Anchoring Of His-Tag-Rab7 Protein On Nanoporous Silicon. Toward Surface Imprinting For Optical Sensing Applications

Molecular imprinting is a universal concept to generate materials with "molecular memory" by performing a polymerization of suitable functional monomers in the presence of a target molecule acting as a template. The subsequent removal of the template creates recognition sites in the molecularly imprinted polymer (MIP) that can, further on, selectively rebind the target1. Although this concept proved to be successful in preparing selective sorbents for compounds of small molecular weight (~200-1200 Da), several specific problems can arise if the target is a macromolecular protein2 as the classical imprinting methodologies fail to address the peculiarities of protein targets. The difficulties are largely attributed to the intrinsic properties of the proteins. Due to their fragility irreversible conformational changes may occur during polymerization; moreover, the large size of the proteins makes them difficult to remove from, or rebind to a highly cross-linked polymeric network. Among the approaches introduced during the past decade to overcome the barriers of protein imprinting2, surface imprinting emerged as the main strategy for macromolecular imprinting. This approach restricts the formation of imprinted binding sites to the surface of a polymer or to a very thin polymer layer with thickness comparable to the size of the protein template. The immobilization of the protein through a self-assembled anchor layer offers additional advantages over polymerization from a proteinmonomer mixture in terms of generating uniformly accessible binding sites. Herein, we propose a new method to prepare Rab7 protein oriented surface-imprinted nanoporous silicon photonic crystals (PhCs) with high template utilization efficiency. Nanoporous silicon PhCs are prepared by controlled electrochemical etching of silicon and subjected to thermal oxidation generating SiO2 to aid surface modification3. A His-tag is used as the anchor to facilitate the protein immobilization/removal, by exploiting a 3-step protocol functionalization of PhC surface involving i) SiO2 silanization with glycidoxypropyltrimethoxysilane (GLYMO) preliminarly reacted with iminodiacetic acid (IDA); ii) Ni2+ attachment by complexation with IDA; iii) Rab7 anchoring due to strong interaction between histidine residues and Ni2+. Rab7 is used as target molecule as it is a small GTPase belonging to the Rab family with a key role on different cellular pathways and processes4: it is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors. Furthermore, Rab7 has specific functions in neurons. Each step of PhC functionalization is monitored by Visible reflectance spectroscopy. Fourier transform of each spectrum affords a peak proportional to effective optical thickness (EOT) of the porous layer3, which is used as analytical signal for monitoring successful functionalization event. Also, protein removal by EDTA and reversibility of functionalization process is checked by determining EOT values. The developed protocol for anchoring his-tag Rab7 protein represents the preliminary step of Rab7 imprinting process, which involves subsequent self-polymerization of dopamine to control the imprinted shell thickness, protein removal by EDTA and rebinding tests monitoring the optical behavior of MIP on PhC, including its imprinting efficiency and selectivity.