Surface plasmon resonance study of g protein/receptor coupling in a lipid bilayer-free system

Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin (Gt) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of Gt following flash or constant light application, fast GTP-dependent dissociation of Gt from Rho, regeneration of Rho, and dependence of Gt binding on light intensity and on concentration of Gt. The apparent KD of the Gt/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.     

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.     

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.     

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.     

Electrochemical evaluation of the interaction between endocrine disrupter chemicals and estrogen receptor using 17,beta-estradiol labeled with daunomycin

A new electrochemical screening method for endocrine disrupting chemicals (EDCs) was developed. To evaluate the binding capacity of EDCs to the estrogen receptor (ER), 17beta-estradiol labeled with daunomycin as an electroactive compound was prepared. The electrochemical sensitivity of the prepared labeled estradiol (LE) was high, as compared with daunomycin. The interaction between LE and ER was observed by the decrease in the electrode response of LE, indicating the specific binding of LE with ER. The competitive reaction between LE and 17beta-estradiol for the limiting binding site on ER produced increases in the peak current of LE. The relative standard deviation at 1 x 10(-8) M 17beta-estradiol was about 10.0% (n = 7). The binding affinity between EDC and ER was also evaluated by comparison with 17beta-estradiol-ER interaction. Bisphenol A, p-nonylphenol and p,p'-DDT was used as a test compound. All test compounds demonstrated some ability to bind with ER. This electrochemical binding assay illustrates a new method for evaluating the binding capacity of EDCs to ER without the need for a separation procedure for the bound and free LE.     

A quantitative assessment of heterogeneity for surface-immobilized proteins

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.     

Surface Plasmon Resonance Biosensor Incorporated in a Michelson Interferometer WithEnhanced Sensitivity

A novel double-pass phase-sensitive surface plasmon resonance (SPR) biosensor based on a Michelson interferometer with differential phase interrogation is presented. The new setup provides an intrinsic resolution enhancement of up to two times in terms of achievable detection sensitivity due to an amplification effect in the SPR phase change when we place the SPR sensor head in the signal arm of the interferometer so that the interrogating optical beam traverses the sensor surface twice. Experimental results obtained from saltwater mixtures and antibody-antigen binding reactions confirmed the expected sensitivity enhancement as compared to the conventional SPR biosensor based on a Mach-Zehnder interferometer     

Selection of ligands for affinity chromatography using quartz crystal biosensor

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.     

An atomic force microscope study of surface roughness of thin silicon films deposited on SiO/sub 2/

Atomic force microscope analysis, with a resolution of /spl lsim/1.1 nm, shows that peak-to-peak surface roughness (/spl Delta/h/sub p-p/) of amorphous silicon films thinner than /spl ap/50 nm on silicon dioxide can be controlled to better than 5 nm. Low-pressure, chemically-vapor-deposited silicon films on silicon dioxide initially show an approximately linear increase in the surface roughness due to growing nuclei as the deposition progresses, followed by a decrease in the surface roughness as growth nuclei coalesce. A simple model based on random nucleation and nuclei growth displays similar trends. Films deposited on rougher substrates show more surface roughness. Surface treatment during the predeposition cleaning process does not significantly affect /spl Delta/h/sub p-p/. As a means of producing smooth surfaces, films thinner than about 20 nm are first deposited more thickly than needed, and then etched back to the desired dimension; the use of a binary HNO/sub 3/ and HF etching process improves roughness control. Boron-ion implanted and subsequently crystallized 45-nm-thick Si films show significant smoothing with /spl Delta/h/sub p-p//spl ap/2.2 nm. Thin amorphous silicon films deposited by source evaporation are attractive because they can be deposited at room temperature, and have smoother surfaces (/spl Delta/h/sub p-p//spl ap/2.5 nm) than comparable films produced by chemical vapor deposition.     

Influence of spinel substrate and over-layer for enhanced SAW and AO properties with KNbO3 thin film

Surface acoustic wave (SAW) propagation characteristics have been studied using modeling calculations for a potassium niobate (KNbO/sub 3/) thin film-layered structure with [001] and [110] orientation on a single crystal spinel (MgAl/sub 2/O/sub 4/) substrate, and a spinel buffer layer on silicon. Variation in the electromechanical coupling and acoustic attenuation has been compared. A significantly high value of coupling factor (k/sub max//sup 2/=23%) is obtained for the [001]KNbO/sub 3//spinel structure by introducing an optimum thickness of spinel over-layer for potential wide bandwidth SAW device applications. The dispersion characteristics with the [110] KNbO/sub 3/ orientation indicate an initial peak in the coupling coefficient value (k/sub max//sup 2/=8.8%) at a relatively low KNbO/sub 3/ film thickness that appears attractive for fabricating devices with thinner films. The KNbO/sub 3/ film with [001] orientation is found attractive for efficient acousto-optic (AO) device application with the formation of a symmetric waveguide structure (spinel(0.5 /spl mu/m)/KNbO/sub 3/(1.0 /spl mu/m)/spinel). A high value of k/sup 2/=23.5% with 50% diffraction efficiency has been obtained for the spinel(0.5 /spl mu/m)/KNbO/sub 3/(1.0 /spl mu/m)/spinel structure at 1 GHz SAW frequency and 633 nm optical wavelength at a very low input drive power of 15.4 mW.     

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.     

Optimization of attenuation estimation in reflection for in vivo human dermis characterization at 20 MHz

In vivo skin attenuation estimators must be applicable to backscattered radio frequency signals obtained in a pulse-echo configuration. This work compares three such estimators: short-time Fourier multinarrowband (MNB), short-time Fourier centroid shift (FC), and autoregressive centroid shift (ARC). All provide estimations of the attenuation slope (/spl beta/, dB.cm/sup -1/.MHz/sup -1/); MNB also provides an independent estimation of the mean attenuation level (IA, dB.cm/sup -1/). Practical approaches are proposed for data windowing, spectral variance characterization, and bandwidth selection. Then, based on simulated data, FC and ARC were selected as the best (compromise between bias and variance) attenuation slope estimators. The FC, ARC, and MNB were applied to in vivo human skin data acquired at 20 MHz to estimate /spl beta//sub FC/, /spl beta//sub ARC/, and IA/sub MNB/, respectively (without diffraction correction, between 11 and 27 MHz). Lateral heterogeneity had less effect and day-today reproducibility was smaller for IA than for /spl beta/. The IA and /spl beta//sub ARC/ were dependent on pressure applied to skin during acquisition and IA on room and skin-surface temperatures. Negative values of IA imply that IA and /spl beta/ may be influenced not only by skin's attenuation but also by structural heterogeneity across dermal depth. Even so, IA was correlated to subject age and IA, /spl beta//sub FC/, and /spl beta//sub ARC/ were dependent on subject gender. Thus, in vivo attenuation measurements reveal interesting variations with subject age and gender and thus appeared promising to detect skin structure modifications.     

Nonregeneration protocol for surface plasmon resonance: study of high-affinity interaction with high-density biosensors

Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.     

The /spl plusmn/45/spl deg/ correlation interferometer as a means to measure phase noise of parametric origin

A radiofrequency interferometric detector is combined with the correlation-and-averaging technique in a new scheme for the measurement of the phase noise of a component. The method relies upon the assumption that the phase noise of the component being tested (DUT) exceeds the amplitude noise, which is consistent with the general experience in the field of wireless engineering. The new scheme is based on the amplification of the DUT noise sidebands and on the simultaneous measurement of the amplified noise by means of two mixers driven in quadrature, /spl plusmn/ 45/spl deg/ off the carrier phase. The /spl plusmn/45/spl deg/ detection has two relevant properties, namely 1) the sensitivity is neither limited by the thermal energy k/sub B/T/sub 0/, nor by temperature uniformity, and 2) the noise of the measurement amplifier is rejected, despite a single amplifier being shared by the two channels of the correlator. The article provides the theoretical background and experimental results. The sensitivity of the first 100-MHz prototype, given in terms of the S/sub /spl phi//(f) floor, is some 12 dB below k/sub B/T/P/sub 0/, where P/sub 0/ is the carrier power. Using a dual carrier suppression scheme, the residual flicker is as low as -168 dBrad/sup 2//Hz at f=1 Hz off the carrier.     

Recent developments in high curie temperature perovskite single crystals

The temperature behavior of various relaxor-PT piezoelectric single crystals was investigated. Owing to a strongly-curved morphotropic phase boundary, the usage temperature of these perovskite single crystals is limited by T/sub R-T/- the rhombohedral to tetragonal phase transformation temperature - which occurs at the significantly lower temperatures than the Curie temperature T/sub c/. Attempts to modify the temperature usage range of Pb(Zn/sub 1/3/Nb/sub 2/3/)O/sub 3/-PbTiO/sub 3/ (PZNT) and Pb(Mg/sub 1/3/Nb/sub 2/3/)O/sub 3/-PbTiO/sub 3/ (PMNT) rhombohedral crystals (T/sub c/ /spl sim/ 150-170/spl deg/C, T/sub R-T/ /spl sim/ 60-120/spl deg/C) using minor dopant modifications were limited, with little success. Of significant potential are crystals near the morphotropic phase boundary in the Pb(Yb/sub 1/2/Nb/sub 1/2/)O/sub 3/-PbTiO/sub 3/ (PYNT) system, with a T/sub c/ > 330/spl deg/C, even though T/sub R-T/ was found to be only half the value at /spl sim/160/spl deg/C. Single crystals in the novel BiScO/sub 3/-PbTiO/sub 3/ system offer significantly higher T/sub c/s > 400/spl deg/C, while exhibiting electromechanical coupling coefficients k/sub 33/ > 90% being nearly constant till the T/sub R-T/ temperature around 350/spl deg/C, which greatly increases the temperature range for transducer applications.     

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.     

Study of x/spl alpha/-Fe/sub 2/O/sub 3/-(1-x)ZrO/sub 2/ solid solution for low-temperature resistive oxygen gas sensors

A noble type of oxygen-sensitive and electrical-conductive material, ZrO/sub 2/-based with /spl alpha/-Fe/sub 2/O/sub 3/ thick-film gas sensor, was investigated for low operating temperature. Amorphous-like solid solutions of x/spl alpha/-Fe/sub 2/O/sub 3/-(1-x)ZrO/sub 2/ powders were derived using the high-energy ball milling technique, and their physical and microstructural properties were characterized from DTA, XRD, TEM, and XPS. The oxygen gas-sensing properties of the screen-printed thick-film gas sensors fabricated from such mechanically-alloyed materials were characterized systematically. Very good sensing properties were obtained with a relative resistance value of 82 in 20% oxygen, and at a low operating temperature of 320/spl deg/C. AC impedance spectra and thermally stimulated current were characterized to investigate the conduction properties of the solid solution, 0.2/spl alpha/-Fe/sub 2/O/sub 3/-0.8ZrO/sub 2/, in air and nitrogen (carrier gas), respectively. It was found that the Arrhenius plots of /spl sigma/T versus 1000/T have two distinct gradients corresponding to two activation energies in the high and low temperature regions. The transition temperature occurs at about 320/spl deg/C that corresponds to an optimal operating temperature of the gas sensor. It is believed that the high oxygen vacancy concentration present in the solid solution, 0.2/spl alpha/-Fe/sub 2/O/sub 3/-0.8ZrO/sub 2/, and the dissociation of the associated oxygen vacancy defect complexes at 320/spl deg/C are the critical factors for the high relative resistance to oxygen gas at low operating temperature.     

金属纳米颗粒在表面等离激元共振生物传感器上的应用

文章研究不同形状和长径比的金属纳米颗粒应用在表面等离激元共振（SPR）生物传感器中对传感信号的影响,自制金属纳米颗粒并进行物理表征,以金纳米颗粒与抗兔IgG进行生物偶联,利用自制角度检测型SPR生物传感器对兔IgG抗体进行检测,结果表明,金属颗粒的形状和长径比对SPR传感器的共振角都有影响,金纳米棒能够明显提高SPR生物传感器的检测灵敏度。     

Improved sensitivity of wavelength-modulated surface plasmon resonance biosensor using gold nanorods

In this report, gold nanorods (GNRs) were used to enhance the sensitivity of the wavelength-modulated surface plasmon resonance (SPR) biosensor. The GNRs were designed and fabricated through seed-medicated growth and surface activation by a layer of a weak polyelectrolyte, poly(acrylic acid) for the attaching antibody. Rabbit anti-goat IgG was immobilized on GNRs, and sandwich assays were carried out to detect goat IgG using a wavelength-modulated SPR biosensor. The detection sensitivity of the nanorod-conjugated antibody is 25-100 times more sensitive than the SPR biosensor without GNRs. Drastic sensitivity enhancement, owing to the electromagnetic interaction between the nanotag and the sensing film, was maximized using the longitudinal plasmonic resonance of the GNRs. GNRs could significantly enhance the sensitivity of the SPR biosensor, and the maximum enhancement effect can be achieved when the longitudinal SPR peak wavelength of GNRs functionally matches the surface plasmon wavelength.