Silanisation de polysaccharides en milieu liquide ionique et réactivité d’un organoalkoxysilane vis-à-vis de nucléophiles simples : vers des hydrogels injectables pour l’ingénierie tissulaire du cartilage

Tissue engineering is a real challenge for the treatment of cartilage pathologies. In this field, biomimetic hydrogels based on natural polymers are among the most commonly used matrices. A hydrogel made of silanized hydroxypropylmethylcellulose (HPMC-Si) is especially promising because it can be injected in cartilaginous lesions by minimally invasive surgery. However, the current synthesis of HPMC-Si is limited by the insolubility of hydroxypropylmethylcellulose (HPMC). This thesis work was focused on finding new synthesis conditions for the design of HPMC-Si hydrogel. In order to obtain a complete solubilization of HPMC and to improve its functionalization by the (3-glycidyloxypropyl) trimethoxysilane (GPTMS), the use of ionic liquids (IL), which are excellent solvents for polysaccharides, was undertaken. The beginning of this study was first devoted to the selection of an IL and then to the development of new reaction conditions. With these new conditions, higher silicon rates were obtained for HPMC modified in ionic liquid medium, however no hydrogel could be formed. The second part was therefore devoted to the synthesis of GPTMS 13C. Indeed, thanks to this radiolabeling, a structural characterization by 13C NMR of the HPMC-Si could be achieved. Finally, the reactivity in organic solvents of three organosilanes, including the GPTMS, was investigated toward nucleophiles representing the common functions found in natural polymers (e.g. -NH2, -OH, -SH). The results of this thesis have provided insights into the GPTMS reactivity in organic medium and thus paves the way to new conditions for the silanization of polysaccharides.

Electrochemical biosensor based on microfabricated electrode arrays for life sciences applications

In developing a biosensor, the utmost important aspects that need to be emphasized are the specificity and selectivity of the transducer. These two vital prerequisites are of paramount in ensuring a robust and reliable biosensor. Improvements in electrochemical sensors can be achieved by using microelectrodes and to modify the electrode surface (using chemical or biological recognition layers to improve the sensitivity and selectivity). The fabrication and characterisations of silicon-based and glass-based gold microelectrode arrays with various geometries (band and disc) and dimension (ranging from 10 μm-100 nm) were reported. It was found that silicon-based transducers of 10 μm gold microelectrode array exhibited the most stable and reproducible electrochemical measurements hence this dimension was selected for further study. Chemical electrodeposition on both 10 μm microband and microdisc were found viable by electro-assisted self-assembled sol-gel silica film and nanoporous-gold electrodeposition respectively. The fabrication and characterisations of on-chip electrochemical cell was also reported with a fixed diameter/width dimension and interspacing variation. With this regard, the 10 μm microelectrode array with interspacing distance of 100 μm exhibited the best electrochemical response. Surface functionalisations on single chip of planar gold macroelectrodes were also studied for the immobilisation of histidine-tagged protein and antibody. Imaging techniques such as atomic force microscopy, fluorescent microscopy or scanning electron microscope were employed to complement the electrochemical characterisations. The long-chain thiol of self-assembled monolayer with NTA-metal ligand coordination was selected for the histidine-tagged protein while silanisation technique was selected for the antibody immobilisation. The final part of the thesis described the development of a T-2 labelless immunosensor using impedimetric approach. Good antibody calibration curve was obtained for both 10 μm microband and 10 μm microdisc array. For the establishment of the T-2/HT-2 toxin calibration curve, it was found that larger microdisc array dimension was required to produce better calibration curve. The calibration curves established in buffer solution show that the microelectrode arrays were sensitive and able to detect levels of T-2/HT-2 toxin as low as 25 ppb (25 μg kg-1) with a limit of quantitation of 4.89 ppb for a 10 μm microband array and 1.53 ppb for the 40 μm microdisc array.

Marine Biosilica as a Tool to Understand the Therapeutic Benefit of Silica in Bone Repair

It is widely accepted that silicon-substituted materials enhance bone formation, yet the mechanism by which this occurs is poorly understood. This work investigates the potential of using diatom frustules to answer on fundamental questions surrounding the role of silica in bone healing. Biosilica with frustules 20m were isolated from Cyclotella meneghiniana a unicellular microalgae that was sourced from the Mississippi River, USA. Silanisation chemistry was used to modify the surface of C. meneghiniana with amine (–NH2) and thiol (–SH) terminated silanes. Untreated frustules and both functionalised groups were soaked in culture medium for 24hrs. Following the culture period, frustules were separated from the conditioned medium by centrifugation and both were tested separately in vitro for cytotoxicity using murine-monocyte macrophage (J774) cell line. Cytotoxicity was measured using LDH release to measure damage to cell membrane, MTS to measure cell viability and live-dead staining. The expression and release of pro-inflammatory cytokines (IL-6 and TNF) were measured using ELISA. Our results found that diatom frustules and those functionalised with amino groups showed no cytotoxicity or elevated cytokine release. Diatom frustules functionalised with thiol groups showed higher levels of cytotoxicity. Diatom frustules and those functionalised with amino groups were taken forward to an in vivo mouse toxicity model, whereby the immunological response, organ toxicity and route of metabolism/excretion of silica were investigated. Histological results showed no organ toxicity in any of the groups relative to control. Analysis of blood Si levels suggests that modified frustules are metabolised quicker than functionalised frustules, suggesting that physiochemical attributes influence their biodistribution. Our results show that diatom frustules are non cytotoxic and are promising materials to better understand the role of silica in bone healing.

A pH optode based on thymol blue: application to determination of CO2 using flow injection analysis system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synthesis and characterisation of photoreactive silanes for the self-assembly on functionalised silica surfaces

The aim of this thesis was to investigate novel techniques to create complex hierarchical chemical patterns on silica surfaces with micro to nanometer sized features. These surfaces were used for a site-selective assembly of colloidal particles and oligonucleotides. To do so, functionalised alkoxysilanes (commercial and synthesised ones) were deposited onto planar silica surfaces. The functional groups can form reversible attractive interactions with the complementary surface layers of the opposing objects that need to be assembled. These interactions determine the final location and density of the objects onto the surface. Photolithographically patterned silica surfaces were modified with commercial silanes, in order to create hydrophilic and hydrophobic regions on the surface. Assembly of hydrophobic silica particles onto these surfaces was investigated and finally, pH and charge effects on the colloidal assembly were analysed. In the second part of this thesis the concept of novel, "smart" alkoxysilanes is introduced that allows parallel surface activation and patterning in a one-step irradiation process. These novel species bear a photoreactive head-group in a protected form. Surface layers made from these molecules can be irradiated through a mask to remove the protecting group from selected regions and thus generate lateral chemical patterns of active and inert regions on the substrate. The synthesis of an azide-reactive alkoxysilane was successfully accomplished. Silanisation conditions were carefully optimised as to guarantee a smooth surface layer, without formation of micellar clusters. NMR and DLS experiments corroborated the absence of clusters when using neither water nor NaOH as catalysts during hydrolysis, but only the organic solvent itself. Upon irradiation of the azide layer, the resulting nitrene may undergo a variety of reactions depending on the irradiation conditions. Contact angle measurements demonstrated that the irradiated surfaces were more hydrophilic than the non-irradiated azide layer and therefore the formation of an amine upon irradiation was postulated. Successful photoactivation could be demonstrated using condensation patterns, which showed a change in wettability on the wafer surface upon irradiation. Colloidal deposition with COOH functionalised particles further underlined the formation of more hydrophilic species. Orthogonal photoreactive silanes are described in the third part of this thesis. The advantage of orthogonal photosensitive silanes is the possibility of having a coexistence of chemical functionalities homogeneously distributed in the same layer, by using appropriate protecting groups. For this purpose, a 3',5'-dimethoxybenzoin protected carboxylic acid silane was successfully synthesised and the kinetics of its hydrolysis and condensation in solution were analysed in order to optimise the silanisation conditions. This compound was used together with a nitroveratryl protected amino silane to obtain bicomponent surface layers. The optimum conditions for an orthogonal deprotection of surfaces modified with this two groups were determined. A 2-step deprotection process through a mask generated a complex pattern on the substrate by activating two different chemistries at different sites. This was demonstrated by colloidal adsorption and fluorescence labelling of the resulting substrates. Moreover, two different single stranded oligodeoxynucleotides were immobilised onto the two different activated areas and then hybrid captured with their respective complementary, fluorescent labelled strand. Selective hybridisation could be shown, although non-selective adsorption issues need to be resolved, making this technique attractive for possible DNA microarrays.