Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture

Three dimensional multicellular co-cultures and anti-cancer drug assays in rapid prototyped multilevel microfluidic devices

This report presents a multilevel microfluidic platform for robust construction of hydrogel scaffold in microchannels and its application to three dimensional (3D) multicellular co-cultures and assays. A new rapid prototyping method based on soft lithography using multi-layered adhesive tapes is also introduced. We have successfully cultured MCF-7 breast cancer cell line more than 11 days with > 98 % viability, and co-cultured MDA-MB-231 breast cancer cells and NIH/3T3 fibroblasts in separate compartments. This multilevel microfluidic device with a cell-laden hydrogel microstructure has also been applied for anticancer drug assays in multicellular niches. Here we tested the effect of estrogen receptor (ER) antagonist drug, tamoxifen, on the growth of ER positive MCF-7 cells in microchannels. The inhibitory effect of tamoxifen on the growth of MCF-7 cells was diminished when they were co-cultured with ER negative MDA-MB-231 cells. The rapid prototyped multilevel microfluidic devices would provide simple, easy to use, low cost, robust, and reproducible cell-based assay platforms for potential end-users such as biologists and pharmacists.close