Catalytic Three-Dimensional Protein Architectures

Abstract

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 microm(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.

Multiphoton fabrication of chemically responsive protein hydrogels for microactuation

“…Taken together, these results imply that a broad range of hydrogel swelling profiles will be possible by judicious selection [or engineering (37)] of protein building blocks. Further, protein matrices composed of avidin, which we previously have demonstrated retain biotin-binding functionality (23)(24)(25), showed attenuated swelling in acid and denaturant solutions in the biotinbound state compared with the unbound state ( Fig. 2d and Fig.…”

“…We and others have shown that direct-write protein matrices can retain ligand-binding and catalytic functionality similar to that of their component proteins (23)(24)(25)(26)(27), implying that native electrostatic and hydrophobic interactions governing protein conformation remain significantly intact within microstructures. However, at pH extremes, unfavorable electrostatic interactions can disrupt the native state and result in protein unfolding (31,32).…”

“…In addition to having the potential to undergo shape and hydration changes in response to chemical triggers, proteins contain a large number of weak acids and bases, similar to polymers used in pH-responsive hydrogels (5). Importantly, matrices composed of functional photocrosslinked proteins can be localized in 3D microenvironments by using multiphoton fabrication (23)(24)(25)(26)(27). Thus, multiphoton-fabricated protein structures could provide high-resolution 3D control over the topography of a stimuliresponsive material-facilitating greater mechanical functionality and incorporation of responsive hydrogels into more complex 3D devices-while potentially providing specificity (e.g., through ligand binding) over volumetric responses.…”

Multiphoton Lithography of Nanocrystalline Platinum and Palladium for Site-Specific Catalysis in 3D Microenvironments

“…We show that site-specific H2O2 decomposition catalyzed by MPL-directed Pt can be used to drive directed fluid flow in three dimensions upon integration within appropriately designed 3D structural components which we envision can be applied broadly for site specific catalysis in microfluidic environments, for instance, toward the design and testing of catalytic micro-pumps and motors. [13][14][15][16][17] The use of MPL to form conductive metallic structures has been explored, [18][19][20][21] however, reports on MPL of catalytic materials are scant 10,22 Information). Using this procedure, Pt and Pd patterns could be printed sequentially on un-modified glass with arbitrary shapes and intrinsic registration using a scanning laser, dynamic-mask based approach ( Figure 1a).…”

Mehrphotonen‐Mikrofabrikation

“…BSA-Voxel wurden mit optischen Pinzetten durch die Plasmamembran einer lebenden Zelle bewegt. [129] Avidinpartikel behielten auch nach dem Festhalten in einer optischen Falle die Fähigkeit zur Biotinbindung.…”