Micromachined Separation Chips with a Precolumn Reactor and End-Column Electrochemical Detector

Abstract

Glass microchips, integrating chemical derivatization reactions, electrophoretic separations, and amperometric detection, have been developed. The performance of the new integrated microfabricated devices is demonstrated for rapid on-chip measurements of amino acids utilizing precolumn reactions of amino acids with o-phthaldialdehyde/2-mercaptoethanol to generate electroactive derivatives that are separated electrophoretically and detected at the end-column electrochemical detector. The influence of the sample/reagent mixing ratio, reagent concentrations, driving voltage, detection potential, and other variables is explored. The integrated microsystem offers a rapid (6 min) simultaneous measurements of eight amino acids, down to approximately 2.5 x 10(-6) M (5 fmol) level, with linearity up to the 2 x 10(-4) M level examined, and good reproducibility (RSD = 2.2-2.7%). A step of the driving voltage is used for decreasing the migration time of late-eluting components and reducing the overall analysis time. The integrated microfabricated device expands the scope of on-chip electrochemical detection to nonelectroactive analytes and holds promise of being a powerful analytical tool.

Microchip Separation and Electrochemical Detection of Amino Acids and Peptides Following Precolumn Derivatization with Naphthalene‐2,3‐dicarboxyaldehyde

“…Detection limits of 3.2 mM arginine, 7.4 mM phenylalanine, and 3.5 mM glycine were estimated from the response for a mixture containing 50 mM of the three amino acids (based on S/N of 3; not shown). Similar detection limits were reported for analogous OPA-based microchip measurements [4], while lower ones (down to 1.7 Â 10 À7 M) were obtained in analogous NDA-based conventional CE systems [6].…”

“…Platinum wires, inserted into the individual reservoirs, served as contacts to the high-voltage power supply. The custom-made microchip with integrated precolumn reaction chamber was fabricated by Micralyne Inc. (Edmonton, Canada) and it was described previously [4]. The chip consisted of a reaction chamber, reagent reservoir, sample reservoir, run buffer reservoir and unused reservoir.…”

“…The electrochemical properties of o-phthaldialdehyde (OPA) derivatives of amino acids have thus been exploited for their quantitation in conventional CE systems [3]. We reported on an on-chip precolumn reactor for the reaction of amino acids with phthaldialdehyde to produce electroactive derivatives [4]. The use of microfluidic devices allows bioassays to be performed more rapidly, easily, and economically [8].…”

Detecção eletroquímica em eletroforese capilar

“…Eletrodos impressos de carbono têm sido uma boa alternativa na construção de detectores eletroquímicos em µchips [267][268][269][270][271][272] . Wang e colaboradores [267][268][269][270][271] demonstraram a potencialidade da utilização de eletrodos impressos removíveis, pela facilidade de troca de eletrodos passivados ou modificados, eliminando a necessidade de troca do sistema completo de separação e reduzindo os custos de confecção do sistema total. Materiais diversos têm sido empregados na construção dos µchips, como polímeros 272,273 e cerâ-mica 274 .…”

“…As aplicações de µchips com ED estendem-se à separação de neurotransmissores [261][262][263][264][265][266][267][272][273][274] , explosivos nitroaromáticos 266,267,275 , acetaminofeno 268 ácidos úrico 268 e ascórbico 268,273 . Estratégias de reação enzimática 268 e derivatização acoplada ao canal de separação 269 foram empregadas na determinação de glicose e aminoácidos, respectivamente. Além disso, foi efetuada a detecção indireta de fragmentos de DNA, separados por eletroforese em gel (CGE), a qual nunca havia sido utilizada com ED 264,276 .…”