Marie-Claude Millot

Functionalization of macroporous organic polymer monolith based on succinimide ester reactivity for chiral capillary chromatography: a cyclodextrin click approach.

Macroporous cross-linked organic polymer based on N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) was prepared inside 75 µm id fused silica capillary as a functionalizable monolithic stationary phase for chromatographic applications. Succinimide groups on the monolith surface provide reactive sites able to react readily through standard electrophile-nucleophile chemistry. Propargylamine was used to prepare alkyne functionalized poly(NAS-co-EDMA). Onto this azido-reactive polymer surface was grafted β-cyclodextrin (CD) via a triazole ring utilizing the copper(I)-catalyzed 1,3-dipolar cyclo-addition reaction. Chemical characterization was performed in situ after each synthetic step by means of Raman spectroscopy. Good enantioseparations of flavanone enantiomers, chosen as test chiral compound, were achieved under reversed phase conditions by both capillary electrochromatography and nano-liquid chromatography (nano-LC) techniques. These results demonstrate the potentiality and usefulness of click chemistry in the preparation of β-CD containing chiral organic polymer monolith.

One hundred spots parallel monitoring of DNA interactions by SPR imaging of polymer-functionalized surfaces applied to the detection of cystic fibrosis mutations

Abstract In the present paper, we report the detection of mutations implicated in human cystic fibrosis (CF). Nine different oligonucleotides are studied, including three possible mutations related to this specific genetic disease: a deletion of three bases, ΔF508, and two single-nucleotide polymorphisms 1540A/G and 1716G/A. We monitor, in real time and in parallel, hybridizations of a solution of unlabeled oligonucleotide targets to a matrix of 100 spots of oligonucleotide probes using surface plasmon resonance (SPR) imaging of a bio-functionalized gold slide. In order to functionalize our gold slide with the DNA probes, we have developed a self-assembled multilayer (SAM) based on electrostatic interactions and formed with 11-mercaptoundecanoic acid (MUA), poly(ethylenimine) (PEI) and ExtrAvidin layers. Probes are then linked to this SAM by the usual strong binding affinity of the avidin–biotin duplex. The 100 spots array deposited by a robot can be addressed either several times, sequentially, with the various oligonucleotide targets, or once, in parallel, with a mixture of some oligonucleotides. The specific response of our system is established along with the possibility of discriminating between a totally complementary sequence and its mutant form, even for a single base mismatch thus demonstrating the capacity of parallel diagnostic using patient like material.

Highly hydrophilic surfaces from polyglycidol grafts with dual antifouling and specific protein recognition properties.

Homopolymer grafts from α-tert-butoxy-ω-vinylbenzyl-polyglycidol (PGL) were prepared on gold and stainless steel (SS) substrates modified by 4-benzoyl-phenyl (BP) moieties derived from the electroreduction of the parent salt 4-benzoyl benzene diazonium tetrafluoroborate. The grafted BP aryl groups efficiently served to surface-initiate photopolymerization (SIPP) of PGL. In similar conditions, SIPP of hydroxyethyl methacrylate (HEMA) permitted the production of PHEMA grafts as model surfaces. Water contact angles were found to be 66°, 15°, and 0° for SS-BP, SS-PHEMA, and SS-PPGL, respectively. The spontaneous spreading of water drops on SS-PPGL was invariably observed with 1.5 μL water drops. PPGL thus appears as a superhydrophilic polymer. Resistance to nonspecific adsorption of proteins of PPGL and PHEMA grafts on gold was evaluated by surface plasmon resonance (SPR) using antibovine serum albumin (anti-BSA). The results conclusively show that PPGL-grafts exhibit enhanced resistance to anti-BSA adsorption compared to the well-known hydrophilic PHEMA. PPGL grafts were further modified with BSA through the carbonyldiimidazole activation of the OH groups providing immunosensing surfaces. The so-prepared PPGL-grafted BSA hybrids specifically interacted with anti-BSA in PBS as compared to antimyoglobin. It is clear that the superhydrophilic character of PPGL grafts opens new avenues for biomedical applications where surfaces with dual functionality, namely, specific protein grafting together with resistance to biofouling, are required.

Biofunctionalized tilted Fiber Bragg Gratings for label-free immunosensing

We present a study aimed at developing a label-free optical fiber biosensor for detection and quantification of biomolecules in real-time. The biosensor based on a Tilted Fiber Bragg Grating (TFBG) transduces a binding event between the probe and target molecules into a change in the refractive index of the medium surrounding the fiber. This work describes the experimental results obtained with three methods for immobilizing biomolecular probes on a TFBG silica cladding surface. Bovine serum albumin (BSA) and anti-BSA are used to assess the performances of the TFBG based biosensor in each configuration.

Zwitterionic polymeric monoliths for HILIC/RP mixed mode for CEC separation applications

Polymer-based monoliths with zwitterionic surface character were synthesized in capillary columns following a two-step approach to provide versatile electrochromatographic stationary phases exhibiting potentiality of both hydrophilic interaction and RP separation modes. UV-initiated free radical copolymerization of N-acryloxysuccinimide and ethylene dimethacrylate was performed using azobisisobutyronitrile as initiator and toluene as porogen. One of the originalities of this approach relies on the dual role of the N-acryloxysuccinimide monomer that is successively used during the preparation protocol to first covalently graft chromatographic selectors on the monolith surface via simple nucleophilic substitution reaction and then to generate negative charges through hydrolysis of remaining N-hydroxysuccinimide units. In this respect, the grafting of hexyldiamine affords potential cationic surface charges. It is shown that it is possible to tune, controlling the pH of the mobile phase, the intensity and direction of the generated EOF. Moreover, the nature of the interfacial interaction process responsible for the observed separations is well governed by the composition of the mobile phase. Polymer backbone hydrophilization is proposed as an efficient way to improve the HILIC behavior of poly(N-acryloxysuccinimide-co-ethylene dimethacrylate) based monolithic CEC columns together with the grafting of an alkyldiamine incorporating a shorter aliphatic segment.

Enantiomeric properties of human albumin immobilized on porous silica supports coated with polymethacryloyl chloride.

Human serum albumin (HSA) was bound to porous silica, using a reactive polymer derived from polymethacryloyl chloride. Two different procedures were used for coating silica with the polymer. In the first method, the polymer was deposited onto amino-silica by reaction between its reactive functions and NH2 groups on silica. In the second method, the monomer was first linked to the amino-silica and copolymerization with the excess of monomer initiated thereafter. The enantiomeric properties of the resulting supports after the coupling of HSA were compared using different mobile phases. The higher amount of HSA bound using the later method, resulted in higher retention of the enantiomers and better enantioselectivity.

The reversible binding of hydrophobically end-capped poly(ethylene glycol)s to poly-β-cyclodextrin-coated gold surfaces: a surface plasmon resonance investigation

Abstract Polymers of β-cyclodextrin (β-CD) bearing amino groups (poly-β-CD-NH2) were grafted to gold surfaces by two different procedures. Hydrophobically end-capped model-polymers were adsorbed onto resulting (poly-β-CD-NH2)-coated sensor chips by formation of inclusion complexes. Their adsorption onto the surface was followed in real time by surface plasmon resonance (SPR). Multilayered structures obtained by inclusion of adamantyl-modified poly(ethylene glycol)s (Ad-PEG) into immobilized β-cyclodextrin cavities were highly stable in aqueous media. Conditions leading to the regeneration of initial (poly-β-CD-NH2)-coated surfaces were studied by SPR. Regeneration by competitors such as polymers of β-cyclodextrin was possible. However, it was found to be a complex mechanism involving two opposite phenomena depending on the structure and composition of the (poly-β-CD-NH2) layer. Complete desorption of the bound Ad-PEG layer was also observed after exposure of the multilayered structures to organic solvents or sodium dodecylsulfate (SDS).

Structural changes of human serum albumin immobilized on chromatographic supports: a high-performance liquid chromatography and Fourier-transform infrared spectroscopy study.

Chiral stationary phases obtained by immobilization of HSA on [C8] and [C18] reversed-phases and on poly(1-vinylimidazole)-coated silica were tested to resolve DL-tryptophan, N-benzoyl-DL-phenylalanine, RS-oxazepam and RS-warfarin racemic mixtures. Parameters of enantioselectivity measured in HPLC are correlated to structural and solvation states for adsorbed HSA, evaluated by FTIR spectroscopy. HSA immobilized on [PVI]-anion-exchangers is highly selective. HSA molecules are not self-associated, only unfolded for a small hydrophobic helix. The HSA-coated reversed-phases have a lower selectivity. Unfolding is larger but the indole-benzodiazepine chiral site is preserved and remains accessible.

Coupling of Antibodies to β-Cyclodextrin-Coated Gold Surfaces via an Intermediate Adamantyl-Modified Carboxymethylated Dextran Layer

Abstractβ-cyclodextrin-coated sensors chips were obtained by grafting amino-β-cyclodextrin or amino-polymers of β-cyclodextrin (poly-β-CD-NH2) to functionalized gold surfaces. An additional carboxymethylated dextran bearing adamantyl groups (Ad-Dex-COOH) was then immobilized onto the surface by formation of inclusion complexes between β-cyclodextrin cavities and adamantyl groups. Such multilayered structures were stable in aqueous media. However, the initial β-cyclodextrin-coated surface could be recovered by using sodium dodecylsulfate solutions (SDS). After activation withN-hydoxysuccinimide, dextran-coated sensor chips were used to bind antibodies. The immunoreactivity of the resulting biosensors was examined. Moreover, conditions leading to the complete regeneration of the initial surface were investigated. Throughout this study, interfacial adsorption and desorption phenomena were followed in real time by an optical technique, Surface Plasmon Resonance (SPR).

A reactive macromolecular matrix for protein immobilization on a gold surface. Application in surface plasmon resonance

A reactive copolymer bearing functions able to react at room temperature with amino groups has been immobilized on a cysteamine-coated gold surface. Polymer immobilization, covalent coupling of rabbit immunoglobulin G and antigen-antibody reactions were studied using a surface plasmon resonance (SPR) technique. The effect of a protein A layer on the antigen-antibody reaction was examined.