Strategies to Improve the Surface Plasmon Resonance-Based Immmunodetection of Bacterial Cells

We have made a comparison of (a) different surface chemistries of surface plasmon resonance (SPR) sensor chips (such as carboxymethylated dextran and carboxymethylated C1) and (b) of different assay formats (direct, sandwich and subtractive immunoassay) in order to improve the sensitivity of the determination of the model bacteria Acidovorax avenae subsp. citrulli (Aac). The use of the carboxymethylated sensor chip C1 resulted in a better sensitivity than that of carboxymethylated dextran CM5 in all the assay formats. The direct assay format, in turn, exhibits the best sensitivity. Thus, the combination of a carboxymethylated sensor chip C1 with the direct assay format resulted in the highest sensitivity for Aac, with a limit of detection of 1.6x106 CFU mL-1. This SPR immunosensor was applied to the detection of Aac in watermelon leaf extracts spiked with the bacteria, and the lower LOD is 2.2x107 CFU mL-1.

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

Orientation effects in copper phthalocyanine films studied by electron paramagnetic resonance spectroscopy

The metallo-phthalocyanines (MPcs) are an interesting group of organic semiconductor materials for applications such as large area solar cells due to their optoelectronic properties coupled with the possibility of easily and cheaply fabricating thin films of MPcs [1, 2]. As for organic semiconductors in general, many of the interesting properties of the MPcs such as magnetism, light absorption and charge transport, are highly anisotropic [2, 3]. To maximise the efficiency of a device based on these materials it is therefore important to study their molecular orientation in films and to assess the influence of different growth conditions and substrate treatments.
X-ray diffraction is a well established and powerful technique for studying texture (and hence molecular orientation) in crystalline materials, but it cannot provide any information about amorphous or nanocrystalline films. In electron paramagnetic resonance (EPR) spectroscopy the signal comes from the spin of unpaired electrons in the material. This technique therefore does not require the sample to be crystalline. It works for any sample with paramagnetic centres such as the MPcs where the unpaired electrons are contributed by the metal. In this paper we present a continuous-wave X-band EPR study using the anisotropy of the EPR spectrum of CuPc [4] to determine the orientation effects in different types of CuPc films. From these measurements we gain insight into the molecular arrangement of films with different spin concentrations, and apply our technique to the study of molecular orientation in photovoltaic cells.

A Theoretical and Experimental Study of Resonance in a High Performance Engine Intake System: Part 2

The unsteady gas dynamic phenomena in a racecar airbox have been examined, and resonant tuning effects have been considered. A coupled 1D/3D analysis, using the engine simulation package Virtual 4-Stroke and the CFD package FLUENT, was used to model the engine and airbox. The models were experimentally validated. An airbox was designed with a natural frequency in the region of 75 Hz. A coupled 1D/3D analysis of the airbox and a Yamaha R6 4 cylinder engine predicted resonance at the single-cylinder induction frequency; 75 Hz at an engine speed of 9000 rpm.

Multi sulfonamide screening in porcine muscle using a surface plasmon resonance biosensor

A binding protein displaying broad-spectrum cross-reactivity within the sulfonamide group was used in conjunction with a sulfonamide specific sensor chip and a surface plasmon resonance biosensor to develop a rapid broad spectrum screening assay for sulfonamides in porcine muscle. Results for 40 samples were available in just over 5 h after the completion of a simple sample preparation protocol. Twenty sulfonamide compounds were detected. Acetylated metabolites were not recognised by the binding protein. Limit of detection (mean-three times standard deviation value when n = 20) was calculated to be 16.9 ng g(-1) in tissue samples. Intra-assay precision (n = 10) was calculated at 4.3 %CV for a sample spiked at 50 ng g(-1) with sulfamethazine, 3.6 %CV for a sample spiked at 100 ng g(-1) with sulfamethazine, 7.2 %CV for a sample spiked at 50 ng g(-1) with sulfadiazine and 3.1 %CV for a sample spiked at 100 ng g-1 with sulfadiazine. Inter-assay precision (n = 3) was calculated at 9.7 %CV for a sample spiked at 50 ng g-1 with sulfamethazine, 3.8 %CV for a sample spiked at 100 ng g(-1) with sulfamethazine, 3.5 %CV for a sample spiked at 50 ng g(-1) with sulfadiazine and 2.8 %CV for a sample spiked at 100 ng g(-1) with sulfadiazine. (C) 2004 Elsevier B.V. All rights reserved.

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: A femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG(3)-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and Fr-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG(6)-COOH and HS-OEG(3)-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-[alpha] 1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-a1-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody–antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1×106–1×102 oocysts ml-1. The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.

Phenomenology of Resonance Reflectance by Intertwined Spiral Arrays

The physical mechanisms underlying the dramatic reduction of the unit cell electrical size along with broadening fractional bandwidths provided by intertwined spiral arrays are discussed. Based upon this insight, a multi-strip transmission line (MTL) model is developed to analytically estimate the equivalent capacitance and inductance of intertwined spiral array elements in terms of their geometrical parameters. The proposed MTL model enables an accurate prediction of the fundamental resonance characteristics and provides a valuable tool for design of the arrays with the specified frequency response.

Analysis of Biological Interactions and Recognitions by Surface Plasmon Resonance

This chapter contains sections titled:

•Introduction to Surface Plasmon Resonance Technology

•Working Principle of SPR

•Sensor Surface Chemistry and Its Fabrications

•Important Factors Impacting on the Performance of SPR-Based Analyses of Biological Interactions on the Nonbiological Transducer Surface

•Localized SPR of Inorganic Nanoparticles for Analyses of Biological Interaction

•References

METHOD OF DETECTING BIOPRODUCTS USING LOCALIZED SURFACE PLASMON RESONANCE SENSOR OF GOLD NANOPARTICLES

(EN)Disclosed is a method of detecting bioproducts using Localized Surface Plasmon Resonance (LSPR) of gold nanoparticles, which can diagnose bioproducts based on changes in the maximum wavelength occurred by an antigen-antibody reaction after immobilization of the gold nanoparticles onto a glass panel. A sensor using such method exhibits high sensitivity, is low in price, and makes quick diagnosis possible, thereby being applicable to various biological fields associated with environmental contaminants, pathogens and the like, as well as diagnosis of diseases. Further, it provides a technology for manufacturing a sensor having higher sensitivity, low price and quick performance, as compared to conventional methods using SPR.

Effects of Gold Nanoparticle and Enzyme Precipitation on Enhancement of Surface Plasmon Resonance Immunoassay: A Super Sensitive Measurement of Anti- Glutamic Acid Decarboxylase Antibody

Introduction: In this study, colloidal gold nanoparticle and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance biosensor.

Methods: The colloidal gold nanoparticle was synthesized as described by Turkevitch et al., and their surface was firstly functionalized with HS(CH2)11(OCH2CH2)3COOH (OEG3¬-COOH) by self assembling technique. Thereafter, those OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti-IgG antibody (specific to the Fc portion of all human IgG subclasses) to form an enzyme-immunogold complex. Characterization was performed by several methods: UV-Vis absorption, dynamic light scattering (DLS), transmission electron microscopy (TEM) and FTIR. The as-prepared enzyme-immunogold complex has been applied in enhancement of SPR immunoassay. A sensor chip used in the experiment was constructed by using 1:10 molar ratio of HS(CH2)11(OCH2CH2)6COOH and HS(CH2)11(OCH2CH2)3OH. The capture protein, GAD65 (autoantigen) which is recognized by anti-GAD antibody (autoantibody) in the sera of insulin-dependent diabetes mellitus patients, was immobilized onto the 1:10 surface via biotin-streptavidin interaction.

Results and conclusions: In the research, we reported the influences of gold nanoparticle and enzyme precipitation on the enhancement of SPR signal. Gold nanoparticle showed its enhancement as being consistent with other previous studies, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. As the results, anti-GAD antibody could be detected at pg/ml level which is far higher than that of commercial ELISA detection kit. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Nuclear magnetic resonance-determined lipoprotein subclass profile in the DCCT/EDIC cohort:associations with carotid intima-media thickness

To relate nuclear magnetic resonance lipoprotein subclass profiles (NMR-LSP) and other lipoprotein-related factors with carotid intima-media thickness (IMT) in Type 1 diabetes.

Lipoprotein subclass profiles of hyperlipidemic diabetic mice measured by nuclear magnetic resonance spectroscopy

Dyslipidemia accelerates vascular complications of diabetes. Nuclear magnetic resonance (NMR) analysis of lipoprotein subclasses is used to evaluate a mouse model of human familial hypercholesterolemia +/- streptozotocin (STZ)-induced diabetes. A double knockout (DKO) mouse (low-density lipoprotein receptor [LDLr] -/-; apolipoprotein B [apoB] mRNA editing catalytic polypeptide-1 [Apobec1] -/-) was studied. Wild-type (WT) and DKO mice received sham or STZ injections at age 7 weeks, yielding control (WT-C, DKO-C) and diabetic (WT-D, DKO-D) groups. Fasting serum was collected when the mice were killed (age 40 weeks) for Cholestech analysis (Cholestech Corp, Hayward, CA) and NMR lipoprotein subclass profile. By Cholestech, fasting triglyceride and total cholesterol increased in DKO-C versus WT-C. Diabetes further increased total cholesterol in DKO. High-density lipoprotein cholesterol (HDL-C) was similar among all groups. NMR revealed that LDL in all groups was present in a subclass the size of large human LDL and was increased 48-fold in DKO-C versus WT-C animals, but was unaffected by diabetes. HDL was found in a subclass equivalent to large human HDL, and was similar among groups. In conclusion, NMR analysis reveals lipoprotein subclass distributions and the effects of genetic modification and diabetes in mice, but lack of particles the size of human small LDL and small HDL may limit the relevance of the present animal model to human disease.

Multiplex biotoxin surface plasmon resonance method for marine biotoxins in algal and seawater samples

A multiplex surface plasmon resonance (SPR) biosensor method for the detection of paralytic shellfish poisoning (PSP) toxins, okadaic acid (and analogues) and domoic acid was developed. This method was compared to enzyme-linked immunosorbent assay (ELISA) methods. Seawater samples (n?=?256) from around Europe were collected by the consortia of an EU project MIcroarrays for the Detection of Toxic Algae (MIDTAL) and evaluated using each method. A simple sample preparation procedure was developed which involved lysing and releasing the toxins from the algal cells with glass beads followed by centrifugation and filtering the extract before testing for marine biotoxins by both multi-SPR and ELISA. Method detection limits based on IC20 values for PSP, okadaic acid and domoic acid toxins were 0.82, 0.36 and 1.66 ng/ml, respectively, for the prototype multiplex SPR biosensor. Evaluation by SPR for seawater samples has shown that 47, 59 and 61 % of total seawater samples tested positive (result greater than the IC20) for PSP, okadaic acid (and analogues) and domoic acid toxins, respectively. Toxic samples were received mainly from Spain and Ireland. This work has demonstrated the potential of multiplex analysis for marine biotoxins in algal and seawater samples with results available for 24 samples within a 7 h period for three groups of key marine biotoxins. Multiplex immunological methods could therefore be used as early warning monitoring tools for a variety of marine biotoxins in seawater samples.