Direct-Write Fabrication of Functional Protein Matrixes Using a Low-Cost Q-Switched Laser

Abstract

We report the use of an inexpensive, small, and "turn-key" Q-switched 532-nm Nd:YAG laser as a source for nonlinear, direct-write protein microfabrication. In this approach, microJoule pulses (pulse widths, approximately 600 ps) are focused using high numerical aperture optics to submicrometer focal spots, creating instantaneous intensities great enough to promote multiphoton excitation of a photosensitizer and subsequent intermolecular cross-linking of protein molecules. By scanning the femtoliter focal volume through reagent solution, extended protein-based structures can be fabricated with precise, three-dimensional topographies. As with earlier studies using a femtosecond titanium:sapphire laser costing more than 100K, physically robust and chemically responsive microstructures can be fashioned rapidly with feature sizes smaller than 0.5 microm, and cross-linking can be achieved using both biologically benign sensitizers (e.g., flavins) and by using the proteins themselves to sensitize cross-linking. We demonstrate in situ fabrication to corral neurite outgrowth and show the ability to functionalize avidin structures with biotinylated reagents, an approach that enables chemical sensing to be performed in specified microenvironments. Characterization of this inexpensive, low-power source will greatly broaden access to direct-write protein microfabrication.

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).…”

“…Moreover, matrices can be cycled rapidly between swelled and condensed states in response to pH steps (Movie S3), indicating (as predicted) that denaturation is reversible. Importantly, the degree to which hydrogel microstructures swell and actuate was found to depend strongly on the crosslinking density of the matrix, a property that can be modulated both by protein concentration within the fabrication solution and laser exposure times (25,26,34) (Fig. S1).…”

“…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.…”

Mask-Directed Multiphoton Lithography

“…This approach allows fabrication of functional gradients ( Figure S1) with arbitrary directionality and slope (by changing the direction and speed of the moving mask) with significantly higher resolution than other techniques that use laser scanning 8 or broadband light sources. 9 Our approach is readily adaptable to existing microscope platforms, is potentially inexpensive, 10 and should facilitate studies of cell development and differentiation.Mask-directed multiphoton lithography enables facile optimization of microfabricated materials for interfacing with cell cultures, such as the capture and incubation of motile cells. By using CAD software and an office printer it was straightforward to rapidly prototype a wide variety of bacterial microchambers, such as those shown in Figure 1C.…”