Abstract

Label-free Nucleic Acid Programmable Protein Array (NAPPA) technology, in combination with protein nanobiocrystallography and its possible future development using anodic porous alumina (APA) along with a cell-free expression system (as summarized here in Figure), appear to form a single approach capable of effectively solving the numerous problems still present in medical diagnosis and therapy. The Anodic Porous Alumina (APA) surface is prepared by a suitable electrolytic process designed to obtain a regular distribution of deep micrometric holes. The high aspect ratio (depth/width ratio) of the pores makes this material also a natural wave guide for any fluorescent molecule present on the bottom of the pores, avoiding crosstalk of many point-light sources too close as frequently in fluorescent NAPPA. The dielectric properties of Al2O3 makes this structure optimal for the realization of an electrically anisotropic system; The application of APA-NAPPA approach as an advanced “on chip laboratory” could result in challenging application - the cell free expressed protein molecules, trapped in the pores, after adding the precipitate can become protein nanocrystals useful for upcoming frontier XFELs technology for protein structure determination. The LB nanotemplate deposited onto the APA surface could be used to triggered protein nanocrystals formation. In principle, the crystallization in the pore could be achieved by means of microdialysis or batch crystallization methods. In the LB approach, nucleation is initiated at the interface between the high surface density LB film and protein/precipitant solution. In situ micro- and nano- GISAXS (Grazing Incidence Small Angle X-ray Scattering) studies confirms the film re-organization when in contact with protein/precipitant solution, resulting in nanocrystal formation, even those unobtainable by classical methods. Previously, LB grown crystals (up to submicron dimensions) were proved to be radiation stable in comparison those grown by classical method. This phenomenon could be fully exploited in serial femtosecond crystallography without cryocooling, reducing the radiation damage effects to the protein structures.

Authors and Affiliations