Michèle Salmain

Carbonylmetalloimmunoassay (CMIA) a new type of non-radioisotopic immunoassay: Principles and application to phenobarbital assay

A new non-radioisotopic immunoassay procedure, which we have termed carbonylmetalloimmunoassay (CMIA), is described. The tracers used in this approach are organometallic carbonyl complexes that can be detected at femtomole levels (300-700 fmol) by Fourier transform infrared (FT-IR) spectroscopy. The validity of the technique has been tested in a phenobarbital assay using as the marker a cyclopentadienylmanganese (I) tricarbonyl (cymantrene) moiety, ethyl acetate extraction to separate the free and bound organometallic fractions, and FT-IR spectroscopy to detect the CO stretching modes of the organometallic label. Typical dilution and standard curves obtained with this CMIA procedure are presented. The method was of comparable sensitivity to a [14C] radioimmunoassay (RIA) for the detection of phenobarbital. A comparison of the results for phenobarbital assays by both CMIA and RIA showed that higher titres were obtained using the CMIA method. The standard curves suggest that CMIA is a reliable and reproducible immunoassay procedure for phenobarbital.

In-depth investigation of protein adsorption on gold surfaces: Correlating the structure and density to the efficiency of the sensing layer

Protein A (PrA), mouse monoclonal anti-IgG antibody (SAb) and deglycosylated avidin (NAV) were adsorbed on gold surfaces to capture the model rabbit IgG and build three immunosensing platforms. The assembling of immunosensors, their specificity, and the receptor accessibility were monitored by polarization modulation reflection-absorption infrared spectroscopy (PM-RAIRS) and quartz crystal microbalance with dissipation measurement (QCM-D) at each step. Combining these two techniques allows us to compare both chemical and structural properties of the sensing layers with the former bringing chemical and semiquantitative information on the grafted protein layers, whereas the latter, in addition to the mass uptake, enables us to take the layer rigidity into account. Grafting of the three capture proteins to the transducer surfaces, covered with appropriate self-assembled monolayers, yielded protein layers with variable properties. NAV formed a dense and rigid molecular layer, likely containing protein aggregates, whereas the amount of PrA was below one monolayer resulting in a flexible layer. The amount of immobilized rabbit IgG was different for the three systems with the densest capture protein layer exhibiting the lowest binding capacity. The accessibility of antibodies on the resulting immunosensors measured by interaction with a secondary antirabbit IgG antibody was found to be closely dependent on their coverage as well as on the rigidity of the protein layer. The overall study provides in-depth information on three of the most common immunosensor recognition interfaces and demonstrates the crucial influence of both structure and density of the protein layer on the efficiency of the molecular recognition phenomena.

Evidence for Targeting Thioredoxin Reductases with Ferrocenyl Quinone Methides. A Possible Molecular Basis for the Antiproliferative Effect of Hydroxyferrocifens on Cancer Cells

Many anticancer compounds are strong inhibitors of thioredoxin reductases (TrxRs), selenoenzymes involved in cellular redox regulation. This study examined the effect of two hydroxyferrocifens (1, FcOH; 2, FcOHTAM) and of their corresponding quinone methides (QMs), 1-QM, and 2-QM, on these enzymes. In vitro, both QMs were more potent TrxR inhibitors (IC50 ≈ 2.5 μM) than the hydroxyferrocifens (IC50 ≈ 15 μM). This inhibition was due to a Michael addition of the penultimate selenocysteine residue of TrxRs to the QMs. In Jurkat cancer cells, both 2 and 2-QM inhibited TrxRs in the same proportion, leading to accumulation of oxidized forms of thioredoxin, while 1 and 1-QM were scarcely effective. This difference of behavior was ascribed to the competitive conversion of 1-QM to an inactive indene in protic medium. This set of experiments confirms for the first time the role played by ferrocenyl quinone methides on several biological targets and gives a molecular basis for these effects. It also highlights differences in the mechanisms of action of 1 and 2 in cancer cells.

Carbonyl metallo immuno assay : a new application for Fourier transform infrared spectroscopy

We describe here the development of a new, non-isotopic immunological assay termed CMIA (carbonyl metallo immunoassay) that uses metal carbonyl complexes as tracers and Fourier transform infrared spectroscopy (FT-IR) as the detection method. This assay is based on the particular spectral features of these complexes, which show very strong absorption bands in the 1,800-2,200 cm(-1) spectral range where proteins and organic molecules do not absorb. In Section 1, the optimisation of the quantitative detection of these tracers is detailed. In Section 2, the implementation of mono-CMIA is described, including the CMIA assays of three antiepileptic drugs (carbamazepine, phenobarbital, phenytoin). Finally, extension to the simultaneous double- and triple-CMIA of these drugs is reported.

Specific binding of avidin to biotin immobilised on modified gold surfaces: Fourier transform infrared reflection absorption spectroscopy analysis

Abstract The immobilisation of biomolecules on gold surfaces has attracted a lot of attention due to the various ways of transducing biorecognition events at such surfaces and of course to its broad field of applications. The current challenge is two-fold: first, to produce a functionalised surface able to bind a given biomolecule in a complex environment, but also resistant to nonspecific adsorption; second, to assess the protein–ligand interaction by a suitable physical method. In our attempt to conceive a new type of biosensor, the biotin/avidin system was chosen as a model of biological (ligand/receptor) interaction. The first task was achieved by developing a two-step procedure, briefly consisting in the chemisorption of the short diamine disulfide cystamine to a gold substrate followed by chemical coupling with the N -succinimidyl ester of biotin. The presence of biotin molecules both specifically and unspecifically bound to the gold surface was assessed by Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) and XPS. Undesired nonspecific binding of biotin was minimised by increasing the chain length of the chemisorbed amine to which was coupled the N -succinimidyl ester of biotin. Chemical characterisation of the adsorbed layer was performed after each step by FT-IRRAS. For the second task, the protein avidin was labelled with an alkyne Co 2 (CO) 6 probe, a transition metal carbonyl entity that yields three characteristic ν CO signals far from the peptide region of absorption, before its interaction with the modified surface. Molecular recognition was checked and quantified by FT-IRRAS. The occurrence of nonspecific adsorption of avidin was measured by exposing the biotinylated substrates to a solution of labelled avidin pre-saturated by biotin. Methods to reduce nonspecific binding of avidin were also shown.

A new application of bioorganometallics: the first simultaneous triple assay by the carbonylmetalloimmunoassay (CMIA) method

The first example of simultaneous triple carbonylmetalloimmunoassay (CMIA), a non-isotopic immunological method, is presented herein. This method, originally developed for mono-assays, uses various transition metal carbonyl complexes as labels and sensitive quantitation of these tracers by Fourier transform infrared (FT-IR) spectroscopy, thanks to the characteristic νCO bands appearing in the 1800–2200 cm−1 region. We show here that this method offers an advance in the challenging field of simultaneous multi-immunoassay, with the example of the assay of three antiepileptic drugs (carbamazepine, phenobarbital and diphenylhydantoin).

Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A (SEA) at the ng level

A direct, label-free immunosensor was designed for the rapid detection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions using quartz crystal microbalance with dissipation (QCM-D) as transduction method. The sensing layer including the anti-SEA antibody was constructed by chemisorption of a self-assembled monolayer of cysteamine on the gold electrodes placed over the quartz crystal sensor followed by activation of the surface amino groups with the rigid homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDITC) and covalent linking of binding protein (protein A or protein G). Four anti-SEA antibodies (two of which from commercial source) have been selected to set up the most sensitive detection device. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1000 or 2000 ngml(-1) with a detection limit of 20 ngml(-1). The total time for analysis was 15 min. Using a sandwich type assay, the response was ca. twice higher and consequently the lowest measurable concentration dropped down to 7 ngml(-1) for a total assay time of 25 min.

Side-chain selective and covalent labelling of proteins with transition organometallic complexes. Perspectives in biology

Abstract This article reviews the current methods available to covalently label peptides and proteins with transition organometallic entities in a side-chain-selective and covalent manner. Almost all these methods take advantage of the reactivity of the nucleophilic groups borne by most proteins, namely the ϵ-amino function of lysine residues and the thiol function of cysteine residues. In this way, the unusual physical and/or spectroscopic properties of transition metals and their organo-complexes may be exploited in different areas of biology and medicine, including analytical biochemistry, protein three-dimensional structure analysis and radio-pharmaceuticals development. To cite this article: M. Salmain, G. Jaouen, C. R. Chimie 6 (2003).

New and efficient routes to biomolecules substituted with cyclopentadienyltricarbonylrhenium and -technetium derivatives.

The small, compact, robust, and nonpolar units of [CpM(CO)3] (M = Re, Tc) coupled with biomolecules may be considered as bioorganometallic entities of potential interest in the field of medicinal chemistry. However, the short half-life of useful radionuclides (186Re t1/2 = 3.7 d, 188Re t1/2 = 16.8 h, 99mTc t1/2 = 6 h), the risks inherent in their use, and their cost have led chemists to search for novel synthetic strategies that allow the rapid introduction of the [CpM(CO)3] moiety as a late step in the course of synthesizing the target molecule. The present paper describes different strategies recently reported in the literature to tackle this problem.

(η6-Arene) ruthenium(II) complexes and metallo-papain hybrid as Lewis acid catalysts of Diels–Alder reaction in water

Covalent embedding of a (eta(6)-arene) ruthenium(II) complex into the protein papain gives rise to a metalloenzyme displaying a catalytic efficiency for a Lewis acid-mediated catalysed Diels-Alder reaction enhanced by two orders of magnitude in water.