In immunology, a standard ELISA has evolved into an ELISpot assaya method used to detect IFN- secreted by T-cells and to determine frequency of antigen-specific CD4 and CD8 T-cells.3,5While enumeration of IFN- producing PD173074 immune cells has become standard in immunology, the quantity and the dynamics of cytokine production in cells are largely unknown. Subsequent incubation with quencher-carrying complementary strands resulted in formation of DNA duplex and caused quenching of fluorescence due to FRET effect. When exposed to IFN-, microwells changed fluorescence from low (quencher hybridized with fluorophore-carrying strand) to high (quenching strand displaced by cytokine molecules). The fluorescence signal was confined to microwells, was changing in real-time and was dependent on the concentration of IFN-. In the future, we plan to co-localize aptamer beacons and cells on micropatterned surfaces in order to monitor in real-time cytokine secretion from immune cells in microwells. Keywords:High-throughput screening, Biosensors, Cell arrays, Aptamers, Surface micropatterning == Introduction == High-throughput screening of cells has become an important a part of drug discovery, cancer research, immune cell analysis and toxicology studies.1,13,14,30The desire to increase throughput is reflected in evolution of a commercial microtiter plate from 96 to 384 to 1536 well format. In addition to increased information content, high-throughput technologies may also offer a better understanding of cellular heterogeneity. In this regard, microfabrication approaches are particularly advantageous for decreasing the size of the microwells to enable capture and culture of single cells.4,6,18,20,21,29,31These single cell arrays may be thought of as providing both the ultimate throughput and the most nuanced view of heterogeneity within a given cell population. An important question that remains to be resolved PD173074 is how to analyze function of cells on micropatterned surfaces? If one is interested in analysis of heterogeneity on cell-by-cell basis then techniques relying on cells secreting into common media (e.g. ELISA) become unsuitable. At the present time, the analysis of cell function within arrays or on micropatterned surfaces is largely limited to intracellular markers,10,22,27,31however, it is advantageous to be able to monitor secreted products in cell arrays. Immunology is usually one area where analysis of secreted products is important. Immune cells are highly specialized and heterogeneous, with phenotype PD173074 being frequently defined by the types of cytokines these cells produce. For example, secretion of IFN-a 17 kDa proteinis used to subcategorize CD4 T-cells into T-helper 1 (Th1) cells (IFN-+) vs. Th2 cells (IFN-).15Production of IFN- in T-cells is associated with vigorous immune response to infections such as HIV and therefore represents an important correlate of immune cell function.16,17Love and co-workers as well as our UDG2 group have been developing micropatterned surfaces that enable detection of proteins secreted by the immune cells to permit high-throughput analysis of immune cell function.2,9,21,24,25,32,33 An approach pursued by our lab has relied on fabrication of poly(ethylene glycol) (PEG) hydrogel microwells on glass and immobilization of monoclonal antibodies (Abs) for capturing single immune cells within the wells.21A more recent variant of this approach was to co-immobilize Abs specific for T-cells and IFN- to be able to capture single CD4 T-cells and to detect secreted IFN- in the same microwells.33This approach allowed to assign cytokine production to specific cells within the array, however, Ab-based sandwich immunoassay employed for detection of IFN- provided only an end-point measurement, revealing no temporal information about cytokine release from cells. DNA or RNA aptamers have emerged as an alternative to Abs for affinity-based biosensors. 7Advantages of aptamers include low cost and chemical stability, but arguably their biggest advantage over Abs is the possibility of engineering beacons that emit signal directly upon analyte binding.8,11,26Recently, our group has developed fluorescence resonance energy transfer (FRET)-based aptamer beacon for detection of IFN-.28In the present study, IFN- aptamer beacons were immobilized inside microwell arrays to create cytokine-sensing micropatterned surfaces. As shown in Fig.1beacons consisted of fluorophore-labeled DNA aptamer hybridized to a complementary quenching strand and attached inside hydrogels via avidinbiotin binding. In the absence of analyte, no signal was observed because of the FRET effect; however, exposure of microwells to IFN- resulted in displacement of the quenching strand and appearance of fluorescence. Importantly, fluorescence signal was dependent on the analyte concentration, was confined to the microwells and was changing as a function of time. This study represents a step towards development of sensing micropatterned surfaces that may be used to both organize cells.