Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for a-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma was, achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFT-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection Was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 mg/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r(2) = 0.981 and slope = 1.03. (c) 2009 Elsevier Inc. All rights reserved.