Detection and accurate quantitation of biomarkers such as alpha-fetoprotein (AFP) can be a key aspect of early stage cancer diagnosis. Microfluidic devices provide attractive analysis capabilities, including low sample and reagent consumption, as well as short assay times. However, to date microfluidic analyzers have relied exclusively on calibration curves for sample quantitation, which can be problematic for complex mixtures such as human serum. We have fabricated integrated polymer microfluidic systems that can quantitatively determine fluorescently labeled AFP in human serum, using either the method of standard addition or a calibration curve. Our microdevices couple an immunoaffinity purification step with rapid microchip electrophoresis separation with laserinduced fluorescence detection system, all under automated voltage control in a miniaturized polymer microchip. In conjunction with laser-induced fluorescence detection, these systems can quantify AFP at ~1 ng/mL levels in ~10 µL of human serum in a few tens of minutes. Our polymer microdevices have been applied in determining AFP in spiked serum samples. These integrated microsystems offer excellent potential for rapid, simple and accurate biomarker quantitation in a point-of-care setting.The two most widely used quantitation tools in traditional analytical chemistry are the calibration curve and the method of standard addition. 1 Micromachined devices for chemical analysis 2, 3 that integrate multiple processes, 4 reduce sample and reagent consumption, 5 and decrease analysis time 6, 7 and instrument footprint, 8, 9 are becoming an attractive alternative to classical separation-based analysis approaches. Although calibration curves have been used in microchip-based chemical analysis,10 , 11 the method of standard addition, which is especially desirable for addressing matrix effects in complex samples 1 such as blood, has seen extremely limited use. Very recently, a serial dilution microfluidic device was applied in standard addition quantitation of mM concentrations of Fe(CN) 6 4− , a model analyte, although the aqueous KCl solution was not one for which matrix effects were anticipated. 12 Alpha-fetoprotein (AFP) is a diagnostic biomarker for Hepatocellular carcinoma (HCC),13 with a reported specificity of 65% to 94%.14 In general, patients with an elevated serum AFP concentration have a higher risk for HCC. Currently, enzyme linked immunosorbent assay (ELISA) is used in the clinical analysis of AFP in human serum.15 With trained personnel, ELISA can provide reliable results, although the multi-hour assay times and microplate format make ELISA best suited for clinical, rather than point-of-care (POC) diagnostics. In contrast, rapid analysis6 , 7 and the ability to combine multiple processing steps4 , 16 on a single device make a microfluidic-based approach very attractive for POC AFP analysis. The analysis and separation of AFP in spiked buffer solutions in a microdevice platform have been reported, [17][18][19] 11,20, 21 However, only calibr...

Microchip capillary electrophoresis of proteins labeled either off-or on-chip with the "chameleon" CE dye 503 using poly(methyl methacrylate) microchips is presented. A simple dynamic coating using the cationic surfactant cetyltrimethyl ammonium bromide prevented nonspecific adsorption of protein and dye to the channel walls. The labeling reactions for both off-and on-chip labeling proceeded at room temperature without requiring heating steps. In off-chip labeling, a 9 ng/mL concentration detection limit for bovine serum albumin (BSA), corresponding to a ~7 fg (100 zmol) mass detection limit, was obtained. In on-chip tagging, the free dye and protein were placed in different reservoirs of the microchip, and an extra incubation step was not needed. A 1 μg/mL concentration detection limit for BSA, corresponding to a ~700 fg (10 amol) mass detection limit, was obtained from this protocol. The earlier elution time of the BSA peak in on-chip labeling resulted from fewer total labels on each protein molecule. Our on-chip labeling method is an important part of automation in miniaturized devices.