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

Abstract

A rapid and highly sensitive CE immunoassay method integrating mixing, reaction, separation, and detection on-chip is described for the measurement of alpha-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.

Rapid hybridization of nucleic acids using isotachophoresis

“…We used six observation points located at distances of 4, 12, 20, 24, 28, and 32 mm from the TE well for 10 nM MBs and 20 nM target hybridization. We used 10 observation points at distances of 8,16,20,24,28,32,36,40,42, and 44 mm (total channel length was 50 mm for this case) for hybridization of 50 pM MBs with 500 pM target.…”

“…Preconcentration with ITP has been applied to antibody-antigen reactions or nucleic acid hybridization reactions for increasing reaction rate or better sensitivity. Kawabata and co-workers showed an on-chip immunoassay method employing ITP for the enhanced sensitivity of immune reaction (24). Persat et al (25) and Bercovici et al (26) each presented the simultaneous extraction and detection of nucleic acids by combining ITP and the hybridization of molecular beacons and target nucleic acids.…”

A multiplexed immunoassay system based upon reciprocating centrifugal microfluidics

“…Generally, the rate of formation of antigen-antibody complexes, which is proportional to the concentration of the antibodies and antigens, dictates the required incubation time for generating a detectable result. This is why it is desirable to introduce other transport mechanisms such as migration and convection into the system to cause turbulence in the antibody solution and/or to generate flow and increase the rate of formation of antigen-antibody complexes 19,20 by employing different innovative methods. A promising methodology that has been examined in recent decades as a candidate for μTAS is the fabrication of microfluidic systems on rotating platforms, or compact discs (CD).…”

“…Beads of different sizes are often used to serve both as solid phases functionalized with capture antigens and to increase the interaction rate by intermittently mixing the reagents on the disk. [16][17][18][19][20][21][22][23] The rate of interaction will also increase by flowing the antibody solution over the array of immobilized antigens and hence increasing the rate of mass transport. [24][25][26] Rotating platforms are especially suitable for flow-through capture systems because the flow can be readily generated by the centrifugal acceleration.…”

Highly sensitive lens culinaris agglutinin-reactive α-fetoprotein is useful for early detection of hepatocellular carcinoma in patients with chronic liver disease

“…The new method uses on-chip electrokinetic reaction and separation by affinity electrophoresis (micro-total (14). In patients with an AFP level of ≥20 µg/ml, µ-TAS AFP-L3% correlated well with LiBASys AFP-L3% (15).…”