Multiphoton lithography of solid protein microstructures

Multiphoton excitation is a process in which two or more photons are absorbed nearly simultaneously by a target molecule in order to excite an electron to a higher energy state. The Shear lab uses a mode-locked, Ti:S, femtosecond pulse laser system to initiate multiphoton excitation of a particular class of molecule called a photosensitizer, which undergoes a chemical reaction in the process of releasing energy. As the laser scans over a small volume of photosensitizer and protein in a buffer solution, the photosensitizer produces high-energy free radicals which diffuse and cause covalent crosslinking between the proteins to occur, creating a crosslinked solid structure within the focal volume. Because this reaction happens only within the focal volume, this method gives precision as small as hundreds of nanometers. These structures can be designed for many studies which require micron-scale precision, environmentally reactive properties (responses to temperature or pH changes), and biocompatibility. The two major projects are discussed in this thesis involve the effect of changing the constituents of the solution: the photosensitizer and the protein. In the first experiment, I synthesized a new type of photosensitizer. A benzophenone dimer was synthesized by running a reaction of 4-benzoylbenzoic acid, succinimidyl esterpowder and 1,3-diaminopropane, filtered using flash column chromatography, and identified using mass spectrometry and NMR analysis. While analysis showed we did create the molecule we intended, the benzophenone dimer was difficult to dissolve and could not fabricate structures. In the second experiment, I used trypsin to hydrolyze bovine serum albumin over a wide range of digestion times, and used the digested fragments to fabricate structures. The structures were analyzed visually using SEM imaging and then through a swelling study, where fabrications were soaked in varying pH phosphate buffers and their dimensions were measured using a graphics software. The most notable result was that digested fragments were much less efficient at crosslinking, and were unable to form fine lines. However, there was no significant change in swelling, even at digestion times of up to twenty-four hours. Further studies on the quantization of the charge, size, and number of protein fragments from our digestion process are proposed.