There is a strong interest in the simultaneous and rapid detection of a multiple biomarkers in a single biological sample. This interest has been one of the driving forces behind the development of microfluidic devices for biomedical applications. The move to these smaller-scale systems has a number of advantages. First, they are capable of analyzing smaller sample volumes. Second, in applications such as capillary electrophoresis the microfluidic system can achieve the same separation resolution in much less time than a larger-scale system. Finally, the reduced size of the analysis setup raises the possibility of developing portable analytical devices.? ? One device under development is capable of measuring eight different electrophoretic runs simultaneously. Another device under development is the miniaturization of a luciferase immunoprecipitation (LIPS) assay that looks for immune response in serum samples. Other interests of our group include the design and development of devices capable of analyzing the secretions and physiology of single cells. Our facilities have now developed to a point where we are able to advise and collaborate with both intramural and extramural investigators on how to produce microfluidic devices that address their specific needs.? ? In collaboration with scientists at NIST, LBPS developed an eight channel plastic device for electrophoretic separation of DNA fragments. Using the microfabrication facilities at NIST, we are able to make micrometer-scale glass-encapsulated microfluidic systems with any desired two-dimensional configuration, as well as templates to stamp thermoplastics or mold elastomers into microfluidic devices. Currently, our focus is to further develop this chip for proteomic applications. One important challenge, key to the successful implementation of these devices, is the ability to achieve real-time detection of fluorescent labels at high sensitivity. We have achieved 10pM, 10 Hz multicolor label detection in eight independent 50u channels in a plastic substrate using a novel ball-lens, fiber optic approach that couples to an imaging spectrograph with CCD detection that allows four labeling dyes to be used simultaneously.? ? This year, we have started a project aimed at miniaturizing the LIPS assay, which uses a fusion protein consisting of Renilla luciferase and an antigen of interest to probe for antibodies in human serum. In its current format, the assay is performed over two hours in a 96-well filter plate, and has been shown effective in detecting a number of autoimmune conditions and infectious diseases. Moving the assay to a microfluidic format could significantly speed the analysis, permit multiplexing, and also facilitate the application of this assay to point-of-care diagnostics. Preliminary experiments using cell extracts containing the fusion protein and commercial anti-CFLAG antibodies in lieu of serum have shown that the positive signal levels are acceptably high even in the miniaturized format. Application of the microfluidic device to serum samples will take place in this next year.
