability to quickly characterize various surface interactions provides knowledge for selecting optimal functionalization for any biosensor.     

Analytical characterization of chiral drug-protein interactions: comparison between the optical biosensor (surface plasmon resonance) assay and the HPLC perturbation method

Two modern, fundamentally different methods were used for a detailed investigation of enantioselective drug-protein interactions, a surface plasmon resonance (SPR)-based Biacore 2000 biosensor assay and the previously validated HPLC perturbation method (HPLC-PM). This is the first time SPR has been used for this purpose. The fundamental features of the two methods were investigated, and the consequences for operation and data evaluation were addressed. With HPLC-PM, chiral data could be obtained directly from the racemic mixture, whereas a separate analysis of each pure enantiomer was required to obtain chiral data with SPR. It was shown that if chirality is not attributed in the SPR analysis, misleading average racemic binding constants will be obtained. Both drug and protein consumption were considerably higher with HPLC-PM. HPLC-PM was found to be best suited for measurements of weak affinity interactions, whereas the SPR method was best for strong interactions. With both methods, the presence of DMSO in the samples severely affected the interactions, introducing errors. The binding of the beta-blockers alprenolol and propranolol to Cel7a cellulase was used as a model system. These methods gave results that agreed quite well qualitatively, but considerable quantitative deviations were sometimes obtained.     

In situ biosensing with a surface plasmon resonance fiber grating aptasensor

Surface plasmon resonance (SPR) biosensors prepared using optical fibers can be used as a cost-effective and relatively simple-to-implement alternative to well established biosensor platforms for monitoring biomolecular interactions in situ or possibly in vivo. The fiber biosensor presented in this study utilizes an in-fiber tilted Bragg grating to excite the SPR on the surface of the sensor over a large range of external medium refractive indices, with minimal cross-sensitivity to temperature and without compromising the structural integrity of the fiber. The label-free biorecognition scheme used demonstrates that the sensor relies on the functionalization of the gold-coated fiber with aptamers, synthetic DNA sequences that bind with high specificity to a given target. In addition to monitoring the functionalization of the fiber by the aptamers in real-time, the results also show how the fiber biosensor can detect the presence of the aptamer's target, in various concentrations of thrombin in buffer and serum solutions. The findings also show how the SPR biosensor can be used to evaluate the dissociation constant (K(d)), as the binding constant agrees with values already reported in the literature.     

Differential impedance spectroscopy for monitoring protein immobilization and antibody-antigen reactions

This work describes the theoretical and experimental approaches for monitoring the interfacial biomolecular reaction between immobilized antibody and the antigen binding partner using novel differential impedance spectroscopy. The prerequisite of any biosensor is the immobilization of macromolecules onto the surface of a transducer. It is clear that the function of most macromolecules changes from what is observed in solution once immobilization has occurred. In the worst case, molecules entirely lose their binding activity almost immediately after immobilization. Certain conditions (e.g., denaturation, interfacial effects based on ionic strength, surface charge, dielectric constants, etc.) at interfaces are responsible for alterations of binding activity; it is not clear whether a combination of such processes is understood. However, these processes in combination must be reliably modeled in order to predict the outcome for most macromolecules. This work presents the theoretical and practical means for elucidating the surface reactivity of biomolecular reagents using ion displacement model with antibody-antigen (Ab-Ag) reaction as the test case. The Ab-Ag reaction was directly monitored using a dual-channeled, impedance analyzer capable of 1 measurement/s using covalent immobilization chemistry and polymer-modified electrodes in the absence of a redox probe. The evidence of Ab-Ag binding was revealed through the evolution of differential admittance. The surface loading obtained using the covalent immobilization chemistry was 9.0 x 10(16)/cm2, whereas with polymer-modified electrodes, the surface loading was 9.0 x 10(15)/cm2, representing a 10 times increase in surface reactivity. The proposed approach may be applicable to monitoring other surface interfacial reactions such as DNA-DNA interactions, DNA-protein interactions, and DNA-small molecule interactions.     

Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance.

Small molecules are difficult to directly detect using commercially available surface plasmon resonance (SPR) instruments. This is because low molecular weight compounds do not have sufficient mass to cause a measurable change in refractive index. Refractive index is sensitive, however, to other properties besides the mass of the analyte. Recently the detection of substantial conformational changes for immobilized proteins using SPR has been reported. However, this property has not yet been exploited for the detection of low molecular weight ligand binding to immobilized protein receptors. Here we demonstrate that ligand-induced conformational changes can be used to monitor the binding of small molecules to immobilized maltose-binding protein and tissue transglutaminase. Ligand binding to a receptor that decreases in hydrodynamic radius yielded a net decrease in refractive index. A net positive change in refractive index was observed for a receptor that increases in hydrodynamic radius. Refractive index changes could not be explained by addition of analyte molecular mass to the surface. These SPR responses were a result of specific receptor-ligand interactions, as judged by the reversibility of the response and the similarities between the SPR-determined equilibrium dissociation constants and reported dissociation constants. Additionally, this technique proved to be effective at detecting specific ligands from a panel of small molecules. This SPR method required no alterations in widely used and commercially available instrumentation yet allowed direct detection of very small molecules such as calcium ions (40 Da). Use of receptor conformation to detect low molecular weight analytes has potential applications in the high-throughput screening of small molecule drug libraries and the development of biosensors.     

Single-molecule assays of calmodulin target binding detected with a calmodulin energy-transfer construct

We have detected single-molecule binding interactions of a target peptide with the calcium-signaling protein calmodulin (CaM) immobilized in an agarose gel, and we have demonstrated the application of a single-molecule binding assay to measure the binding strength of CaM with the CaM-binding domain of calmodulin-dependent protein kinase II (CaMKII). The results demonstrate the potential for ultrasensitive assays of CaM-target interactions and the measurement of a picomolar dissociation constant. To detect single-molecule protein interactions, single-molecule assays require that the analyte molecule be confined to the focal spot of the objective for the time scale of the measurement. We demonstrate the deleterious effect of surface immobilization on CaM. As an alternative to surface immobilization, we have constructed a CaM/maltose binding protein fusion protein, which renders CaM translationally immobile in a low weight percent agarose gel. The target binding functionality of CaM assayed in agarose gels is in good agreement with solution assays. The utility of the construct for detecting interactions with CaM targets was demonstrated in a single-molecule assay of binding interactions of MBP-CaM with the CaMKII CaM-binding domain peptide. A value of 103 +/- 35 pM for the dissociation constant of this interaction was determined by simple counting of fluorescent molecules.     

纳米材料在SPR生物传感器中的应用进展

目的研究纳米材料在SPR生物传感器中的应用进展。方法介绍表面等离子共振(SPR)原理和近几年来纳米材料在SPR生物传感器中的应用研究现状,并重点综述常见的几种纳米材料(包括金纳米粒子、金纳米棒、银纳米粒子、磁纳米粒子、石墨烯和量子点)在SPR生物传感器中的应用新进展。结果 SPR生物传感器是对表面折射率变化敏感的一项分析技术,然而,传统的SPR传感器无法检测极小的折射率变化,这就阻碍了其在超灵敏检测中的应用。结论采用纳米材料提高SPR检测灵敏度和特异性是SPR技术发展的必然趋势。     

ssDNA aptamer-based surface plasmon resonance biosensor for the detection of retinol binding protein 4 for the early diagnosis of type 2 diabetes

Retinol binding protein 4 (RBP4) is a useful biomarker in the diagnosis of type 2 diabetes since its level in the serum is higher in insulin-resistant states. Accurate measurement of the serum RBP4 levels is hampered by conventional immunologic methods, such as enzyme-linked immunosorbent assay (ELISA). In this study, therefore, we have developed an aptamer-based surface plasmon resonance (SPR) biosensor that can be used to sense for RBP4 in serum samples. A single-stranded DNA (ssDNA) aptamer that showed high affinity (Kd = 0.2 +/- 0.03 microM) and specificity to RBP4 was selected. This RBP4-specific aptamer was immobilized on a gold chip and used in a label-free RBP4 detection using SPR. Analysis of RBP4 in artificial serum using SPR was compared with ELISA and Western blot analysis. Our results indicated that the RBP4-specific aptamer-based SPR biosensor gave better dose-dependent responses and was more sensitive than ELISA assays. As such, this RBP4 aptamer-based SPR biosensor can be potentially used to monitor the RBP4 levels within the serum as an indicator of type 2 diabetes.