Autonomous Polymer Loading and Sample Injection for Microchip Electrophoresis

Abstract

We have developed an extremely simple method for microchip electrophoresis. Loading of a sieving polymer solution and injection of a sample solution are autonomously executed by a microchip fabricated in poly(dimethylsiloxane) (PDMS). In advance, the energy for the fluid pumping is stored in bulk PDMS by evacuating air dissolved in PDMS, and the information for the sample plug regulation is coded into the microchannel design. Besides the simplicity, our method brings about an advantageous effect: sample compaction due to the discontinuous electrophoretic mobility at the sample/polymer interface. The sample compaction effect was moderate in ordinary size-dependent separation for double-stranded DNA and was extreme in affinity electrophoresis for single-stranded DNA (ssDNA). In the latter separation mode, ssDNA components were sequence-specifically separated by difference in affinity to a probe oligonucleotide immobilized to the sieving polymer matrix. We separated up to 60-mer ssDNA mixtures based on single-base substitutions. The separation processes included typically 100-fold sample compaction and were completed within 15-30 s. This technology provides easy, simple, and sensitive method for detection of gene point mutations and typing of single-nucleotide polymorphisms.

Phosphate‐affinity electrophoresis on a microchip for determination of protein kinase activity

“…Design and fabrication of the microchip were described elsewhere [17]. Briefly, the microchip consisted of a 30 Â 30 Â 2 mm PDMS part, containing four identical T-shaped microchannel patterns, and a 76 Â 52 Â 1 mm glass slide.…”

“…1A). Autonomous filling of the solutions into the microchannel [17] was confirmed, and mPAE was carried out at room temperature ($251C) by applying voltages to platinum wires (0.5 mm diameter) dipped into the microchip reservoirs using a home-made high-voltage power supply. The voltages were À250 V for the plain PDMA reservoir and 0 V for the other two reservoirs.…”

“…Figure 1A schematically shows the microchannel just prior to the mPAE. The PDMA, the sample, and the Phostag-PDMA conjugate solutions have been introduced by the autonomous sample injection technique [17]. The two passive stop valves define three regions of the microchannel for the individual solutions.…”

“…We previously demonstrated an autonomous sample injection technique for electrophoresis of DNA on a PDMS-glass microchip [17]. With this technique, the sample and the sieving polymer solutions are autonomously introduced into the microchannel by absorption of air into the PDMS channel walls, and the sample plug is defined by two passive stop valves.…”

“…When the sample is electrophoresed into the ligand-immobilized polymer region, we can instantaneously observe the binding behavior of the target species to the ligand. This type of affinity electrophoresis has successfully been applied to detection of ssDNA using the complementary oligonucleotide as the affinity ligand [17][18][19].…”