Nucleation of sub-micrometer protein crystals in square-shaped macroporous silicon structures

Macroporous silicon substrates, with square-shaped pores, have been used to crystallize hen egg white lysozyme by the sitting drop vapor diffusion method. The X-ray diffraction technique was used to determine the tetragonal structure of the crystals. Use of an asymmetric anodization procedure to produce pore size gradients in porous structure, ranging from 400 nm to 1 m, resulted in the formation of sub-micrometer-sized protein crystals within the macroporous structure. The presence of the crystals was observed by field emission scanning electron microscopy and confirmed by Raman and infrared spectroscopy. The present work provides experimental evidence of sub-micrometer crystal growth from pore corners and rough sides of the pore walls, attributed to the reduction of the potential energy for nucleation, in accordance with the different mathematical models developed so far.