|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
40. Integrated Electrochemical Detection with Microfabricated Capillary Electrophoresis Chips
Pankaj Singhal1, Jin
Xie2, Alexander N. Glazer1, and Richard A. Mathies2
Microfabrication technology has enabled the development of miniaturized capillary electrophoresis (CE) chips or microdevices that can perform preparation, amplification and electrophoretic separation of a wide variety of analytes on a very short time scale1-3. However, nearly all microchip analyses to date have utilized laser excited fluorescence detection. While fluorescence detection is very effective, it is not easy to integrate the laser and optical system into the microfabricated chip to make a completely miniaturized analysis system. We have therefore been exploring the development of microfabricated electrochemical detection systems for microchip CE analyses because of the high sensitivity of this method and the ease of integration.
In our first studies, platinum electrodes were microfabricated on glass CE-chips to demonstrate the feasibility of integrated electrochemical detection4. Although, redox-active neurotransmitters were detected directly with high sensitivity, non-electroactive DNA could only be detected using indirect detection. In order to make electrochemical detection more universal for chip-based analyses, redox-active labels can be attached to inherently non-electroactive compounds. Specifically, we have synthesized an M-13 primer with hydroquinone and ferrocene labels to demonstrate the feasibility of attaching labels to DNA. We have worked out the synthetic routes to prepare active N-hydroxysuccinimide esters of these labels. Activated esters of 1, 4-dihydroxy-2-naphthoic acid or a ferrocene were coupled with a 5'-aminohexyl terminated M-13(-40) universal primer sequence to make two different electroactive DNA primers. We have been able to detect these labeled DNA primers down to zeptomole levels using our micro-fabricated CE-chips with integrated electrochemical detection.
As a number of different labels are available for attachment to various analytes, simultaneous detection of multiple samples is conceivable with very high selectivity. To demonstrate this concept, we selected various labels which exhibit different redox-properties and are therefore readily distinguishable. These labels were detected with high selectivity using CE-chips with integrated electrochemical detection. A matrix-coding method was developed to collect the electrochemical signals from each label. This method also uniquely addresses each signal, so that the labels were detected without any overlap from each other. CE-chip designs using this approach for multiplex analyses in a single separation will also be presented. To further highlight the potential of integrated electrochemical detection, we will present a fully portable version of our microchip based system. This instrument validates that integrated electrochemical detection allows CE-chip based analyses to be miniaturized and portable.
1 Woolley, A. T. and Mathies, R. A.; (1994) Proc. Natl. Acad. Sci. U.S.A. 91 11348-11352.
2 Woolley, A. T., Sensabaugh, G. F. and Mathies, R. A.; (1997) Anal. Chem. 69 2256-2261.
3 Simpson, P. C., Roach, D., Woolley, A. T., Thorsen, T., Johnston, R., Sensabaugh, G. F. and Mathies, R. A.; (1998) Proc. Natl. Acad. Sci. U.S.A. 95 2256-2261.
4 Woolley, A. T., Lao, K., Glazer, A. N. and Mathies, R. A.; (1998) Anal. Chem. 70 684-688.
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