Marie Vandeput

Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination.

A chip format sandwich-type immunoassay based on Nanobodies(®) (Nbs) with the Human Epidermal Growth Factor Receptor (HER2) extracellular domain as antigen model has been developed. The HER2 is considered as an important biomarker because its overexpression causes an aggressive type of breast cancer. Nbs are single domain antigen-binding fragments derived from camelid heavy-chain antibodies. The strategy of the presently developed sandwich immunoassay takes advantage of the small size of Nbs for the detection of the electroactive redox tracer onto the screen printed electrode (SPE). A capture anti HER2 Nb was covalently immobilized onto the SPE, and the detection Nb, raised against another epitope of HER2, was labeled with horseradish peroxidase (HRP). The biosensor signal corresponded to the electroreduction of para-quinone generated at the SPE by the HRP in the presence of hydroquinone and hydrogen peroxide. The best performing and optimized immunoassay conditions consisted of 2 and 20 min for the first and the second incubation times, respectively. The amperometric signal obtained was proportional to the logarithm of HER2 concentration between 1 and 200 µg/mL and the modified SPE storage stability lasted for at least three weeks. Determination of HER2 in human cells has been realized.

Liquid Chromatography with Amperometric Detection at a Silver Based Detector for the Determination of Thiocompounds: Application to the Assay of Thiopurine Antimetabolites in Urine

A silver amperometric detector coupled to liquid chromatography (LC) was used for the determination of 6-thioguanine (6-TG) and two of its metabolites, thiouric acid (TU) and 2-amino-6-mercaptopurine riboside (6-TGR). The silver detector coupled to LC operated at a low applied potential (0.08 V vs Ag/AgCl) and offered a chromatogram with peak responses corresponding to molecules interacting with silver, namely, chloride ions and small soluble biothiols in addition to the organothiol drug compounds investigated. Online electrochemical surface cleaning permitted the improvement of the repeatability and peak shape of the recorded signal compared to direct current amperometric detection (AD) when operating in chloride containing media. The studied molecules were eluted isocratically within 5 min on a reversed-phase C18 column without interference from endogenous biothiols present in urine samples. Diluted urine samples (1:1) were directly injected in the LC setup; a linear calibration curve was obtained between peak area and analyte concentration between 0.1 and 10 μM for all the studied molecules. Limits of detection (LODs) were 0.03, 0.008, and 0.01 μM, and the limits of quantification (LOQs) were 0.1, 0.02, and 0.03 μM for TU, 6-TG, and 6-TGR, respectively. Within-day RSDs were 2%, 0.8%, and 1% and between-day RSDs were 2%, 0.9%, 2% for TU, 6-TG, and 6-TGR, respectively. Recoveries in spiked urine were 99.8%, 99.9%, and 99.0% for TU, 6-TG, and 6-TGR, respectively.

An experimental design approach to optimize an amperometric immunoassay on a screen printed electrode for Clostridium tetani antibody determination.

An immunoassay for the determination of anti-tetani antibodies has been developed using a screen printed electrode (SPE) as solid support for toxoid (antigen) immobilization. The assay was performed in guinea pig serum. The immunoreaction and the subsequent amperometric detection occurred directly onto the SPE surface. The assay consisted of spiking the anti-tetani sample directly onto the toxoid modified SPE, and then a second antibody, i.e. a HRP-labeled anti-immunoglobulin G, was deposited onto the biosensor. Subsequent amperometric detection was realized by spiking 10 µL of a hydroquinone (HQ) solution into 40 µL of buffer solution containing hydrogen peroxide. An experimental design approach was implemented for the optimization of the immunoassay. The variables of interest, such as bovine serum albumin (BSA) concentration, incubation times and labeled antibody dilution, were optimized with the aid of the response surface methodology using a circumscribed central composite design (CCCD). It was observed that two factors exhibited the greatest impact on the response, i.e. the anti-tetani incubation time and the dilution factor of the labeled antibody. It was discovered that in order to maximize the response, the dilution factor should be small, while the anti-tetani antibody incubation time should be long. The BSA concentration and the HRP-anti-IgG incubation had very limited influence. Under the optimized conditions, the immunoassay had a limit of detection of 0.011 IU/mL and a limit of quantification of 0.012 IU/mL. These values were below the protective human antibody limit of 0.06 IU/mL.

Flow-through enzyme immobilized amperometric detector for the rapid screening of acetylcholinesterase inhibitors by flow injection analysis.

A commercially available thin-layer flow-through amperometric detector, with the sensing block customized in an original design, was applied to the screening of drug compounds known as acetylcholinesterase (AChE) inhibitors. AChE from electric eel was covalently immobilized onto a cysteamine modified gold disk adjacent to a silver disk working electrode. On-line studies were performed by flow injection analysis (FIA) in PBS buffer pH 7.4. Seven commercially available AChE inhibitors used in the medical field, namely neostigmine, eserine, tacrine, donepezil, rivastigmine, pyridostigmine and galantamine as well as two natural compounds, quercetin and berberine, were investigated. The same trend of inhibitory potency as described in the literature was observed. Of particular interest and in addition to the determination of the IC50 values, this flow-through system allowed the study of both, the stability of the enzyme-inhibitor complex and the kinetic of the enzyme activity recovery.

Liquid chromatography-electrochemical detection for the determination of ethoxyquin and its dimer in pear skin and salmon samples

Ethoxyquin (EQ) is widely used as a synthetic antioxidant in animal feed, an antiscalding agent in apples and pears and as a color preservative in some spices. Since the presence of EQ in food products could cause negative health effects it is necessary to develop reliable analytical methods to evaluate the risk of human exposure. In this work, a sensitive, selective and accurate method based on solid-liquid extraction followed by clean-up with solid sorbent and liquid chromatography-electrochemical detection analysis with boron doped diamond electrode (LC-EC) for the determination of ethoxyquin and its dimer (EQDM) in pear skin and salmon samples, was developed. The method was validated according to the European Commission guidelines. The main variables of extraction were accurately optimized. The amounts of solid sorbents for clean-up procedure were optimized by using experimental design. A Box-Behnken design to obtain the optimum conditions was applied. For validation, a matrix-matched calibration was established and a recovery assay with spiked samples was carried out. The limits of detection (LODs) found were 0.05 and 0.1mgkg-1 for EQ and its dimer, respectively. The precision (as relative standard deviation, RSD) was lower than 15% with recoveries of compounds close to 100% in spiked samples.

Contribution of Electrochemistry to the Biomedical and Pharmaceutical Analytical Sciences.

All analytical techniques have experienced major progress since the last ten years and electroanalysis is also involved in this trend. The unique characteristics of phenomena occurring at the electrode-solution interface along with the variety of electrochemical methods currently available allow for a broad spectrum of applications. Potentiometric, conductometric, voltammetric and amperometric methods are briefly reviewed with a critical view in terms of performance of the developed instrumentation with special emphasis on pharmaceutical and biomedical applications.