Henry Garrett Wada

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

Implementation of the on-chip immunoassay for alpha-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 10min. 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, AFP-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 0.1ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922ng/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.

“Electrokinetic Analyte Transport Assay” for α-fetoprotein immunoassay integrates mixing, reaction and separation on-chip

A rapid and highly sensitive CE immunoassay method integrating mixing, reaction, separation, and detection on‐chip is described for the measurement of α‐fetoprotein (AFP), a liver cancer marker in blood. Antibody‐binding reagents, consisting of 245‐bp DNA coupled anti‐AFP WA1 antibody (DNA‐WA1) and HiLyte dye‐labeled anti‐AFP WA2 antibody (HiLyte‐WA2), and AFP‐containing sample were filled into adjacent zones of a chip channel defined by the laminar flow lines of the microfluidic device using pressure‐driven flow. The channel geometry was thus used to quantitatively aliquot the reagents and sample into the chip. DNA‐WA1 was electrokinetically concentrated in the channel and sequentially transported through the AFP‐sample zone and HiLyte‐WA2 zone by ITP in such a manner that the AFP sandwich immune complex formation took place in the sample and HiLyte‐WA2 zones. The sandwich AFP immune complex was then detected by LIF after CGE in a separation channel that was arranged downstream of the reaction channel. AFP was detected within 136 s with a detection sensitivity of 5 pM. The on‐chip immunoassay described here, applying ITP concentration, in‐channel reaction, and CGE separation, has the potential of providing a rapid and sensitive method for both clinical and research applications.

On-chip quantitative PCR using integrated real-time detection by capillary electrophoresis

Quantitative PCR (qPCR) has been widely used for the detection and monitoring of a variety of infectious diseases. PCR and CE were integrated into a microfluidic chip that was designed to achieve rapid real‐time amplicon sampling, separation, and quantitation without requiring various probes. A novel chip design allows the overlapped execution of PCR and CE, minimizing the time required for CE analysis after each PCR cycle. The performance of the on‐chip qPCR method was demonstrated using a 45‐minutes model assay protocol for the phiX174 bacteriophage, and the multiplexing capability of the method was demonstrated by adding a second target, E. coli genomic DNA, to the model assay. The results indicate good sensitivity, reproducibility, and linearity over the tested assay range, 50 to 2 × 104 copies/25 μL reaction. Based on this performance, the on‐chip qPCR method should be applicable to a wide variety of infectious disease detection and monitoring assays with the addition of suitable sample preparation protocols.