We have developed an acrylic microfluidic device that sequentially couples liquid-phase isoelectric focusing (IEF) and free solution capillary electrophoresis (CE). Rapid separation (<1 min) and preconcentration (73x) of species were achieved in the initial IEF dimension. Using full-field fluorescence imaging, we observed nondispersive mobilization velocities on the order of 20 microm/s during characterization of the IEF step. This transport behavior allowed controlled electrokinetic mobilization of focused sample bands to a channel junction, where voltage switching was used to repeatedly inject effluent from the IEF dimension into an ampholyte-based CE separation. This second dimension was capable of analyzing all fluid volumes of interest from the IEF dimension, as IEF was 'parked' during each CE analysis and refocused prior to additional CE analyses. Investigation of each dimension of the integrated system showed time-dependent species displacement and band-broadening behavior consistent with IEF and CE, respectively. The peak capacity of the 2D system was approximately 1300. A comprehensive 2D analysis of a fluid volume spanning 15% of the total IEF channel length was completed in less than 5 min.

Chip-based microcolumn separation systems often require serpentine channels to achieve longer separation lengths within a compact area. However, analyte bands traveling through curved channels experience an increased dispersion that can reduce the benefit of increased channel length. This paper presents analytical solutions for dispersions, numerical models for minimizing dispersion in microchannel turns, and experiments used to validate numerical models and to demonstrate the effectiveness of dispersion−reduction schemes. An analytical solution for the geometric dispersion caused by a constant radius turn is presented. We also propose metrics for characterizing the performance of miniaturized electrophoresis systems that utilize dispersion-introducing turns. The analytical solution and metrics can be used to determine when compensating turns should be used and when these turns are either not necessary or ineffective. For situations where a constant radius turn introduces significant geometric dispersion, numerical shape optimization routines were used to determine optimal geometries that minimize geometric dispersion while limiting reductions in channel width. Experiments using photobleached-fluorescence and caged-fluorescence visualization were conducted to validate the employed numerical models and to verify the turn designs proposed here.

There is a need in surgical oncology for contrast agents that can enable real-time intraoperative visualization of solid tumors that can enable complete resections while sparing normal surrounding tissues. The Tumor Paint™ agent BLZ-100 is a peptide-fluorophore conjugate that can specifically bind solid tumors and fluoresce in the near-infrared range, minimizing light scatter and signal attenuation. In this study, we provide a preclinical proof of concept for use of this imaging contrast agent as administered before surgery to dogs with a variety of naturally occurring spontaneous tumors. Imaging was performed on excised tissues as well as intraoperatively in a subset of cases. Actionable contrast was achieved between tumor tissue and surrounding normal tissues in adenocarcinomas, squamous cell carcinomas, mast cell tumors and soft tissue sarcomas. Subcutaneous soft tissue sarcomas were labeled with the highest fluorescence intensity and greatest tumor-to-background signal ratio. Our results establish a foundation that rationalizes clinical studies in humans with soft tissue sarcoma, an indication with a notably high unmet need.

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A photobleached-fluorescence imaging technique for visualizing microscale flow fields and obtaining molecular diffusion and advection information has been developed. The technique tracks fluorophores in the region of a photobleached line in a planar microdevice and yields quantitative diffusive and advective transport data. Visualizations of two-and weakly three-dimensional electroosmotically and pressure-driven fluid flow fields are demonstrated using the photobleaching of fluorescein and fluorescein-dextran conjugates. Photobleached-fluorescence imaging tracks undisturbed fluorophores, functions in polymer and glass microfluidic devices, can take advantage of fluorescent conjugates present in biochemical assays, and has a photobleached region that is flow independent.