PEPTIMPRINT

Raquel Gutiérrez-Climente, Giang Ngo, Margaux Clavié, Jérémie Gouyon, Yoann Ladner, Pascal Etienne, Pascal Dumy, Catherine Perrin, Ahmad Mehdi, Pierre Martineau, Martine Pugnière and Gilles Subra 

Abstract

The development of artificial receptors able to selectively recognize a target protein is of particular interest in separation, diagnostics, and therapeutics fields. Herein, we disclose a method to prepare biomimetic and functionalized protein imprints in biocompatible conditions avoiding any protein denaturation. For that purpose, a set of different hybrid silylated amino acid derivatives were synthesized and used without tetraethyl orthosilicate to prepare our molecularly imprinted polymers, allowing to reduce to a minimum of the silicon amount, in order to obtain imprints made almost entirely of amino acids to mimic paratope surfaces of antibodies. Such functional building blocks were polymerized on the surface of magnetic silica nanoparticles at pH 8.5 in ultrapure water in the presence of two globular proteins: cytochrome C or lysozyme. The resulting imprinted hybrid materials were evaluated for their adsorption capacity, specificity, and selectivity by quartz-crystal microbalance with dissipation and magnetic enzyme-linked immunosorbent assay (ELISA) assays. High imprinting factors of 8.7 were measured for these biomimetic hybrid materials (corresponding to approximately 4000 and 450 ng of protein per cm² immobilized on molecularly imprinted polymers and non-imprinted polymer nanoparticles, respectively), representing a significant breakthrough in sol-gel-based molecular imprinting materials. Moreover, competition experiments performed by magnetic ELISA (mELISA) show very good specificity of our imprints at the usual concentrations of ELISA measurements.

Raquel Gutiérrez-Climente, Margaux Clavié, Jérémie Gouyon, Giang Ngo, Yoann Ladner, Pascal Etienne, Pascal Dumy, Pierre Martineau, Martine Pugnière, Catherine Perrin, Gilles Subra and Ahmad Mehdi

Abstract

Ordered mesoporous materials and their modification with multiple functional groups are of wide scientific interest for many applications involving interaction with biological systems and biomolecules (e.g., catalysis, separation, sensor design, nano-science or drug delivery). In particular, the immobilization of enzymes onto solid supports is highly attractive for industry and synthetic chemistry, as it allows the development of stable and cheap biocatalysts. In this context, we developed novel silylated amino acid derivatives (Si-AA-NH₂) that have been immobilized onto SBA-15 materials in biocompatible conditions avoiding the use of toxic catalyst, solvents or reagents. The resulting amino acid-functionalized materials (SBA-15@AA) were characterized by XRD, TGA, EA, Zeta potential, nitrogen sorption and FT-IR. Differences of the physical properties (e.g., charges) were observed while the structural ones remained unchanged. The adsorption of the enzyme lysozyme (Lyz) onto the resulting functionalized SBA-15@AA materials was evaluated at different pHs. The presence of different functional groups compared with bare SBA-15 showed better adsorption results, for example, 79.6 nmol of Lyz adsorbed per m² of SBA-15@Tyr compared with the 44.9 nmol/m² of the bare SBA-15.

Raquel Gutiérrez-Climente,   Margaux Clavié,   Pascal Dumy,   Ahmad Mehdi  and  Gilles Subra

Abstract

Proteins play a central role for the signal transmission in living systems since they are able to recognize specific biomolecules acting as cellular receptors, antibodies or enzymes; or being themselves recognized by other proteins in protein/protein interactions, or displaying epitopes suitable for antibody binding. In this context, the specific recognition of a given protein, unlocks a range of interesting applications in diagnosis and in targeted therapies. Obviously, this role is already fulfilled with antibodies with unquestionable success. However, the design of synthetic artificial systems able to endorse this role is still challenging with a special interest to overcome limitations of antibodies, in particular their production and their stability. Molecular Imprinted Polymers (MIPs) are attractive recognition systems which could be an alternative for the specific capture of proteins in complex biological fluids. MIPs can be considered as biomimetic receptors or antibodies mimics displaying artificial paratopes. However, MIPs of proteins remains a challenge due to their large size and conformational flexibility, their complex chemical nature with multiple recognition sites and their low solubility in most organic solvents. Classical MIP synthesis conditions result in large polymeric cavities with and unspecific binding sites on the surface. In this review, the potentiality of sol-gel process as inorganic polymerization strategy to overcome the drawbacks of protein imprinting is highlighted. Thanks to the mild and biocompatible experimental conditions required and the use of water as solvent, the inorganic polymerization approach suits better to proteins than organic polymerization. Through numerous examples and applications of MIPs, we proposed a critical evaluation of the parameters that must be carefully controlled to achieve sol-gel protein imprinting (SGPI), including the choice of the monomers taking part in the polymerization.