Applications of multiphoton-excited photochemistry to microsecond capillary electrophoresis, photolithography, and the development of smart materials

textLaser-based techniques have become essential tools for probing biological molecules in systems that demand high spatial and temporal control. This dissertation presents the development of micro-analytical techniques based on multiphoton excitation (MPE) to promote highly localized, three-dimensional (3D) photochemistry of biologically relevant molecules on submicron dimensions. Strategies based on capillary electrophoresis (CE) have been developed for the rapid separation and spectroscopic analysis of short-lived photochemical reaction products. High-speed separation and analysis are achieved through a combination of very high electric fields and a laser-based optical system that uses MPE for both the generation and detection of hydroxyindole photoproducts on the time scale of microseconds. MPE was also used for the development of photolithographic techniques for the creation of microstructured protein-based materials with highly defined three-dimensional (3D) topographies. Specifically, a multiphoton lithographic (MPL) technique was developed that used a low-cost microchip laser for the rapid prototyping of 3D microarchitectures when combined with dynamic optical masking. Furthermore, MPL was used to create novel “smart” biomaterials that reproducibly respond with tunable actuation to changes in the local chemical and thermal environment. The utility of these materials for creating biocompatible cellular microenvironments was demonstrated and presents a novel approach for studying small populations of microorganisms. Finally, through the development of a multifocal approach that used multiple laser beams to promote the photocrosslinking of biological molecules, the speed and versatility of MPL was extended to allow both the parallel fabrication of 3D microstructures and the rapid creation of large-scale biomaterials with highly defined spatial features.Chemistr

Three-Dimensional Printing of Photoresponsive Biomaterials for Control of Bacterial Microenvironments

Advances in microscopic three-dimensional (μ3D) printing provide a means to microfabricate an almost limitless range of arbitrary geometries, offering new opportunities to rapidly prototype complex architectures for microfluidic and cellular applications. Such 3D lithographic capabilities present a tantalizing prospect for engineering micromechanical components, for example, pumps and valves, for cellular environments composed of smart materials whose size, shape, permeability, stiffness, and other attributes might be modified in real time to precisely manipulate ultralow-volume samples. Unfortunately, most materials produced using μ3D printing are synthetic polymers that are inert to biologically tolerated chemical and light-based triggers and provide low compatibility as materials for cell culture and encapsulation applications. We previously demonstrated feasibility for μ3D printing environmentally sensitive, microstructured protein hydrogels that undergo volume changes in response to pH, ionic strength, and thermal triggers, cues that may be incompatible with sensitive chemical and biological systems. Here, we report the systematic investigation of photoillumination as a minimally invasive and remotely applied means to trigger morphological change in protein-based μ3D-printed smart materials. Detailed knowledge of material responsiveness is exploited to develop individually addressable “smart” valves that can be used to capture, “farm”, and then dilute motile bacteria at specified times in multichamber picoliter edifices, capabilities that offer new opportunities for studying cell–cell interactions in ultralow-volume environments

Building on the foundation of daring hypotheses: Using the MKK4 metastasis suppressor to develop models of dormancy and metastatic colonization

AbstractThe identification of a novel metastasis suppressor function for the MAP Kinase Kinase 4 protein established a role for the stress-activated kinases in regulating the growth of disseminated cancer cells. In this review, we describe MKK4’s biological mechanism of action and how this information is being used to guide the development of new models to study cancer cell dormancy and metastatic colonization. Specifically, we describe the novel application of microvolume structures, which can be modified to represent characteristics similar to those that cancer cells experience at metastatic sites. Although MKK4 is currently one of many known metastasis suppressors, this field of research started with a single daring hypothesis, which revolutionized our understanding of metastasis, and opened up new areas of exploration for basic research. The combination of our increasing knowledge of metastasis suppressors and such novel technologies provide hope for possible clinical interventions to prevent suffering from the burden of metastatic disease