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Two hydrolytic enzymes, namely lysozyme and trypsin, were covalently immobilized onto stainless steel surfaces using wet chemistry processes. The immobilization strategy took advantage of the spontaneous physisorption of the polymer poly(ethylene imine) (PEI) onto stainless steel to yield a firmly attached, thin organic layer containing a high density of primary amine functions. Both enzymes were then covalently grafted to the surface via a glutaraldehyde cross-linker. Alternatively, a thicker underlayer of PEI was chemisorbed by cross-linking two PEI layers by glutaraldehyde. The effective presence of both enzymes on the stainless steel surfaces and their relative amount were assessed by immunochemical assays employing specific anti-enzyme antibodies. Eventually, the hydrolytic activity of the immobilized enzymes was evaluated by local enzymatic tests with suitable substrates. This work demonstrates that, although the amount of enzymes did not vary significantly with the underlayer thickness, their hydrolytic activity could be much improved by increasing the distance from the oxide surface and, likely, by favoring their accessibility. Our data suggest that the immobilization of enzymes on solid oxide surfaces is feasible and efficient, and that the enzymes retain catalytic activity. It may thus provide a promising route towards biofilm-resistant materials., (Copyright 2009. Published by Elsevier Inc.)

A new type of sensor was built by synthesizing a long-chain thiol functionalized with an aromatic head group and grafting it onto a gold surface. The synthesis route is here described, together with the IR, MS, and RMN analysis of the new product. Adsorption of the latter onto gold was assessed by a combination of RAIRS and XPS data. Those reveal that a monolayer of thiol is adsorbed and oriented with the benzene groups toward the external part of the layer. Detection tests were performed in various atmospheres by QCM. The response shows good sensitivity to 2,4-dinitrotrifluoromethoxybenzene as a model of nitroaromatic compound.

Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) was successively used to monitor the covalent immobilization of biotin molecules onto a planar gold substrate covered with a self-assembled monolayer of cystamine and to transduce the molecular recognition of avidin and biotin. This detection was greatly facilitated and made selective by the labeling of avidin and of biotin with various transition metal carbonyl probes. The binding of avidin to the surface was optimized by blocking the nonspecific binding sites by adsorption of an unrelated protein, bovine serum albumin. This work exemplifies the feasibility of detecting biomolecular associations involving molecules of any size at a liquid/solid interface by using a simple and accessible surface analysis technique.

As part of our project of developing a new IR-based immunosensor, we investigated the functionalization of gold substrates with thin organic films containing biotin ligands. A two-step procedure was developed consisting of the chemisorption of short amine-terminated organosulfur compounds, followed by their reaction at the solid liquid interface with an activated ester derivative of biotin. Covalent binding of biotin to these attachment layers was assessed by Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy (XPS). The interaction of activated biotin with alcohol- and carboxylic acid-terminated monolayers was also investigated, and, as expected, no binding occurred. Moreover, mixed layers of short alcohol- and amine-terminated thiolates were successfully constructed at the gold surfaces and were shown to be the most efficient for the covalent binding of biotin thanks to the blocking effect of the thioalcohol, which prevented direct adsorption of biotin to the gold surface. Copyright 2001 Academic Press.