Elaboration of silica colloid/polymer hybrid support for oligonucleotide synthesis.

One way to increase the sensitivity of DNA diagnostic assays developed on microarrays is to improve the solid phase that allows the extraction of the target from a biological sample, before detection. Two parameters are influencing the performances of this capture step: (i) the specific surface area being offered for the capture and (ii) the number and the accessibility of oligonucleotide probes immobilized on the surface. In this context, we have developed an attractive approach which fulfills these two points. Our strategy was to elaborate a new material of high specific surface area, suitable to serve as support for both solid-phase oligonucleotide synthesis and in vitro diagnostic assay. This material has consisted of aggregates of colloidal amino-silica nanoparticles covalently linked by poly(ethylene oxide) (PEO) arms. The aggregation of amino-silica particles in the presence of reactive bis-isocyanate PEO was achieved in a controlled manner. The aggregate size and structure were examined by microscopy. The specific surface area of this material was measured by nitrogen adsorption technique. The composition of aggregate was studied by thermogravimetry and X-ray photoelectron spectroscopy. Then, this material has been successfully used as support for oligonucleotide synthesis of high yield and purity. The resulting system will be further evaluated in a diagnostic assay on a microarray.

The use of poly(ethylene oxide) for the efficient stabilization of entrapped alpha-chymotrypsin in silicone elastomers: a chemometric study.

The enzyme alpha-chymotrypsin, a model for catalytic proteins, was entrapped in different silicone elastomers that were formed via the condensation-cure room temperature vulcanization (CC-RTV) of silanol terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, in the presence of different poly(ethylene oxide) oligomers that were functionalized with triethoxysilyl groups. The effects of various chemical factors on both the activity and entrapping efficiency of proteins (leaching) were studied using a 2-level fractional factorial design--a chemometrics approach. The factors studied include the concentration and chain length of poly(ethylene oxide), enzyme content, and crosslinker (TEOS) concentration. The study indicated that poly(ethylene oxide) can stabilize the entrapped alpha-chymotrypsin in silicone rubber: the specific activity can be maximized by incorporating a relatively high content of short chain, functional PEO. Increased enzyme concentration was found to adversely affect the specific activity. The effect of TEOS was found to be insignificant when PEO was present in the elastomer, however, it does affect the activity positively in the case of simple elastomers.

Highly active, lipase silicone elastomers.

Lipase Candida rugosa was entrapped in silicone rubber via condensation-cure room temperature vulcanization of silanol-terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, to give a highly active silicone-enzyme elastomer. The effect on enzyme activity of addition of water and hydrophilic polymeric moieties based on poly(ethylene oxide) 2 was examined, as were crosslinker concentration, enzyme concentration, and elastomer thickness. It was demonstrated that lipase is most active in silicone elastomers and more active in silicone oils than simple hydrocarbons. Crosslink density in these elastomers was not an important factor in the reactivity of the rubber. However, the addition of hydrophilic species prior to elastomer formation decreased the efficiency both of the dispersion of the enzyme and the resulting activity of the elastomer. This effect could be moderated by prior exposure of the lipase to silicone oil. Thus, hydrophobic silicones play a protective/activating role for lipase.