表面等离子共振-质谱法对相互作用的生物分子在10-15mol水平的微量鉴定

利用生物传感芯片质谱法(BIA/MS)对微球蛋白及其抗体的相互作用进行分析和鉴定.将微球蛋白抗体偶联到芯片上,让微球蛋白溶液流过芯片表面,然后使用“三明治”结构的微再生方法把结合的微球蛋白从芯片上洗脱下来,再对其进行酶解及质谱鉴定,在10-15mol水平得到了明确的结果.     

Surface characterization and efficiency of a matrix-free and flat carboxylated gold sensor chip for surface plasmon resonance (SPR)

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM–MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen–antibody interaction. For both interactions, the well characterized densely functionalized SAM–MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.     

表面等离子体共振生物传感器连续检测莱克多巴胺

利用表面等离子体共振生物传感器对莱克多巴胺抗体与固定在芯片表面的莱克多巴胺衍生物的相互作用进行了分析,解离常数为2.56×10-6s-1。根据一定范围内相对响应值和时间近似呈线性关系的动力学特性,建立了连续检测的方法,从而简化了实验步骤,有利于提高芯片的使用寿命。检测莱克多巴胺采用抑制法,将莱克多巴胺衍生物固定在芯片的表面,莱克多巴胺抗体与样品混合后流过芯片的表面,所得相对响应值与样品中莱克多巴胺的浓度成反比。单个样品的检测时间设定为15min,对应的检出限小于4μg/L。     

Simultaneous Determination of β2-Microglobulin and Ig E Using Real-Time Biospecific Interaction Analysis (BIA)

Abstract

Real-time biospecific interaction analysis (real time BIA) based on Surface Plasmon Resonance (SPR) has been used to measure the concentration of β2-microglobulin (β2-μ) and Ig E, used as a model system, simultaneously in buffer and plasma. The method relies on specific binding of β2-microglobulin and Ig E to a sensor chip surface where the monoclonal antibodies anti-β2-microglobulin and anti-Ig E were covalently immobilized. The primary binding of the analytes to the surface is followed by the injection, in sequence, of secondary antibodies, each one specific for a different epitope of β2-microglobulin and Ig E, respectively. The binding between the antibodies and the analytes is recorded as an increase in the SPR signal expressed in Resonance Units (RU). The SPR signal is directly related to the amount of β2-microglobulin and Ig E bound to the surface, and depends upon the concentration of the analytes in the sample. The analysis was performed in buffer and serum to show any non-specific binding due to serum proteins. The concentration range 0.35-5.55 nM for both analytes is covered, with good precision and close correlation with an independent standard concentration measurement.     

水凝胶微流控芯片的快速加工及在细胞培养检测中的应用

采用具有紫外光聚合性能的聚乙二醇(PEG)基水凝胶材料, 通过紫外光聚合作用快速加工双层水凝胶微流控芯片, 并验证了其对肿瘤细胞代谢液进行检测的可行性. 与传统微流控芯片材料相比, 该水凝胶芯片材料具有更好的生物相容性及可操控性, 可直接加工成形, 在生物学领域特别是细胞培养过程控制方面具有良好的应用前景. 实验结果表明, 该水凝胶微流控芯片可在微尺度空间有效模拟细胞生长环境, 并实现对细胞连续捕获后的原位培养. 将该芯片与卟啉可视阵列传感器系统结合, 经代谢特征分析可有效区分不同种类肿瘤细胞, 实现芯片细胞培养平台上的细胞代谢指纹快速可视化传感检测.     

Multichannel surface plasmon resonance imaging and analysis of micropatterned self-assembled monolayers and protein affinity interactions

Multichannel images of 11-mercaptoundecanoic acid and 11-mercapto-1-undecanol self-assembled monolayers together with a biospecific interferon-gamma (IFN-gamma)/anti-IFN-gamma antibody immunoaffinity interaction were observed by the two-dimensional surface plasmon resonance (2D-SPR) imaging system. With the fabricated 2D-SPR imaging system, adopting a white light source in combination with a narrow band-pass filter, sharp images were resolved, minimizing the diffraction patterns on the resulting images. Micropatterning of self-assembled monolayers was acheived by exploiting the UV photolysis of thiol bonding, instead of conventional photolithography. The line profile calibration of the image contrast with ellipsometric analysis enabled us to discriminate the change in monolayer thickness within a sub-nanometer scale. For the protein interactions on the surface, the biospecific affinity recognition reaction of IFN-gamma antigen with surface-immobilized antibody was analyzed. Through the signal amplification strategy based on the enzyme-catalyzed precipitation reaction in a sandwich-type immunoassay, biospecific antigen binding was found detectable down to a concentration of 1 ng/mL.     

A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental effects

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ER) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. Both cases are analyzed: unliganded as well as liganded. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate signaling and signaling deleterious to health) may be better controlled. The fractal approach is applied to analyte-ER interaction data available in the literature. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D(f)) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. For example, the binding rate coefficient, k, exhibits a 4.60 order of dependence on the fractal dimension, D(f), for the binding of unliganded and liganded VDR mixed with GST-RXR in solution to Spp-1 VDRE (1,25-dihydroxyvitamin D(3) receptor element) DNA immobilized on a sensor chip surface (Cheskis and Freedman, Biochemistry 35 (1996) 3300-3318). A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. A predictive relationship is also presented for the ratio K(A)(=k/k(d)) as a function of the ratio of the fractal dimensions (D(f)/D(fd)). This has biomedical and environmental implications in that the dissociation and binding rate coefficients may be used to alleviate deleterious effects or enhance beneficial effects by selective modulation of the surface. The K(A) exhibits a 112-order dependence on the ratio of the fractal dimensions for the ligand effects on VDR-RXR interaction with specific DNA.     

表面等离子体共振法检测人血清白蛋白抗体活性

表面等离子体共振法是研究生物大分子间相互作用的有效方法之一,和非直接方法（如酶联免疫）相比,具有实时、快速和免标记等特点。我们在甲羧基化葡聚糖修饰的传感片表面直接交联固一人血清白蛋白（ＨＳＡ）,用于ａｎｔｉ－ＨＳＡ抗体活性的检测,并用Ｈ３ＰＯ４（０．１ｍｏｌ／Ｌ）溶液再生。结果表明表面等离子体共振（ＳＰＲ）生物传感器能快速实时检测ａｎｔｉ－ＨＳＡ抗体的活性,且传感片能够重复使用１００次以上。     

Surface plasmon resonance immunosensor for IgE analysis using two types of anti-IgE antibodies with different active recognition sites.

A simple and novel method for the determination of an IgE antibody based on a surface plasmon resonance immunosensor for the diagnosis of an allergy is described. The method involves the use of an anti-IgE(D) antibody and an anti-IgE(H) antibody, which reacts with the Ce2 domain and the Ce3 domain of the IgE antibody. The anti-IgE(D) antibody was immobilized on the gold surface of a sensor chip by physical adsorption. An IgE antibody sample was incubated by adding it to an anti-IgE(H) antibody solution to form an anti-IgE(H) immunocomplex through a reaction of the Ce3 domain of the IgE antibody. The incubated solution was introduced onto the sensor chip and the immunocomplex of the IgE-anti-IgE(H) then reacted with the anti-IgE(D) antibody immobilized on the sensor chip through the Ce2 domain of the IgE antibody part of the IgE-anti-IgE(H) immunocomplex. The detection limit of the present method for the determination of the IgE antibody was about 10 ppb. The affinity constants for the anti-IgE(H) antibody immunocomplex with the IgE antibody in solution and that of the anti-IgE(H) antibody immunocomplex with the IgE antibody immobilized on the sensor chip by a biotin-streptavidin interaction were estimated to be 4.1 x 10(7) M(-1) and 5.8 x 10(6) M(-1), respectively. The affinity constant for the immunocomplex of the anti-IgE(H) antibody with the IgE antibody with the anti-IgE(D) immobilized on the sensor chip was estimated to be 4.9 x 10(7) M(-1), 20-times larger than the affinity constant for the IgE antibody immunocomplex with the anti-IgE(D) antibody immobilized on the sensor chip, based on a direct immunoassay method of the IgE antibody under the same experimental conditions.     

Modification of aluminum chips for LDI mass spectrometry of proteins

Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI TOFMS) combined with affinity chromatography on immobilized phenylboronic acid agarose gels was used for selective enrichment and detection of specifically modified proteins such as glycated proteins in complex biological samples. Physicochemical grafting of hydrophilic polymers on aluminum surface was developed to reduce nonspecific protein sorption and to create a proper support layer for a three-dimensional affinity hydrogel. Grafted agarose allowed the fixation of three-dimensional agarose hydrogel on the chip surface. Both pinched polymers and hydrogels were effectively derivatized. 3-Aminophenylboronic acid (mPBA) was covalently immobilized as an affinity ligand to achieve specific binding of glycated plasma proteins. Alternatively, the affinity sorbent was immersed into the hydrogel to increase binding capacity. MALDI TOFMS was used to evaluate binding efficiency and molecular mass changes of human serum albumin due to glycation. Glycated proteins were captured directly on the chip with high selectivity and efficacy, and low nonspecific binding. Thus they could easily be characterized by MALDI TOFMS.     

Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg⁻¹) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.     

Convection, diffusion and reaction in a surface-based biosensor: modeling of cooperativity and binding site competition on the surface and in the hydrogel

We study theoretically the transport and kinetic processes underlying the operation of a biosensor (particularly the surface plasmon sensor "Biacore") used to study the surface binding kinetics of biomolecules in solution to immobilized receptors. Unlike previous studies, we concentrate mainly on the modeling of system-specific phenomena rather than on the influence of mass transport limitations on the intrinsic kinetic rate constants determined from binding data. In the first problem, the case of two-site binding where each receptor unit on the surface can accommodate two analyte molecules on two different sites is considered. One analyte molecule always binds first to a specific site. Subsequently, the second analyte molecule can bind to the adjacent unoccupied site. In the second problem, two different analytes compete for one binding site on the same surface receptor. Finally, the third problem considers the case of positive cooperativity among bound molecules in the hydrogel using a simple mean-field approach. The transport in both the flow channel and the hydrogel phases of the biosensor is taken into account in this case (with few exceptions, most previous studies assume a simpler model in which the hydrogel is treated as a planar surface with the receptors). We consider simultaneously diffusion and convection through the flow channel together with diffusion and cooperativity binding on the surface and in the hydrogel. In each case, typical results for the concentration contours of the free and bound molecules in the flow channel and hydrogel regions are presented together with the time-dependent association/dissociation curves and reaction rates. For binding site competition, the analysis predicts overshoot phenomena.     

Surface acoustic wave biosensor as a tool to study the interaction of antimicrobial peptides with phospholipid and lipopolysaccharide model membranes

Surface acoustic wave biosensors are a powerful tool for the study of biomolecular interactions. The modulation of a surface-confined acoustic wave is utilized here for the analysis of surface binding. Phase and amplitude of the wave correspond roughly to mass loading and viscoelastic properties of the surface, respectively. We established a procedure to reconstitute phospholipid and lipopolysaccharide bilayers on the surface of a modified gold sensor chip to study the mode of action of membrane-active peptides. The procedure included the formation of a self-assembled monolayer of 11-mercaptoundecanol, covalent coupling of carboxymethyl-dextran, and subsequent coating with a poly- l-lysine layer. The lipid coverage of the surface is highly reproducible and homogeneous as demonstrated in atomic force micrographs. Ethanol/triton treatment removed the lipids completely, which provided the basis for continuous sequences of independent experiments. The setup was applied to investigate the binding of human cathelicidin-derived peptide LL32, as an example for antimicrobial peptides, to immobilized phosphatidylserine membranes. The peptide-membrane interaction results in a positive phase shift and an increase in amplitude, indicating a mass increase along with a loss in viscosity. This suggests that the bilayer becomes more rigid upon interaction with LL32.     

Molecular recognition in a supramolecular hydrogel to afford a semi-wet sensor chip

This communication describes a new molecular recognition chip using a semi-wet microenvironment provided by a self-assembled hydrogel. On the basis of the evidence that the molecular recognition capability of artificial chemosensors are practically retained even in the hydrogel compared to those in aqueous solution, we miniaturized the functionalized hydrogel to produce an unprecedented molecular recognition chip. We believe that the present noncovalent immobilization method is generally applicable to many chemosensors, which leads to a unique semi-wet sensor chip suitable to convenient and high-throughput assay to plural analytes.     

On-chip photoactivation of heterologously expressed rhodopsin allows kinetic analysis of G-protein signaling by surface plasmon resonance spectroscopy

Surface plasmon resonance spectroscopy allows the study of protein interaction dynamics in real-time. Application of this technique to G-protein coupled receptors, the largest family of receptors involved in signal transduction, has been complicated by their low level of expression and the critical dependence of their native conformation on the hydrophobic transmembrane lipid environment. Here, we investigate and compare three different strategies to immobilize rhodopsin, a prototypical G-protein coupled receptor on a sensor chip surface using antibodies and a lectin for receptor capturing. By further probing of different experimental conditions (pH, detergent type) we identified the optimal factors to maintain rhodopsin in a functional conformation and extended this approach to recombinant rhodopsin that was heterologously expressed in COS cells. Functional operation of rhodopsin on the sensor chip surface was proven by its activation and subsequent light-stimulated G-protein coupling. The influence of these experimental parameters on the association and dissociation kinetics of G-protein receptor coupling was determined. Thereby, we found that the kinetics of G_t interaction were not changed by the strategy of immobilization or the type of detergent. Regeneration of opsin directly on a chip allowed recycling of the immobilized native and recombinant receptor. Thus, the approach provides an experimental framework for choosing the most suitable conditions for the solubilization, immobilization, and for functional tests of rhodopsin on a biosensor surface.     

Surface plasmon resonance immunosensor for histamine based on an indirect competitive immunoreaction

The use of a surface plasmon resonance immunosensor for the analysis of histamine (β-imidazole ethylamine) is described. The method is based on an indirect competitive reaction of an anti-histamine antibody in a sample solution with histamine immobilized on a sensor chip and with histamine in the sample solution. A sensor chip immobilized with histamine was prepared using a self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA) as an anchor membrane, followed by an amino-coupling reaction with histamine after activation of the 11-MUA layer on the sensor chip by treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide. The sensor chip can be reused, after regeneration with a 10 mM HCl solution, which dissociates the anti-histamine antibody complex from histamine on the sensor chip. The affinity constants for the immunocomplex of the anti-histamine antibody with histamine in the solution and for that of the anti-histamine antibody with histamine immobilized on the sensor chip were calculated to be 1.5 × 10⁷ and 7.2 × 10⁵ M⁻¹, respectively, by assuming a Langmuir-type adsorption of the anti-histamine antibody to histamine immobilized on the sensor chip. The detection limit of the method was determined to be 3 ppb.     

Immunobiosensor detection of domoic acid as a screening test in bivalve molluscs: comparison with liquid chromatography-based analysis

A rapid and sensitive immuno-based screening method was developed to detect domoic acid (DA) present in extracts of shellfish species using a surface plasmon resonance-based optical biosensor. A rabbit polyclonal antibody raised against DA was mixed with standard or sample extracts and allowed to interact with DA immobilized onto a sensor chip surface. The characterization of the antibody strongly suggested high cross-reactivity with DA and important isomers of the toxin. The binding of this antibody to the sensor chip surface was inhibited in the presence of DA in either standard solutions or sample extracts. The DA chip surface proved to be highly stable, achieving approximately 800 analyses per chip without any loss of surface activity. A single analytical cycle (sample injection, chip regeneration, and system wash) took 10 min to complete. Sample analysis (scallops, mussels, cockles, oysters) was achieved by simple extraction with methanol. These extracts were then filtered and diluted before analysis. Detection limits in the ng/g range were achieved by the assay; however, the assay parameters chosen allowed the test to be performed most accurately at the European Union's official action limit for DA of 20 microg/g. At this concentration, intra- and interassay variations were measured for a range of shellfish species and ranged from 4.5 to 7.4% and 2.3 to 9.7%, respectively.     

A novel method to prepare SPR sensor chips based on photografting molecularly imprinted polymer

A novel method to prepare surface plasmon resonance(SPR) sensor chips based on grafted imprinted polymer is explored. Benzophenone photografting system is used to grow molecularly imprinted polymer(MIP) films from the modified surface of gold substrate.The surface morphology and thickness of MIP films were investigated by scanning electronic microscope(SEM).The adsorption properties of sensor chip were studied by SPR spectroscopy.The results demonstrate that nano-MIP films can be constructed on the surface of gold substrate with the good adsorption of template molecules.     

Determination of sulfamethazine in milk by biosensor immunoassay

A biosensor based on surface plasmon resonance (SPR) measurement was developed for use in an immunoassay for detection of sulfamethazine (SMZ) in milk. The biospecific surface was a carboxymethyl dextran-modified gold-surface sensor chip to which SMZ was covalently bound. The assay was based on inhibition of the binding of polyclonal antibodies to immobilized SMZ by SMZ in the sample. The SPR response changed inversely in relation to the antibiotic concentration in the sample. Calibration curves were constructed for SMZ in buffer and in milk at a concentration which included the maximum residue limit (0 to 200 micrograms/kg). The analysis time per sample varied from 8 to 30 min. Different flow rates and antibodies were modified alternatively during the study to assess their influence on the performance of the assay. The active antibody concentration was calculated at approximately 1880 and 180 nM for the antibody anti-SMZ 1 and the antibody anti-SMZ 2, respectively. No cross-reactivity of antibodies with other antibiotics was found. Under optimal conditions, the detection limits in milk for SMZ were 8 and 1.7 micrograms/kg, respectively, for antibody 1 and antibody 2, at a flow rate of 20 microL/min.     

Enantioselective analysis of melagatran via an LSPR biosensor integrated with a microfluidic chip

The impact of chiral compounds on pharmacological and biological processes is well known. With the increasing need for enantiomerically pure compounds, effective strategies for enantioseparation and chiral discrimination are in great demand. Herein we report a simple but efficient approach for the enantioselective determination of chiral compounds based on a localized surface plasmon resonance (LSPR) biosensor integrated with a microfluidic chip. A glass microfluidic chip with an effective volume of ～0.75 μL was fabricated for this application. Gold nanorods (AuNRs) with an aspect ratio of ～2.6 were self-assembled onto the surface of the inner wall of the chip to serve as LSPR transducers, which would translate the analyte binding events into quantitative concentration information. Human α-thrombin was immobilized onto the AuNR surface for enantioselective sensing of the enantiomers of melagatran. The proposed sensor was found to be highly selective for RS-melagatran, while the binding of its enantiomer, SR-melagatran, to the sensor was inactive. Under optimal conditions, the limit of detection of this sensor for RS-melagatran was found to be 0.9 nM, whereas the presence of 10?000-fold amounts of SR-melagatran did not interfere with the detection. To the best of our knowledge, this is the first demonstration of an LSPR-based enantioselective biosensor.