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.     

SERS quantitative detection of trace human chorionic gonadotropin using a label‐free Victoria blue B as probe in the aggregated immunonanogold sol substrate

Nanogold particles (NG) were modified by anti‐rabbit antibody (RAb) against human chorionic gonadotropin to obtain an immunonanogold probe (ING). In pH 7.0 Na₂HPO₄‐citrate buffer solution containing KCl, ING probes formed large aggregates in which Victoria blue B (VBB) molecules were adsorbed on the surface and which exhibited strong surface‐enhanced Raman scattering (SERS) at a peak of 1612 cm^–1. After addition of human chorionic gonadotropin (hCG) an immune reaction with the ING probe occurred to form dispersive ING–hCG complexes with non‐SERS activity that led to a decreased SERS peak at 1612 cm^–1. The decreased SERS intensity was linear to the concentration of hCG over 2.4–73.2 ng/mL. The ING reaction was studied in detail by SERS, scanning electron microscope (SEM), resonance Rayleigh scattering (RRS), surface plasmon resonance (SPR) absorption and laser scattering techniques. SERS quenching was observed and discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.     

Label-free colorimetric sensor for ultrasensitive detection of heparin based on color quenching of gold nanorods by graphene oxide

A novel label-free colorimetric strategy was developed for ultrasensitive detection of heparin by using the super color quenching capacity of graphene oxide (GO). Hexadecyltrimethylammonium bromide (CTAB)-stabilized gold nanorods (AuNRs) could easily self-assembly onto the surface of GO through electrostatic interaction, resulting in decrease of the surface plasmon resonance (SPR) absorption and consequent color quenching change of the AuNRs from deep to light. Polycationic protamine was used as a medium for disturbing the electrostatic interaction between AuNRs and GO. The AuNRs were prevented from being adsorbed onto the surface of GO because of the stronger interaction between protamine and GO, showing a native color of the AuNRs. On the contrary, in the presence of heparin, which was more easily to combine with protamine, the AuNRs could self-assembly onto the surface of GO, resulting in the native color disappearing of AuNRs. As the concentration of heparin increased, the color of AuNRs would gradually fade until almost colorless. The amounts of self-assembly AuNRs were proportional to the concentration of heparin, and thereby the changes in the SPR absorption and color had been used to monitor heparin levels. Under optimized conditions, good linearity was obtained in a range of 0.02-0.28 μg/mL (R=0.9957), and a limit of detection was 5 ng/mL. The simultaneous possession of high sensitivity and selectivity, simplicity, rapidity, and visualization enabled this sensor to be potentially applicable for ultrasensitive and rapid on-site detection toward trace heparin.     

Comparison of different hepatocyte cell lines for use in a hybrid artificial liver model

Selection of a cell line suitable for a hybrid artificial liver model employing cellulose porous beads (CPBs) was investigated. Hep G2 cells grown in a culture dish exhibited appreciably higher ureogenesis and gluconeogenesis activities than those grown in CPBs. SEM observation of CPBs revealed marked difference in the distribution of attached cells from one bead to another, and showed that almost all the cell-bearing micropores were completely packed with cells. With the aim of selecting a cell line not prone to excessive aggregation and which grows moderately so as not to fill up the micropores, cells of 6 cell lines, HLE, HLF, Hep 3B, PLC/PRF/5, Huh 7 and Hep G2, were cultivated in dishes. Hep G2, HLE, and HLF increased to 5 × 10⁵ cells/cm², whereas PLC/PRF/5 grew only to 5 × 10⁴, and Hep 3B and Huh 7 up to 2 × 10⁴ cells/cm². The specific activities of ureogenesis and gluconeogenesis of Huh 7 were the highest among the lines tested - 42- and 7-fold those of Hep G2, respectively. When the 6 cell lines were grown in a submerged culture with 0.6 g/l of CPBs, Huh 7 had the lowest cell concentration of 0.54 × 10⁶ cells/ml, and the highest activities of ammonia consumption and urea and glucose production (1.38 μ mol NH₃, 99 nmol urea, and 14.5 nmol glucose/10⁶cells/h). Consequently, Huh 7 is considered to be a suitable cell line for use in the development of an artificial liver model employing porous beads. This revised version was published online in July 2006 with corrections to the Cover Date.     

新型锌多糖抗凝血涂层修饰聚氯乙烯导管

目的：提高体外循环聚氯乙烯导管的血液相容性。方法：采用层层自组装的方法在PVC表面形成锌离子和多糖（肝素或硫酸葡聚糖）的复合涂层来提高PVC的血液相容性。结果：傅立叶红外光谱表明锌多糖复合物成功的沉积到PVC管表面,与未修饰的PVC管相比,修饰后的PVC管具有较长的部分活化的凝血酶原时间和很少数量的血小板黏附。体系中引入硫酸葡聚糖后,表面涂层具有更好的稳定性。结论：锌多糖抗凝血涂层很好的提高了聚氯乙烯导管的血液相容性。     

Salivary protein adsorption onto hydroxyapatite and sds‐mediated elution studied by in situ ellipsometry

Whole unstimulated saliva from two donors was investigated both with respect to adsorption characteristics and SDS‐induced elutability. Salivary protein adsorption onto hydroxyapatite (HA) discs was studied by means of in situ ellipsometry in the concentration range 0.1–20% saliva. The adsorbed amounts on HA were found to be similar to those on silica, but the rates of adsorption were lower. Protein adsorption was virtually unaffected by the presence of Na⁺, whereas Ca²⁺ induced nucleation of calcium phosphate at the surface, the deposition rate being influenced by the pellicle age but not by the presence of saliva in bulk solution. The SDS elutability of adsorbed pellicles was determined on HA as well as on silica surfaces. Desorption from both surfaces was found to occur in the same SDS concentration range, although a residual layer was observed on HA. The slight net positive charge and lower charge density of HA as compared to the strongly negatively charged silica, may, at least partly, account for this observation by causing a reduction in the repulsive force between protein‐surfactant complexes and the surface. Inter‐individual differences, observed in the adsorption as well as elution experiments, are thought to relate to the compositional differences observed by SDS‐PAGE.     

Antifouling poly(β-peptoid)s

A new type of polymer highly resistant to nonspecific protein adsorption is reported. Poly(N-methyl-β-alanine) (PMeA) and poly(N-ethyl-β-alanine) (PEtA) synthesized via cobalt-catalyzed carbonylative polymerization of N-methylaziridine and N-ethylaziridine were end-functionalized with thiol groups and grafted onto Au surfaces. Protein adsorption was studied by the surface plasmon resonance (SPR) method. The amounts of representative single proteins adsorbed onto the PMeA- and PEtA-grafted surfaces were below the detection limit of SPR at the pg/mm(2) level. After exposure to full blood plasma and serum for 10 min, protein adsorption was at the level of ～ 100 pg/mm(2), similar to the level of protein adsorption on poly(ethylene glycol) surfaces subjected to identical conditions. These poly(β-peptoid)s therefore provide excellent protein resistance comparable to the best antifouling materials known to date. The strong proton-accepting ability when forming hydrogen bonds is suggested to be an important attribute for these poly(β-peptoid)s as well as other poly(tertiary amide)s as antifouling materials.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

Cluster analysis of protein fourier transform infrared spectra

Cluster analysis techniques were used to examine a set of Fourier transform infrared (FT-IR) spectra of bovine serum albumin (BSA) in the adsorbed and nonadsorbed states. The region from 1480 to 1600 cm^?1, comprising the amide II band, was used. Spectra were preprocessed to compensate for linear baseline variation, and the single linkage method of cluster analysis was applied. As expected, the spectra of adsorbed and nonadsorbed BSA fell into two distinct clusters. However, no further clustering was observed among the adsorbed BSA spectra on the basis of surface type, suggesting that surface specificity of the spectral changes induced in BSA by adsorption is not detectable above experimental variation. This work illustrates the value of using cluster analysis in the FT-IR study of proteins as a complement to other data analysis methods.     

Simulation study of methanol and ethanol adsorption on graphitized carbon black

GCMC simulations are applied to the adsorption of sub-critical methanol and ethanol on graphitized carbon black at 300 K. The carbon black was modelled both with and without carbonyl functional groups. Large differences are seen between the amounts adsorbed for different carbonyl configurations at low pressure prior to monolayer coverage. Once a monolayer has been formed on the carbon black, the adsorption behaviour is similar between the model surfaces with and without functional groups. Simulation isotherms for the case of low carbonyl concentrations or no carbonyls are qualitatively similar to the few experimental isotherms available in the literature for methanol and ethanol adsorption on highly graphitized carbon black. Isosteric heats and adsorbed phase heat capacities are shown to be very sensitive to carbonyl configurations. A maximum is observed in the adsorbed phase heat capacity of the alcohols for all simulations but is unrealistically high for the case of a plain graphite surface. The addition of carbonyls to the surface greatly reduces this maximum and approaches experimental data with carbonyl concentration as low as 0.09 carbonyls/nm².     

Adhesion of murine cells to glass microbeads: substrate-adsorbed serum proteins.

Small (1 mm diameter) glass beads treated with acid and alkali provided a satisfactory substratum for the attachment and growth of normal and SV-40 transformed Balb/c 3T3 murine cells. Cells attached to the beads with similar, though slower kinetics as to flat glass surfaces, and spread and grew with their usual morphology; when detached by EGTA treatment, they left behind cellular substrate-attached material (SAM) which was similar electrophoretically to that obtained from tissue culture plastic. Because of their small size and rough etched surfaces, the beads have a large surface area and high adsorptive capacity, and so are a useful tool to isolate specific serum proteins adsorbed from the culture medium that may be important for cell attachment and spreading. The adsorbed serum proteins were solubilized with SDS and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. They included all reduced species adsorbed to tissue culture plastic, and only small amounts of one other major protein extracted from a “bacteriological” polystyrene surface on which cells could not grow. Profiles of unreduced samples differed considerably. The profile of adsorbed proteins varied little with tiem (5 minutes–4 days), temperature (4°–37°C), pH (5–9), presence of the protease inhibitor PMSF, or serum concentration (0.1–10%). Much of the adsorbed protein, qualitatively similar to the SDS-extracted material, could be eluted with H₂O or phosphate-buffered saline. Purified albumin and fibrinogen bound avidly to the beads; the material adsorbed from serum contained a large amount of albumin, however, little fibrinogen and no cold-insoluble globulin (as a 220 K protein) could be detected by Coomassie blue stained SDS-PAGE gels.     

Detection of inducible nitric oxide synthase using a suite of electrochemical, fluorescence, and surface plasmon resonance biosensors

A suite of biosensors for rapid detection of inducible nitric oxide synthase (iNOS) is described. First, a metal-enhanced electrochemical detection (MED) sensor, which relied on the redox properties of a silver monolayer, was developed. The linear detection range was between 8.64 × 10⁻² and 5.4 × 10¹ ng/ml with a detection limit of 1.69 × 10⁻⁴ ng/ml. This method was compared with surface plasmon resonance (SPR) biosensors in which polyclonal mouse anti-iNOS was covalently immobilized onto a gold surface using an iNOS antigen. The linear detection range recorded was between 3.37 × 10¹ and 5.4 × 10⁻² ng/ml with a detection limit of 2 × 10⁻³ ng/ml. Finally, an ultrasensitive portable capillary (UPAC) fluorescence immunosensor, in which a mouse anti-iNOS antibody was covalently immobilized onto the inner surface of a capillary and a rabbit anti-iNOS antibody was employed as the secondary antibody, was developed. The resulting signals were found to be directly proportional to iNOS concentrations between 1.52 × 10⁻¹ and 1.52 × 10⁻² ng/ml with a detection limit of 1.05 × 10⁻³ ng/ml. These immunosensors exhibit low cross-reactivity toward potential interferents such as human serum albumin and ovalbumin. The SPR and UPAC biosensors were validated using simulated blood spiked with recombinant iNOS, resulting in recoveries of 85% and 88.5%, respectively. The research presented in this article could potentially provide new ways of detecting NO for diagnostic and biomarker purposes in medical research.     

Potential mechanisms for the regulation of growth factor binding by heparin

Heparin and heparan sulfate proteoglycans (HSPG) bind many soluble growth factors and this binding is now recognized as an important mechanism for modulation of cell activity. Fibroblast growth factor-2 (FGF-2) is one of the best characterized of the heparin-binding growth factors and it has been shown experimentally that heparin regulation of FGF-2 activity is dependent on the level of cell HSPG and the concentration of heparin. In this paper, we explore, using mathematical modeling, proposed mechanisms for heparin regulation and determine how they impact FGF receptor binding. We demonstrate that the experimentally observed receptor binding phenomena can be reproduced if cells (1) express heparin-binding cell surface molecules and if either (2) these heparin binding sites are FGFR and bind heparin and FGF-2-heparin complexes or (3) are surface molecules able to bind FGF-2 and couple with FGF-2 receptors to form high-affinity FGF-2-bound surface complexes. The ability of heparin to directly interact with the FGFR and bind FGF-2 in the absence of this coupling function was not sufficient to explain heparin activity. These findings have implications with regard to regulation of heparin-binding growth factors and could help guide the development of highly specific growth regulatory molecules through specific regulation by heparin and HSPG.     

Formation and characterization of three dimensional human hepatocyte cell line spheroids on chitosan matrix for in vitro tissue engineering applications

Chitosan was used as a matrix to induce three-dimensional spheroids of HepG2 cells. Chitosan films were prepared and used for culturing Hep G2 cells. Attachment kinetics of the cells was studied on the chitosan films. The optimum seeding density of the Hep G2 cells, required for three-dimensional spheroid formation was determined and was found to be 5 × 10⁴/ml. The growth kinetics of Hep G2 cells was studied using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, and morphology of the cells was studied through optical photographs taken at various days of culture. The liver cell functions of the spheroids were determined by measuring albumin and urea secretions. The results obtained from these studies have shown that the culture of Hep G2 cells on chitosan matrix taking appropriate seeding density resulted in the formation of three-dimensional spheroids and exhibited higher amount of albumin and urea synthesis compared to monolayer culture. These miniature “liver tissue like” models can be used for in vitro tissue engineering applications like preliminary evaluation of the toxicity of drugs and chemicals.     

Requirement for heparan sulphate proteoglycans to mediate basic fibroblast growth factor (FGF-2)-induced stimulation of Leydig cell steroidogenesis

This study reports that, in contrast to previous findings, basic fibroblast growth factor (FGF-2) stimulates immature Leydig cell steroidogenesis in the absence of luteinizing hormone (LH). Heparan sulphate proteoglycans (HSPGs) are essential for this action of FGF-2 and the data suggest that HSPG/FGF-2 interactions have a significant role in the maintenance of immature Leydig cell steroidogenesis. Culture conditions were established for the maintenance of immature rat Leydig cells steroidogenesis in vitro for at least 2 days. Under these conditions the effect of exposure to FGF-2 at doses ranging from 0.1–10 ng/ml was shown to cause a significant stimulation of basal, but not LH-stimulated, 5-androstane 3,17β-diol production over 24h in culture. This stimulatory action on basal steroidogenesis is mediated through HSPG, as it was blocked by the addition of heparin (100 μg/ml), sodium chlorate (25mM) and protamine sulphate (5 μg/ml). These data demonstrate the involvement of HSPG in regulating FGF-2 action on Leydig cells and a potential role for Leydig cell HSPG in mediating paracrine regulatory actions of other heparin binding growth factors.     

Selective binding of polymyxin B to negatively charged lipid monolayers

It is demonstrated by direct measurement of surface radioactivity that the cationic polypeptide antibiotic polymyxin B is specifically adsorbed to negatively charged lipid monolayers. The latter attracted the following amounts of the biologically active $\text{mono}\text{-N[}{}^{14}\text{C]}\text{acetylpolymyxin B}$ derivative (PX): lipid A from Proteus mirabilis, 0.17; phosphatidic acid, 0.12; phosphatidylglycerol and phosphatidylserine, 0.11; dicetylphosphate, 0.107; sulfoquinovosyldiglyceride, 0.104; phosphatidylinositol and cardiolipin, 0.095; and phosphatidylethanolamine, 0.017 μg/cm². Adsorption of PX to phosphatidylcholine, monogalactosyldiglyceride and stearylamine was almost or completely zero. Total lipids from Escherichia coli adsorbed 0.057 in comparison to 0.051 μg PX/cm² of an artificial mixture of phosphatidylethanolamine/phosphatidylglycerol/cardiolipin in the proportions 75 : 25 : 5. The concentration of the surface active PX at the air/water interphase was 0.091 μg/cm². These saturation surface concentrations of PXat lipid monolayers were reached at 1 μg/ml bulk concentrations in 2 mM NaCl/1 mM Tris · HCl, pH 7.2. They decreased with decreasing surface charge density of the adsorbing monolayer. In an experiment with cardiolipin/phosphatidylethanolamine mixtures it was shown that two molecules of cardiolipin induced adsorption of one molecule PX giving a 1 : 1 ratio with regard to positive and negative charges. This could be due to a similar charge density of about one charge per 40–50 Ų in PX and lipid bilayers composed of phospholipids. The electrostatic PX-lipid interaction was severely inhibited by 10^?2 and 10^?1 M Ca²⁺ and Na⁺, respectively. It is discussed that the specificity of PX against Gram-negative bacteria is caused by the occurrence of lipid A, phosphatidylglycerol and cardiolipin at the cell surface of these microorganisms.     

Isolation of thecal cells: an assessment of purity and steroidogenic potential

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E(2)) and progesterone (P(4)) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E(2) could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P(4) in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E(2) was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P(4) (P<0.05) in the serum-free cell culture media. The stimulation of P(4) production was greater at higher LH concentration (50 ng/ml) (P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs. EC(50)>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.     

The morphology of adsorbed extracellular matrix assemblies is critically dependent on solution calcium concentration.

The adsorption of proteins to surfaces may alter their biological properties. Understanding and controlling these interactions is important in ultrastructural, biochemical and cellular studies. We have previously demonstrated that both the morphology and biological function of extracellular matrix assemblies such as fibrillin and type VI collagen microfibrils are influenced by surface chemistry. In this study we have employed atomic force microscopy to determine if the morphology of extracellular matrix microfibrils is influenced by solution chemistry. Microfibrils were adsorbed to mica or poly-L-lysine modified mica (mica-PLL) in the presence of 31 microM-1000 microM Ca(2+). Although both microfibrillar species adsorbed to mica and mica-PLL at all calcium concentrations, maximal adsorption was observed on mica at 125-250 microM. On mica surfaces fibrillin microfibril morphology varied continuously with calcium concentration from laterally diffuse assemblies at high concentrations to compact assemblies at low concentrations. In contrast, distinct type VI collagen microfibril morphologies were observed at high, intermediate and low calcium concentrations. Similar calcium dependent microfibrillar morphologies were evident on mica-PLL. Therefore physiologically relevant concentrations of solution calcium, independent of surface charge, profoundly influenced both the adsorbed amount and morphology of native extracellular assemblies. These studies highlight the importance not only of surface chemistry but also of solute composition and concentration in influencing the morphology and hence biological function of adsorbed proteins.     

Nano-scale probe fabrication using self-assembly technique and application to detection of<Emphasis Type="Italic">Escherichia coli</Emphasis> O 157∶H7

A self-assembled monolayer of protein G was fabricated to develop an immunosensor based on surface plasmon resonance (SPR), thereby improving the performance of the antibody-based biosensor through immobilizing the antibody molecules (IgG). As such, 11-mercaptoundecanoic acid (11-MUA) was adsorbed on a gold (Au) support, while the non-reactive hydrophilic surface was changed through substituting the carboxylic acid group (-COOH) in the 11-MUA molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholide (EDAC). The formation of the self-assembled protein G layer on the Au substrate and binding of the antibody and antigen were investigated using SPR spectroscopy, while the surface topographies of the fabricated thin films were analyzed using atomic force microscopy (AFM). A fabricated monoclonal antibody (Mab) layer was applied for detectingE. coli O157∶H7. As a result, a linear relationship was achieved between the pathogen concentration and the SPR angle shift, plus the detection limit was enhanced up to 10² CFU/mL.