Marie Vandeput

Research in Drug Development (RD3), Pharmacognosy Bioanalysis and Drug Discovery

RESEARCH ACTIVITIES

Her research fields deal with the development of innovative analytical methods looking for new technologies and at nano/microfluidics scale. Her main research is focused on methods using chromatography liquid coupled to mass spectrometry to characterize therapeutic proteins (biotherapeutic drugs) and biological proteins to study posttranslational modifications, especially glycosylation. Her research was also devoted to the development of “proof-of-concept” fluidic systems coupled to electrochemical detector which integrated a reactor such as an immobilized enzyme to study pharmaceutical and agri-food compounds. These customized systems were applied on drug screening looking at therapeutical inhibitors but also on the determination of xenobiotics

Selected publications

Articles dans des revues avec comité de lecture

2023

Synthesis and biological activity of iron(II), iron(III), nickel(II), copper(II) and zinc(II) complexes of aliphatic hydroxamic acids

Aliphatic hydroxamic acids (HA) with varying numbers of carbon atoms, C2, C6, C8, C10, C12 and C17, and corresponding Fe(II), Fe(III), Ni(II), Cu(II) and Zn(II) complexes have been synthesized and characterized by various methods, including structural determination by single crystal X-ray diffraction and theoretical calculations. The biological activities of HA and their complexes have been assessed on a panel of pathogens, including eight bacteria strains and one fungus. The C12 aliphatic HA displayed the lowest minimum inhibitory concentrations (MIC) towards several microbial strains and a selective antifungal activity. This antifungal selectivity was further improved considering the Ni(II), Cu(II) and Zn(II) complexes of C12 HA showed higher IC50 values, thus less impact on the SiHa human cell line viability. This work warrants further investigation to understand the underlying mechanism of action and potential biological applications of HA and the derived metal complexes.

2021

Fc Glycosylation Characterization of Human Immunoglobulins G Using Immunocapture and LC-MS.

Immunoglobulins G (IgG) are proteins produced by the immune system of higher life forms that play a central role in the defense against microbial pathogens. IgG bind pathogens with the hypervariable Fab component and mediate a diversity of effector functions by binding to immune effector cells via their crystallizable (Fc) component. All IgG Fc carry a polymorphic N-glycan that regulates its binding properties and thereby its effector functions. The glycosylation profile of IgG Fc is modulated by physiological and pathological conditions, including infectious diseases and inflammatory disorders. Characterization of IgG Fc glycosylation profiles is a promising approach to understand the pathogenesis of diseases involving the immune system and to develop novel biomarkers of disease activity. Measuring the proportion of the different IgG Fc glycoforms remains an analytical challenge, that requires a sensitive and reproducible analytical approach.This chapter describes an optimized approach for the preparation and the analysis of Fc N-glycans from total serum or plasma IgG using magnetic beads, RapiFluor MS label©, and LC-MS.

2019

Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation.

Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.

Isoniazid bactericidal activity involves electron transport chain perturbation

2018

Electrochemical studies of ethoxyquin and its determination in salmon samples by flow injection analysis with an amperometric dual detector

Ethoxyquin (EQ) is an antioxidant widely used in the food industry. Some concerns for human health have been reported since its utilization in animal feed may lead to residues in human food such as salmon samples. This work aimed to investigate the electrochemical behavior of EQ and its major oxidation products namely a dimer of EQ (EQDM) and ethoxyquin quinone imine (EQI) by cyclic voltammetry (CV) at a carbon screen printed electrode (cSPE). The stability of these products and their reactivity against glutathione (GSH) were also studied. Oxidized products and thiol adducts were identified by a microelectrolysis of an EQ solution onto a cSPE in the absence and in the presence of GSH. The products were analyzed off-line, by liquid chromatography coupled to a mass spectrometer (LC-MS). One of the generated product (EQI) was shown to be highly reactive towards GSH. Based on the redox pattern of EQ, a flow injection analysis with a dual cSPE was developed in order to detect in a rapid manner EQ in salmon samples. Since matrix effects were observed, matrix-matched calibration curves with spiked samples were built. A linear response was obtained between 20-100 μM and a limit of detection (LOD) of 7.5 μM (8.2 mg/kg of salmon). Trueness was assessed with recovery assays at three levels of concentration. The recovery was close to 100 %. Precision was determined as RSD (%) with values lower than 6 % in all cases.

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 0.1 mg kg−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.

2017

Electrochemical detectors in liquid chromatography: Recent trends in Pharmaceutical and Biomedical Analysis

Liquid chromatography (LC) coupled to an electrochemical (EC) detector is a complementary analytical tool compared to LC coupled with optical or mass spectrometry detectors (LC-MS). LC-EC can be applied to the determination of molecules difficult to be analyzed by other commercially available detectors. New EC detector design and new working electrode material have extended the scope of application in the field of pharmaceutical compounds analysis. Combining EC with LC-MS offers additional advantages compared to optical detectors in terms of drug stability and drug metabolism mimicry studies. Selected literature devoted to pharmacologically active compounds in their dosage forms, herbal drugs in natural products, drug residues in feed and/or in biological samples are reported in this review.

A Reduced Graphene Oxide-based Electrochemical DNA Biosensor for the Detection of Interaction between Cisplatin and DNA based on Guanine and Adenine Oxidation Signals

A glassy carbon electrode (GCE) was modified by electrochemically reduced graphene oxide (ERGO) for subsequent dsDNA immobilization. The interaction of cisplatin with dsDNA was studied at this modified electrode. Quantitative investigations were performed by adsorptive transfer stripping voltammetry (AdTSV) using differential pulse voltammetry (DPV). The morphology and structure of graphene oxide (GO) and ERGO modified GCEs (GO/GCE and ERGO/GCE, respectively) were characterized by UV-vis, FT-IR, Raman spectroscopy and cyclic voltammetry. Compared with the bare GCE and the GO/GCE, the ERGO/GCE exhibited excellent electrocatalytic activity towards the oxidation of dsDNA due to guanine and adenine groups, testified by high oxidation peak currents and decreased oxidation potentials. The interaction of micromolar concentrations of cisplatin with surface confined dsDNA was readily detected as inferred from the decrease of the voltammetric oxidation peaks of guanine and adenine. This trend was significantly greater at the ERGO/GCE compared to the GO/GCE. The interaction of cisplatin with dsDNA was also studied in solution phase by AdTSV with detection at the ERGO/GCE.

Application of a tyrosinase microreactor - detector in a flow injection configuration for the determination of affinity and dynamics of inhibitor binding

An original self-contained enzymatic microreactor − detector in a thin layer flow-through configuration has been developed for the screening of tyrosinase inhibitors. The microreactor- detector consisted of a gold disk for tyrosinase immobilization adjacent to a glassy carbon disk as working electrode (GCE). Using L-tyrosine as substrate, the enzymatic product formed (dopaquinone) was detected amperometrically at the GCE. Experimental parameters most affecting the L-tyrosine response were optimized with a central composite design. Measurements were performed in phosphate buffer pH 6.0 with an applied potential of +50 mV vs Ag/AgCl and a flow rate of 100 μL/min. The determined Kmapp was 1.9 ± 0.1 mM. The setup permitted the determination of the efficiency and the duration of inhibition of twelve compounds known as tyrosinase inhibitors namely kojic acid, gallic acid, azelaic acid, benzoic acid, L-ascorbic acid, hydroquinone, glabridine, resveratrol, quercetin dihydrate, captopril, L-cysteine and L-glutathione reduced. L-Ascorbic acid and kojic acid were found to be the most potent inhibitors for the two studied criteria with IC50 of 32 μM and 156 μM, respectively.

2016

Antibodies as target for affinity biosensors

Antibodies or immunoglobulins (Ig) are proteins produced by the immune system to protect the body by identifying and neutralizing pathogens. The determination of antibodies is an important area in bioanalysis because their presence provides information about pathologies such as infections, allergies, auto-immune diseases and cancers. Antibodies can be readily detected and quantified by using immunosensors. This review provides an up-to-date overview of immunosensors for the determination of antibodies which can be implemented in the clinical area, for point-of care applications or in routine laboratories.

2015

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.

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 10µL of a hydroquinone (HQ) solution into 40µL 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 0.011IU/mL and a limit of quantification of 0.012IU/mL. These values were below the protective human antibody limit of 0.06IU/mL.

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

(Graph Presented). 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.

2014

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.

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. © 2014 Elsevier B.V.