This proposal aims at further advancement of Flow Injection (FI) methodology by exploiting the recent developments in Bead Injection (BI) technique. BI, in addition to having well proven benefits in microfluidic manipulation through FI instrumentation, uses microspheres as carrier/support for stimulants, reagents and fluorescent probes as well as for cells. It has been demonstrated in our past work, that through automation, precision control of reagents and solutions can work synergistically with real-time analysis to provide novel information on important biological functions. Increasingly, microscopy and cytometry have focused on the investigation of real-time activities of living cells. However, real-time analyses have been restricted by four critical factors. (1) Precision in microfluidic control for reagent addition and solution manipulation. (2) Biological responses that are inconsistent owing to physiological stress and changes in the viability of a cell line. (3) The necessity for automation in the execution and data collection of an experiment. (4) An instrument/software platform that is easy to use and readily adaptable to the researcher's needs. FI is a well-established technique that is highly flexible and uniquely adaptable to those biomedical research areas that require precise fluidic manipulations and/or temporal resolution of kinetic data. BI is a FI technique that uses fluidic handling of eucaryotic cells that are immobilized on microcarrier beads, allowing for the automated renewal of cells at the microscopic level. Because the cell population is replaceable, these micro-beads serve as a renewable biosensor, an important element in improving assay reliability. Likewise, these micro-beads can also serve as a carrier for reagents or immobilized probes that can be used in a variety of cellular studies and assays.
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