Photochimie dans le champ proche: application à la photoimmobilisation de biomolécules à l'échelle nanométrique

In view of future generations of biosensors, the immobilization of biomolecules onto selected materials with a well controlled topical addressability is of primary importance. Photoimmobilization is a technique allowing the immobilization of a biomolecule through a photochemical reaction. It induces a covalent bonding between the biomolecule and the substrate, by means of a photoreagent. The aryldiazirine, used as photoreagent, is photoactivated at 350 nm, a wavelength that does not usually degrade the biological functionalities of biomolecules. Mask assisted illumination is used to immobilize biomolecules on spatially well defined domains. This technique's lateral resolution is limited by light diffraction, a problem that can be solved with an illumination in the optical near field. Scanning near field optical microscopy (SNOM) is a technique using an optical probe with sub wavelength dimension that scans the sample a few nanometers above its surface. The probe interacts with the sample via the near electromagnetic field either emitted or scattered by the probe or by the sample. The resolution of SNOM is not limited by diffraction, but rather by the size of the optical probe and the distance between the probe and the sample. This work demonstrates that SNOM can be used not only as an observation tool, but also as a structuring tool. Indeed, when light is injected into the optical probe, it also becomes a nanometer size light source. The SNOM can then induce photochemical reactions in the near field, leading to very localized photoimmobilization of biomolecules. The potential of direct near field photoactivation is first demonstrated on photoresists, a well known system commonly used in photolithography. Features with line width between 220 nm and 300 nm and depth between 45 nm and 50 nm are obtained with standard processes using positive resists deposited 1.5 μm thick. Maleimidoaryldiazirine (MAD) photoimmobilization in the near field, as well as in the far field, demonstrates the topical addressability of light induced immobilization with a small molecule that forms a thin and homogenous layer on the sample surface. The photoimmobilization of MAD is performed on silicon substrates. SNOM illumination is then applied to the photoimmobilization of a biomolecule onto a glass substrate. A protein, bovine serum albumin (BSA), modified with diazirine-based photoreagent is used. BSA is labelled with fluorescein and its characterization is subsequently done by fluorescence observations. The smallest structure photoimmobilized and observed with the SNOM has a feature size of 470 nm. On average, the feature size is 800 nm, which demonstrates that SNOM is five times better in resolution than far field illumination techniques. This work also shows the influence of the optical probe's thermal behavior. With an injected light intensity of 1.8 mW, the probe's temperature has been measured up to 140°C. Diazirine, the photoreagent used for BSA photoimmobilization can be thermally activated at 85-90°C. It is, therefore, shown that the heating of the probe can contribute to the degradation of the photoimmobilized structure's lateral resolution.

    Thèse École polytechnique fédérale de Lausanne EPFL, n° 2300 (2000)
    Faculté des sciences et techniques de l'ingénieur
    Institut d'imagerie et optique appliquée
    Jury: Hans Jörg Mathieu, Philippe Renaud, Ronald Shelden, Hans Sigrist

    Public defense: 2001-2-2


    Record created on 2005-03-16, modified on 2016-08-08


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