Catalytic three-dimensional protein architectures

We demonstrate a strategy for microfabricating catalytically active, three-dimensional matrixes composed of cross-linked protein in cellular and microfluidic environments. In this approach, a pulsed femtosecond laser is used to excite photosensitizers via multiphoton absorption within three-dimensionally defined volumes, a process that promotes cross-linking of protein residue side chains in the vicinity of the laser focal point. In this manner, it is possible to fabricate protein microparticles with dimensions on the order of the multiphoton focal volume (less than 1 mu m(3)) or, by scanning the position of a laser focal point relative to a specimen, to generate surface-adherent matrixes or cables that extend through solution for hundreds of micrometers. We show that protein matrixes can be functionalized either through direct cross-linking of enzymes, by decoration of avidin matrixes with biotinylated enzymes, or by cross-linking biotinylated proteins that then are linked to biotinylated enzymes via an avidin couple. Several formats are explored, including microparticles that can be translocated to desired sites of action (including cytosolic positions), protein pads that generate product gradients within cell cultures, and on-column nanoreactors for microfluidic systems. These biomaterial fabrication technologies offer opportunities for studying a variety of cell functions, ranging from single-cell biochemistry and development to perturbation and analysis of small populations of cultured cells.