Antibody regeneration on degenerate Si electrodes for calibration and reuse of impedance biosensors

Mild denaturing agents were studied for antibody regeneration atop degenerate (highly doped) Si and Au electrodes using comparable 11-carbon chain linker reagents onto which the mouse monoclonal antibody to peanut protein Ara h 1 is covalently immobilized. Of the reagents studied, only 200 mM KSCN is effective for antibody regeneration and detection of Ara h 1 atop Au electrodes, allowing 15 days of sensor usage after daily antibody unfolding and refolding, while 200 mM KSCN +10 mM HF is effective atop degenerate Si electrodes, allowing 30 days of sensor usage. The addition of HF is required for antibody regeneration atop Si electrodes, as demonstrated by cyclic voltammetry in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 at pH 7.3, where the oxidation/reduction peaks can be observed only in the presence of HF. The impedance spectrum for detection of Ara h 1 gradually degrades during these multi-day regeneration trials, but calibration experiments performedwithin one day illustrate that sensor electrodes can be calibrated on the day of use towithin about 2%

Amperometric Bioelectronic Tongue for glucose determination

An amperometric Bioelectronic Tongue is reported for glucose determination that contains eight sensor electrodes constructed using different metal electrodes (Pt, Au), oxidoreductase enzymes (glucose oxidase, ascorbate oxidase, uricase), and membrane coatings (Nafion, chitosan). The response to varying concentrations of glucose, ascorbic acid, uric acid, and acetaminophen was tested for two models, concentration determination by current density measurements at individual electrodes and concentration determination by a linear regression model for the entire electrode array. The reduced chi-squared for the full array model was found to be about one order of magnitude lower than that for the individualelectrode model. Discrimination of glucose from chemical interference by the other three species is accomplished through a combination of enzyme catalysis, metal electrocatalysis, and membrane surface charge. The benefit of incorporating enzyme electrodes into the sensor array is illustrated by the lower correlation coefficients between different enzyme electrodes relative to non-enzyme coated electrodes. This approach can be more generally applied to detection of other substrates of oxidoreductase enzymes

Low Temperature Electrochemical Deposition of Highly Active Elements

Electrochemical methods are attractive for thin film deposition due to their simplicity, conformal and high rate deposition, the ability to easily make multilayers of different composition, ease of scale-up to large surface areas, and applicability to wide variety of different shapes and surface geometries. However, many elements from periodic table of commercial importance are too active to be electrodeposited from aqueous solution. Recent advances are briefly reviewed for room temperature methods for electrochemical deposition, including electrodeposition from ionic liquids, electrodeposition from organic solvents, combined electrodeposition and precipitation on liquid metal cathodes, and galvanic deposition. Recent studies of electrodeposition from ionic liquids include deposition of thick (40 µm) Al coatings on high-strength steel screws in a manufacturing environment; deposition of continuous Si, Ta and Nb coatings; and numerous interesting mechanistic studies. Recent studies of electrodeposition from organic solvents include Al coatings from the AlCl3-dimethylsulfone electrolyte, which demonstrate that additives can be employed to suppress impurity incorporation and to improve the deposit quality, and thick (5-7 µm) and continuous Si coatings from SiCl4 in acetonitrile. Galvanic deposition of Ti, Mo and Si coatings onto Al alloys has recently been reported, which is potentially much simpler and less expensive than electrodeposition from ionic liquids and organic solvents, but has complications associated with substrate consumption and coating adhesion

Immersion/Electroless Deposition of Cu on Ta

Continuous Cu films are deposited on Ta by a two-step process; galvanic displacement of Ta by Cu from ammonium fluoride solutions and subsequent electroless Cu deposition from a formaldehyde-containing bath. The conditions necessary for good adhesion are discussed. The extent of oxide film removal in the HF pretreatment solution is studied by electrochemical impedance spectroscopy. The charge-transfer resistance of about 57 Ω-cm2 is several orders of magnitude lower than that measured for the Ta native oxide. These results are consistent with the removal of the Ta2O5 portion of the native oxide

Impedance Biosensor Utilizing a Si Substrate Deposited by Wet Methods

We report an impedance biosensor utilizing a Si electrode created by wet chemical deposition atop 6061 Al alloy. The sensor electrode is created by galvanic/electroless Si deposition from an electrolyte containing 10 mM HF and 20 mM Na2SiF6 in 80 wt% formic acid, followed by antibody immobilization. The impedance response of the sensor electrode to increasing concentrations of peanut protein Ara h 1, a common food allergen, can be fit to an equivalent circuit containing three RC loops. The circuit element most sensitive to antigen binding is the charge transfer resistance, yielding a detection limit of 4 ng/mL

Electrodeposition of MoS2 for Charge Storage in Electrochemical Supercapacitors

Mo sulfide thin films were cathodically electrodeposited onto glassy carbon electrodes (GCE) from aqueous electrolytes containing 10 mM (NH4)2MoS4 and 0.2 M KCl. Film adhesion was adequate only for electrodes pretreated by potential cycling in 1.0 M HNO3 and 0.1 M NaF to enhance the surface roughness and partially oxidize the GCE. Previous studies report direct cathodic electrodeposition of MoS2, but energy dispersive x-ray spectroscopy and x-ray diffraction suggest that the as-deposited film is closer in stoichiometry to MoS3, which can be converted to MoS2 by annealing in Ar at 600°C for one hour. The charge storage capability of electrodeposited Mo sulfide films is studied here for the first time in 1.0 M Na2SO4 over the thickness range 50 nm to 5 µm, and before and after high temperature annealing. The highest capacitance is obtained for 50 nm thick MoS2 films is 330 F/g measured by galvanostatic charge discharge at 0.75 A/g, and 360 F/g measured by cyclic voltammetry at 10 mV/sec. The capacitance per unit mass decreases with increasing film thickness due to reduced electrochemical accessibility. MoS2 film formed by high temperature annealing in Ar have a charge storage capability about 40x higher than the as-deposited Mo sulfide films

Impedance Biosensor Incorporating a Carboxylate-Terminated Bidentate Thiol for Antibody Immobilization

An impedance biosensor is reported that employs the bidentate thiol, 16-[3,5-bis(mercaptomethyl)phenoxy]-hexadecanoic acid (BMPHA), as a bifunctional reagent for antibody immobilization atop an Au electrode, and the results are compared to those obtained for the analogous monodentate reagent, 16-mercaptohexadecanoic acid (16 MHA). The detection limit for peanut protein Ara h 1 on the BMPHA bidentate thiol- coated Au electrode is approximately 0.71 ng/mL (0.01 nM), about 3x lower than that obtained on the comparable monodentate (16-MHA) thiol-coated Au electrode. Daily impedance measurements were employed to study antibody regeneration with a mild denaturing agent, 0.2 M KSCN at pH 7.3. The antibody-coated electrodes retained activity towards Ara h1 for 10 and 20 days of regeneration of the monodentate- and BMPHA-coated Au electrodes, respectively, illustrating the superior stability of protein films atop the BMPHA bidentate thiol

Standardization of cytokine flow cytometry assays

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4(+)cytokine(+ )cells and CD8(+)cytokine(+ )cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays