Technical Notes Surface Plasmon Resonance Detection for Capillary Electrophoresis Separations Rebecca J. Whelan and Richard N. Zare* Department of Chemistry, Stanford University, Stanford, California 94305-5080 A miniaturized surface plasmon resonance sensor has been used as an on-line detector for capillary electro- phoresis separations. The capillary was modified slightly to shield the sensor electronics from the high voltages applied during the separation. A three-component mixture of high refractive index materials was separated and detected at the millimolar level by an untreated gold- sensing surface. A simple protein immobilization proce- dure was used to functionalize the surface for selective protein detection. A hybrid buffer system was developed, in which both the deposition of immobilized protein layers and the electrophoretic delivery of protein analytes were optimized. The detection system has a reproducibility of 15%, a dynamic range of 3 orders of magnitude, and a detection limit for IgG of 2 fmol. Capillary electrophoresis (CE) 1,2 can achieve rapid and high- resolution separations of many different classes of analytes. It has been applied to a diverse range of analytical challenges, from the investigation of biomolecules and biological function, 3,4 to the screening of clinical and environmental samples, 5,6 to genome sequencing. 7 It is particularly powerful for biological applications, because it normally uses aqueous buffers and requires miniscule amounts of injected sample, enabling the chemical analysis of single biological entities and the screening of samples available in small amounts. Although excellent detection methods exist for CE, many of these methods rely on the presence of inherent chromophores, fluorophores, or electrochemically active groups, or else they require that the molecule of interest be labeled in order to become spectroscopically or electrochemically accessible. Refractive index (RI) detection has the advantage of being universally applicable and nondestructive, and it has found wide use in high-performance liquid chromatography (HPLC) to detect otherwise elusive ana- lytes such as carbohydrates. 8 The application of RI to microscale separation techniques such as μHPLC and CE has been limited by the challenges of miniaturizing a bulk property detector for nanoliter volumes. As a result, RI detection has not gained the extensive use in CE that it enjoys in HPLC, although RI detectors for CE have been developed, 9-11 and detection limits as low as 10 -8 RIU have been achieved. 12 Another detection scheme that relies on the sensing of refractive index is surface plasmon resonance (SPR) spectroscopy. In SPR the refractive index of a dielectric medium (such as an aqueous solution) is monitored indirectly by measuring the angle at which the resonant excitation of a surface plasmon wave in a metal adjacent to the dielectric occurs. The principles of SPR have been extensively reviewed. 13-15 By immobilizing molecules on the metal surface with affinity for the analyte(s) of interest, a sensitive and selective sensor can be created. In this way, SPR has been fruitfully applied to biological interaction analysis. This technology has been made commercially available in the BIAcore system (Biacore AB, Uppsala, Sweden). 16 More recently, a miniaturized and integrated SPR sensor has been developed (Spreeta, Texas Instruments, Dallas, TX). 17,18 Using either BIAcore or laboratory- built instrumentation, SPR has been used as a detector for flow injection analysis (FIA) and HPLC 19-24 as well as a means for * Corresponding author. E-mail: zare@ stanford.edu. (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981 , 53, 1298-1302. (2) Jorgenson, J. W.; Lukacs, K. D. Science 1983 , 222, 226-272. (3) Kennedy, R. T. Anal. Chim. Acta 1999 , 400, 163-180. (4) Hu, S.; Dovichi, N. J. Anal. Chem. 2002 , 74, 2833-2850. (5) Thormann, W.; Lurie, I. S.; McCord, B.; Marti, U.; Cenni, B.; Malik, N. Electrophoresis 2001 , 22, 4216-4243. (6) Dabek-Zlotorynska, E.; Aranda-Rodriguez, R.; Keppel-Jones, K. Electro- phoresis 2001 , 22, 4262-4280. (7) Dovichi, N. J.; Zhang, J. Angew. Chem., Int. Ed. 2000 , 39, 4463-4468. 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