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.

Fluctuations of the Stokes and anti-Stokes surface-enhanced resonance Raman scattering intensities in an electrochemical environment

Stokes and anti-Stokes SERRS intensity fluctuations were observed from a roughened silver electrode immersed in diluted solutions of Brilliant Green (BG), a behaviour linked to single-molecule events. The distributions of the anti-Stokes to Stokes ratios were obtained and their shape showed a strong dependence on the applied potential.

Results of a phase IIa clinical trial of an anti-inflammatory molecule, chaperonin 10, in multiple sclerosis

Background Chaperonin 10 (Cpn10) is a mitochondrial molecule involved in protein folding. The aim of this study was to determine the safety profile of Cpn10 in patients with multiple sclerosis (MS). Methods A total of 50 patients with relapse-remitting or secondary progressive MS were intravenously administered 5 mg or 10 mg of Cpn10 weekly for 12 weeks in a double-blind, randomized, placebo controlled, phase II trial. Clinical reviews, including Expanded Disability Status Scale and magnetic resonance imaging (MRI) with Gadolinium, were undertaken every 4 weeks. Stimulation of patient peripheral blood mononuclear cells with lipopolysaccharide ex vivo was used to measure the in vivo activity of Cpn10. Results No significant differences in the frequency of adverse events were seen between treatment and placebo arms. Leukocytes from both groups of Cpn10-treated patients produced significantly lower levels of critical proinflammatory cytokines. A trend toward improvement in new Gadolinium enhancing lesions on MRI was observed, but this difference was not statistically significant. No differences in clinical outcome measures were seen. Conclusions Cpn10 is safe and well tolerated when administered to patients with MS for 3 months, however, a further extended phase II study primarily focused on efficacy is warranted.

Magnetization, magnetotransport and electron magnetic resonance studies of nanoparticles and nanowires of Pr0.5Sr0.5MnO3

In this paper, we present the preparation and characterization of nanoparticles and nanowires of Pr0.5Sr0.5MnO3 (PSMO). The main results of this investigation are as follows: (a) a comparison with the properties of the bulk material shows that the ferromagnetic (FM) transition at 270 K remains unaffected but the anti-ferromagnetic (AFM) transition at TN = 150 K disappears in the nanoparticles, (b) the size induced ground state magnetic phase (below 150 K) is predominantly FM, coexisting with a residual AFM phase, and (c) the temperature dependence of magnetic anisotropy shows complex behaviour, being higher in the nanoparticles at high temperatures and lower at moderately lower temperatures in comparison with the bulk. The results obtained from the extensive magnetization, magnetotransport and electron magnetic resonance studies made on various samples are presented and discussed in detail.

The ATHEROMA (Atorvastatin Therapy: Effects on Reduction of Macrophage Activity) Study. Evaluation using ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging in carotid disease

Objectives: The aim of this study was to evaluate the effects of low-dose (10 mg) and high-dose (80 mg) atorvastatin on carotid plaque inflammation as determined by ultrasmall superparamagnetic iron oxide (USPIO)-enhanced carotid magnetic resonance imaging (MRI). The hypothesis was that treatment with 80 mg atorvastatin would demonstrate quantifiable changes in USPIO-enhanced MRI-defined inflammation within the first 3 months of therapy. Background: Preliminary studies indicate that USPIO-enhanced MRI can identify macrophage infiltration in human carotid atheroma in vivo and hence may be a surrogate marker of plaque inflammation. Methods: Forty-seven patients with carotid stenosis >40% on duplex ultrasonography and who demonstrated intraplaque accumulation of USPIO on MRI at baseline were randomly assigned in a balanced, double-blind manner to either 10 or 80 mg atorvastatin daily for 12 weeks. Baseline statin therapy was equivalent to 10 mg of atorvastatin or less. The primary end point was change from baseline in signal intensity (ΔSI) on USPIO-enhanced MRI in carotid plaque at 6 and 12 weeks. Results: Twenty patients completed 12 weeks of treatment in each group. A significant reduction from baseline in USPIO-defined inflammation was observed in the 80-mg group at both 6 weeks (ΔSI 0.13; p = 0.0003) and at 12 weeks (ΔSI 0.20; p < 0.0001). No difference was observed with the low-dose regimen. The 80-mg atorvastatin dose significantly reduced total cholesterol by 15% (p = 0.0003) and low-density lipoprotein cholesterol by 29% (p = 0.0001) at 12 weeks. Conclusions: Aggressive lipid-lowering therapy over a 3-month period is associated with significant reduction in USPIO-defined inflammation. USPIO-enhanced MRI methodology may be a useful imaging biomarker for the screening and assessment of therapeutic response to "anti-inflammatory" interventions in patients with atherosclerotic lesions. (Effects of Atorvastatin on Macrophage Activity and Plaque Inflammation Using Magnetic Resonance Imaging [ATHEROMA]; NCT00368589).

Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to or 70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

Structural studies of analgesics and their interactions. XII. Structure and interactions of anti-inflammatory fenamates. A concerted crystallographic and theoretical conformational study

A theoretical conformational analysis of fenamates, which are N-arylated derivatives of anthranilic acid or 2-aminonicotinic acid with different substituents on the aryl (phenyl) group, is reported. The analysis of these analgesics, which are believed to act through the inhibition of prostaglandin biosynthesis, was carried out using semi-empirical potential functions. The results and available crystallographic observations have been critically examined in terms of their relevance to drug action. Crystallographic studies of these drugs and their complexes have revealed that the fenamate molecules share a striking invariant feature, namely, the sixmembered ring bearing the carboxyl group is coplanar with the carboxyl group and the bridging imino group,the coplanarity being stabilized by resonance interactions and an internal hydrogen bond between the imino and carboxyl groups. The results of the theoretical analysis provide a conformational rationale for the observed invariant coplanarity. The second sixmembered ring, which provides hydrophobicity in a substantial part of the molecule, has limited conformational flexibility in meclofenamic, mefenamic and flufenamic acids. Comparison of the conformational energy maps of these acids shows that they could all assume the same conformation when bound to the relevant enzyme. The present study provides a structural explanation for the difference in the activity of niflumic acid, which can assume a conformation in which the whole molecule is nearly planar. The main role of the carboxyl group appears to be to provide a site for intermolecular interactions in addition to helping in stabilizing the invariant coplanar feature and providing hydrophilicity at one end of the molecule. The fenamates thus provide a good example of conformation- dependent molecular asymmetry.

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis sigma/anti-sigma factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the sigma factor/anti-sigma factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of sigma factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. (C) 2010 Elsevier Inc. All rights reserved.

Measuring Binding Kinetics Of Surface-Bound Molecules Using The Surface Plasmon Resonance Technique

Surface plasmon resonance (SPR) technology and the Biacore biosensor have been widely used to measure the kinetics of biomolecular interactions in the fluid phase. In the past decade, the assay was further extended to measure reaction kinetics when two counterpart molecules are anchored on apposed surfaces. However, the cell binding kinetics has not been well quantified. Here we report development of a cellular kinetic model, combined with experimental procedures for cell binding kinetic measurements, to predict kinetic rates per cell. Human red blood cells coated with bovine serum albumin and anti-BSA monoclonal antibodies (mAbs) immobilized on the chip were used to conduct the measurements. Sensor-grams for BSA-coated RBC binding onto and debinding from the anti-BSA mAb-immobilized chip were obtained using a commercial Biacore 3000 biosensor, and analyzed with the cellular kinetic model developed. Not only did the model fit the data well, but it also predicted cellular on and off-rates as well as binding affinities from curve fitting. The dependence of flow duration, flow rate, and site density of BSA on binding kinetics was tested systematically, which further validated the feasibility and reliability of the new approach. Crown copyright (c) 2008 Published by Elsevier Inc. All rights reserved.

Studies of dinucleoside monophosphates and monomer-polynucleotide interactions by proton magnetic resonance

The nature of the intra- and intermolecular base-stacking interactions involving several dinucleoside monophosphates in aqueous solution have been investigated by proton magnetic resonance spectrosocopy, and this method has been applied to a study of the interaction of polyuridylic acid with purine and adenosine monomers.

The pmr spectra of adenylyl (3' → 5') cytidine (ApC) and cytidylyl (3' → 5') adenosine (CpA) have been studied as a function of concentration and temperature. The results of these studies indicate that the intramolecular base-stacking interactions between the adenine and cytosine bases of these dinucleoside monophosphates are rather strong, and that the stacking tendencies are comparable for the two sequence isomers. The chemical shifts of the cytosine H₅ and adenine H₂ protons, and their variations with temperature, were shown to be consistent with stacked conformations in which both bases of the dinucleoside monophosphates are preferentially oriented in the anti conformation as in similar dApdC, and dCpdA (dA = deoxyadenosine; dC = deoxycytidine) segments in double helical DNA. The intramolecular stacking interaction was found to have a pronounced effect on the conformations of the ribose moieties, and these conformational changes are discussed. The concentration studies indicate extensive self-association of these dinucleoside monophosphates, and analysis of the concentration data facilitated determination of the dimerization constant for the association process as well as the nature of the intermolecular complexes.

The dependence of the ribose conformation upon the extent of intramolecular base-stacking was used to demonstrate that the base-base interaction in cytidylyl (3' → 5') cytidine (CpC) is rather strong, while there appears to be little interaction between the two uracil bases of uridylyl (3' → 5') uridine (UpU).

Studies of the binding of purine to several ribose and deoxyribose dinucleoside monophosphates show that the mode of interaction is base-stacking, and evidence for the formation of a purine-dinucleoside monophosphate intercalated complex is presented. The purine proton resonances are markedly broadened in this complex, and estimates of the purine linewidths in the complex and the equilibrium constant for purine intercalation are obtained.

A study of the interaction of unsubstitued purine with polyuridylic acid at 29°C by pmr indicated that purine binds to the uracil bases of the polymer by base-stacking. The severe broadening of the purine proton resonances observed provides strong evidence for the intercalation of purine between adjacent uracil bases of poly U. This interaction does not result in a more rigid or ordered structure for the polymer.

Investigation of the interaction between adenosine and polyuridylic acid revealed two modes of interaction between the monomer and the polymer, depending on the temperature. At temperatures above 26°C or so, monomeric adenosine binds to poly U by noncooperative A-U base stacking. Below this temperature, a rigid triple-stranded 1A:2U complex is formed, presumably via cooperative hydrogen-bonding as has previously been reported.

These results clearly illustrate the importance of base-stacking in non-specific interactions between bases, nucleosides and nucleotides, and also reveal the important role of the base-stacking interactions in cooperatively for med structures involving specific base-pairing where both types of interaction are possible.

Proton magnetic resonance studies of ribonucleic acid complexes. I. Complexes of biological bases and oligonucleotides with RNA. II. Template recognition and the degeneracy of the genetic code

Part I. Complexes of Biological Bases and Oligonucleotides with RNA

The physical nature of complexes of several biological bases and oligonucleotides with single-stranded ribonucleic acids have been studied by high resolution proton magnetic resonance spectroscopy. The importance of various forces in the stabilization of these complexes is also discussed.

Previous work has shown that purine forms an intercalated complex with single-stranded nucleic acids. This complex formation led to severe and stereospecific broadening of the purine resonances. From the field dependence of the linewidths, T₁ measurements of the purine protons and nuclear Overhauser enhancement experiments, the mechanism for the line broadening was ascertained to be dipole-dipole interactions between the purine protons and the ribose protons of the nucleic acid.

The interactions of ethidium bromide (EB) with several RNA residues have been studied. EB forms vertically stacked aggregates with itself as well as with uridine, 3'-uridine monophosphate and 5'-uridine monophosphate and forms an intercalated complex with uridylyl (3' → 5') uridine and polyuridylic acid (poly U). The geometry of EB in the intercalated complex has also been determined.

The effect of chain length of oligo-A-nucleotides on their mode of interaction with poly U in D₂0 at neutral pD have also been studied. Below room temperatures, ApA and ApApA form a rigid triple-stranded complex involving a stoichiometry of one adenine to two uracil bases, presumably via specific adenine-uracil base pairing and cooperative base stacking of the adenine bases. While no evidence was obtained for the interaction of ApA with poly U above room temperature, ApApA exhibited complex formation of a 1:1 nature with poly U by forming Watson-Crick base pairs. The thermodynamics of these systems are discussed.

Part II. Template Recognition and the Degeneracy of the Genetic Code

The interaction of ApApG and poly U was studied as a model system for the codon-anticodon interaction of tRNA and mRNA in vivo. ApApG was shown to interact with poly U below ~20°C. The interaction was of a 1:1 nature which exhibited the Hoogsteen bonding scheme. The three bases of ApApG are in an anti conformation and the guanosine base appears to be in the lactim tautomeric form in the complex.

Due to the inadequacies of previous models for the degeneracy of the genetic code in explaining the observed interactions of ApApG with poly U, the "tautomeric doublet" model is proposed as a possible explanation of the degenerate interactions of tRNA with mRNA during protein synthesis in vivo.

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays. (c) 2007 Elsevier B.V. All rights reserved.

ac gate-induced Fano resonance and peak splitting of conductance in a quantum dot

We have investigated the conductance of a quantum dot system suffering an anti-symmetric ac gate voltage which induces the transition between dot levels in the linear regime at zero temperature in the rotating wave approximation. Interesting Fano resonances appear on one side of the displaced resonant tunnelling peaks for the nonresonant case or the peak splitting for the resonant case. The line shape of conductance (vs Fermi energy) near each level of the quantum dot can be decomposed into two profiles: a Breit-Wigner peak and a Fano profile, or a Breit-Wigner peak and a dip in both cases.

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes.

The detection of paralytic shellfish poisoning (PSP) toxins in contaminated shellfish is essential for human health preservation. Ethical and technical reasons have prompted the search for new detection procedures as an alternative to the mouse bioassay. On the basis of the detection of molecular interactions by surface plasmon resonance (SPR) biosensors, an inhibition assay was developed using an anti-GTX2/3 antibody (GT13-A) and a saxitoxin-CM5 chip. This assay allowed for quantification of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), gonyautoxin 2,3 (GTX2/3), decarbamoyl gonyautoxin 2,3 (dcGTX2/3), gonyautoxin 5 (GTX5), and C 1,2 (C1/2) at concentrations from 2 to 50 ng/mL. The interference of five shellfish matrixes with the inhibition assay was analyzed. Mussels, clams, cockles, scallops, and oysters were extracted with five published methods. Ethanol extracts and acetic acid/heat extracts (AOAC Lawrence method) performed adequately in terms of surface regeneration and baseline interference, did not inhibit antibody binding to the chip surface significantly, and presented STX calibration curves similar to buffer controls in all matrixes tested. Hydrochloric acid/heat extracts (AOAC mouse bioassay method) presented surface regeneration problems, and although ethanol-acetic acid/dichloromethane extracts performed well, they were considered too laborious for routine sample testing. Overall the best results were obtained with the ethanol extraction method with calibration curves prepared in blank matrix extracts. STX recovery rate with the ethanol extraction method was 60.52 ± 3.72%, with variations among species. The performance of this biosensor assay in natural samples, compared to two AOAC methods for PSP toxin quantification (mouse bioassay and HPLC), suggests that this technology can be useful as a PSP screening assay. In summary, the GT13-A-STX chip inhibition assay is capable of PSP toxin detection in ethanol shellfish extracts, with sufficient sensitivity to quantify the toxin in the range of the European regulatory limit of 80 g/100 g of shellfish meat.