Anti‐Clostridium tetani Antibody Determination in Serum Samples by Amperometric Immunosensing

An electrochemical (EC) immunosensing assay for anti‐Clostridium tetani antibody determination in serum has been developed. The antigen tetanus toxoid was immobilized on superparamagnetic nanobeads. The immunoreaction occurred in Eppendorf minitubes. The anti‐tetani antibody was incubated in the presence of the toxoid functionalized nanobeads, then reacted with horseradish peroxidase‐labeled anti‐IgG. The resulting immunobeads were retained onto the carbon paste working electrode with a magnet. Hydroquinone served as redox label. The level of anti‐Clostridium tetani antibody in guinea pig serum samples was determined by amperometry using a carbon based screen‐printed electrode (cSPE) housed onto a magnetic support. The EC response was proportional to the logarithm of the antibody concentration comprised between 0.0046 IU/mL and 0.175 IU/mL with a limit of detection of 0.0046 IU/mL. In order to minimize the matrix effect, the standard addition method was applied. The assay was validated by comparing the EC immunosensing data with those obtained by applying the ELISA method described in the European Pharmacopoeia.     

Microwave-Triggered Metal-Enhanced Chemiluminescence (MT-MEC): Application to Ultra-fast and Ultra-sensitive Clinical Assays

In this rapid communication we describe a new approach to protein detection with chemiluminescence. By combining common practices in protein detection with chemiluminescence, microwave technology, and metal-enhanced chemiluminescence, we show that we can use low power microwaves to substantially increase enzymatic chemiluminescent reaction rates on metal substrates. As a result, we have found that we can in essence trigger chemiluminescence with low power microwave (Mw) pulses and ultimately, perform on-demand protein detection assays. Using microwave triggered metal-enhanced chemiluminescence (MT-MEC), we not only improve the sensitivity of immunoassays with enhanced signal-to-noise ratios, but we also show that we can accurately quantify protein concentrations by integrating the photon flux for discrete time intervals.     

Enzymatic/Immunoassay Dual‐Biomarker Sensing Chip: Towards Decentralized Insulin/Glucose Detection

Performing bioassay formats based on enzyme and antibody recognition reactions with a single detection chip remains an unmet challenge owing to the different requirements of such bioassays. Herein, we describe a dual‐marker biosensor chip, integrating enzyme and antibody‐based assays for simultaneous electrochemical measurements of insulin (I) and glucose (G). Simultaneous G/I sensing has been realized by addressing key fabrication and operational challenges associated with the different assay requirements and surface chemistry. The I immunosensor relies on a peroxidase‐labeled sandwich immunoassay, while G is monitored through reaction with glucose oxidase. The dual diabetes biomarker chip offers selective and reproducible detection of picomolar I and millimolar G concentrations in a single microliter sample droplet within less than 30 min, including direct measurements in whole blood and saliva samples. The resulting integrated enzymatic‐immunoassay biosensor chip opens a new realm in point‐of‐care multiplexed biomarker detection.     

Feasibility Study of Biosensors Based on Polymelamine‐modified Screen‐printed Carbon Electrodes

We herein report the use of melamine and a low‐cost screen‐printed carbon electrode (SPCE) as the base matrices for the preparation of an electrochemical biosensor. Following the electrochemical polymerization of melamine, the resulting polymelamine was deposited on the SPCE surface to give layers bearing –NH₂ functional groups, which allowed the attachment of anti‐IgE (immunoglobulin E) antibodies. The resulting anti‐IgE‐labeled SPCEs were then incubated with IgE solutions of various concentrations prior to analysis by chronoamperometry using Ru(NH₃)₆³⁺ as an electrochemical mediator. A logarithmic relationship was observed between the chronoamperometric current and the IgE concentration between 5.3 and 530 fM (i. e. over 2 orders of magnitude). In addition, a detection limit of 0.64 fM was achieved in addition to a recovery of 114 ± 14 % for a fetal bovine serum sample spiked with 16 fM IgE. Furthermore, only a small quantity of sample was required for analysis, and the IgE assay was suitable for use in a complex serum matrix without interference. We therefore expect that this novel system will be useful for monitoring the changes in blood IgE levels during the clinical treatment of allergic asthma and rhinitis.     

Disposable microfluidic ELISA for the rapid determination of folic acid content in food products

A micro-analytical system for rapid and quantitative analysis by inhibition immunoassay is presented and applied to the detection of folic acid. Eight polymer microchannels of 65-nL volume each and containing microelectrodes are embedded in a cartridge so that they can be operated simultaneously. All fluidic steps as well as the amperometric detection in the channels are operated by an instrument and software developed in-house. The fluidic steps of the immunoassay occur through hydrodynamic loading of the different solutions through the channels. The speed and duration of the flow and incubation parameters can thus be adapted to the biological and testing requirements. The effectiveness of the system was demonstrated by analysing folic acid concentrations in real infant formula samples within 5 min. In an effort to get a fully monitored assay, each fluidic step is monitored thanks to continuous amperometric detection of oxygen in the microchannel.     

Determination of cyclosporine A in whole blood: comparison of a chromatographic method with three different immunological methods

Cyclosporine A is potent immunosuppressive agent characterized by a narrow therapeutic range, inter- and intra-individual variability and a lack of correlation between drug dosage and blood levels. In view of these facts, blood levels of CyA should be routinely monitored to assess organ rejection and toxicity.

We evaluated CyA as well as its metabolites (AM9, AM19, AMl, and AM4N) in whole blood samples from 117 patients using commercially available immunological assays (AxSYM, EMIT, Dimension) and HPLC.

Cross-reactivity of the immunological assays was evaluated using different concentrations of the CyA metabolites (in vitro cross-reactivity) and by statistical analysis of patient data (in vivo cross-reactivity). Cross-reactivity was seen in all immunological assays, with differences in in vitro and in vivo cross-reactivity.

The statistical analysis showed a classical correlation between HPLC and AxSYM of r² = 0.89, HPLC versus EMIT of r² = 0.93, and HPLC versus Dimension of r² = 0.93.

The percentage metabolite cross-reactivity (%) by immunological assays for four metabolites at two concentrations each (250 and 1000 ng ml⁻¹) was lowest with the Dimension assay.

Of the immunological methods examined, the new Dimension for CyA determination can be relied on to produce results comparable to HPLC; other advantages are its simplicity, practicability and ease of handling.     

Voltammetric Immunoassay for the Detection of Protein Biomarkers

A strategy for a fast (ca. 20 min), specific, electrochemical immunoassay for the cardiac biomarker creatine kinase (CK) and the human cytokine interleukin 10 (IL10) has been developed in this paper. The polyaniline modified gold surface formed from electrochemical reduction of diazonium salt supplies a solid substrate to link the activated carboxylic acid groups from the antibodies, which were labelled with ferrocene. The direct electrochemistry of ferrocene allows the analysis of protein markers with good sensitivity. The creatine kinase sensor demonstrates limit of detection of 0.5 pg mL^?1 in a physiological Krebs‐Henseleit solution. The anti‐IL10 antibody retained fluorescence activity after further coupling to ferrocene and covalent immobilization on to a gold electrode, showing a linear detection range for IL‐10 from 0.001 ng mL^?1 to 50 ng mL^?1 in PBS. We attribute the high sensitivity to the well‐controlled modified surface which results in end–on antibodies that can specifically capture the antigen with ease.     

Flow methods in chiral analysis

The methods used for the separation and analytical determination of individual isomers are based on interactions with substances exhibiting optical activity. The currently used methods for the analysis of optically active compounds are primarily high-performance separation methods, such as gas and liquid chromatography using chiral stationary phases or chiral selectors in the mobile phase, and highly efficient electromigration techniques, such as capillary electrophoresis using chiral selectors. Chemical sensors and biosensors may also be designed for the analysis of optically active compounds.     

Microwave-Triggered Chemiluminescence with Planar Geometrical Aluminum Substrates: Theory,Simulation and Experiment

Previously we combined common practices in protein detection with chemiluminescence, microwave technology, and metal-enhanced chemiluminescence to demonstrate that we can use low power microwaves to substantially increase enzymatic chemiluminescent reaction rates on particulate silvered substrates. We now describe the applicability of continuous aluminum metal substrates to potentially further enhance or "trigger" enzymatic chemiluminescence reactions. Furthermore, our results suggest that the extent of chemiluminescence enhancement for surface and solution based enzyme reactions critically depends on the surface geometry of the aluminum film. In addition, we also use FDTD simulations to model the interactions of the incident microwave radiation with the aluminum geometries used. We demonstrate that the extent of microwave field enhancement for solution and surface based chemiluminescent reactions can be ascribed to "lightning rod" effects that give rise to different electric field distributions for microwaves incident on planar aluminum geometries. With these results, we believe that we can spatially and temporally control the extent of triggered chemiluminescence with low power microwave (Mw) pulses and maximize localized microwave triggered metal-enhanced chemiluminescence (MT-MEC) with optimized planar aluminum geometries. Thus we can potentially further improve the sensitivity of immunoassays with significantly enhanced signal-to-noise ratios.