Comparison of different silylation protocols for the functionalization of silicon microsystems for (bio)sensing: uniform and reproducible layers for reliable device

Surface functionalization of silicon-based materials is a mandatory step for the realization of a wide variety of (bio)analytical devices, and the quality of the coverage of the sensor surface with the sensing molecule can definitely improve the analytical performance of bio- and chemo-sensors. The functionalization of silicon surfaces with organofunctional silanes allows the exposure of several functional groups (amino-, thiol-, epoxy-, carboxy-) and is the first step for the immobilization of recognition elements (biological or not). For micro-sized and ultra-sensitive devices based on Micro(Opto)ElectromechanicalSystems -M(O)EMS- this requirement of a uniform and reproducible sensing layer is even more necessary. A thin, uniform and reproducible silane layer is a prerequisite for a uniform and reproducible sensing layer. The main objective of this study was the comparison of different silylation strategies in terms of homogeneity, stability and reactivity of the silane layer. Silicon surfaces, mimicking the surface of M(O)EMS, were silylated with different silanes according with different experimental conditions and the resulting coatings were characterized by AFM, SEM and fluorescence microscopy measurements. A vast array of complex silanes is used commonly in industrial processes, and most surfaces were modified by means of functionalized monoalkylsilanes (R-SiX3) with three leaving groups (X= alogen or alkoxy) and a R group which is generally an alkyl chain with the desired end group. The silanes hydrolyze in solution to form silanols and once the silanols are formed, they readily attach to the surface. These silanes can polymerize to form Si-O-Si (siloxane) linkages, that can form a 2D silane network on the surface if it happens after the coverage of the surface, but can also produce 3D silane networks that then bind the surface. The latter is an unwanted, unpredictable and unreproducible reaction that generate non-uniform surface. Silanes (RR'-SiX) with only one leaving group cannot polymerize (on plane or in solution) giving a monolayer, without any networking. By tuning the silylation conditions in terms of silane, concentrations and reaction time, different coverages can be achieved. For a reliable comparison of the functionalization protocols, flat silicon samples were prepared and on each of them a photoresist deposition was performed. This approach allows the silylation of a defined area of the sample and the resist-covered area, once the silylation is accomplished, can be easily removed, providing a bare, uncovered area, as internal control for the measurements. The silane layers were characterized by AFM and SEM techniques, using gold nanoparticles (30 nm diameter) that bind the end-groups of silanes to improve reliability of surface imaging. On silylated surfaces also fluorescent molecules were covalently immobilized and a map of the functionalized areas was performed by fluorescence microscopy. This approach makes possible the evaluation of the reactivity of the end-group of the organosilane layer. Combining the information from the different techniques, a comparison of the different silylation protocols was accomplished and the immobilization protocols optimized on model surfaces will be then applied to the microdevice surfaces. This systematic approach allowed the definition of a reliable silylation protocol, from surface pretreatment, to silylation and rinsing, that gives a smooth, reactive and homogeneous silane layer, to be exploited for the realization of silicon based microsensors. We would like to thank the Italian Minister of University and Research (MIUR) Futuro in Ricerca (FIR) programme under the grant N. I51J12000310001 (SENS4BIO)