Technical Notes Electrochemical Coding for Multiplexed Immunoassays of Proteins Guodong Liu, Joseph Wang,* ,† Jeonghwan Kim, and M. Rasul Jan ‡ Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003 Greg E. Collins Naval Research Laboratory, Chemistry Division, Washington D.C. 20375 An electrochemical immunoassay protocol for the simul- taneous measurements of proteins, based on the use of different inorganic nanocrystal tracers is described. The multiprotein electrical detection capability is coupled to the amplification feature of electrochemical stripping transduction (to yield fmol detection limits) and with an efficient magnetic separation (to minimize nonspecific adsorption effects). The multianalyte electrical sandwich immunoassay involves a dual binding event, based on antibodies linked to the nanocrystal tags and magnetic beads. Carbamate linkage is used for conjugating the hydroxyl-terminated nanocrystals with the secondary an- tibodies. Each biorecognition event yields a distinct vol- tammetric peak, whose position and size reflects the identity and level, respectively, of the corresponding antigen. The concept is demonstrated for a simultaneous immunoassay of  2 -microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively. These nanocrystal labels exhibit similar sensitivity. Such elec- trochemical coding could be readily multiplexed and scaled up in multiwell microtiter plates to allow simulta- neous parallel detection of numerous proteins or samples and is expected to open new opportunities for protein diagnostics and biosecurity. As research moves into the era of proteomics, scientists are faced with the challenge of developing effective methods for identifying and quantitating proteins. 1 Such new techniques are essential for the diagnosis of various disease states, for defense against biological threats, and for improving drug discovery. The ability to measure simultaneously multiple proteins in a single assay holds an enormous potential for meeting the growing demands of these diagnostic and biodefense applications. Immu- noassays are highly suitable for high-throughput screening, as they require minimal sample manipulations, are compatible with multiwell or microchip formats, and require small amounts of target analytes. 2 Most efforts for multianalyte immunoassays have focused on multicolor fluorescent detection (in connection with different organic dyes). 2 However, such multicolor fluorescence- linked immunoassays are often complicated by the requirement of an elaborate excitation and detection scheme and by the broad emission bands. 3 Electrochemical immunoassays have evolved dramatically over the past two decades 4 and are ideally suited for meeting the portability requirements of decentralized point-of-care testing or field detection of bioagents. A dual-analyte immunoassay using electrochemical detection of metal ion labels was proposed by Hayes et al. 5 More recently, Karube and co-workers 6 described an antibody-based array electrochemical biosensor for the simul- taneous identification of multiple antigens. Here we report on an electrical immunoassay coding protocol for the simultaneous measurements of multiple proteins based on the use of different inorganic nanocrystal tracers. Colloidal nanocrystals have been used recently for the simultaneous fluorescent immunoassay of four toxins 3 and for the electrical hybridization detection of multiple DNA targets. 7 In our new bioassay (Figure 1), the target antigens are captured using magnetic beads conjugated with the corresponding antibodies (A, B). The bound antigens are then detected by reactions with a pool of nanocrystal-antibody pairs (C) and stripping voltammetric measurement of the corresponding metals (D). Each individual protein recognition event thus yields a distinct voltammetric peak, * Corresponding author. E-mail: joewang@nmsu.edu. Tel.: 505-646-2140. † Permanent address: Department of Chemical and Material Engineering, Arizona State University, Tempe, AZ 85287. ‡ Permanent address: Department of Chemistry, University of Peshawar, Pakistan. (1) Zhu, H.; Bilgin, M.; Snyder, M. Annu. Rev. Biochem. 2003, 72, 783. (2) (a) Swartzman, E. E.; Miraglia, S. J.; Mellentin-Michelotti, J.; Evangelista, L.; Yuan, P. M. Anal. Biochem. 1999, 271, 143. (b) Luminex100 IS total system, http: // luminexcorp.com. (3) Goldman, E. R.; Clapp, A. R..; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684. (4) Cousino, M.; Jarbawi, T.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1997, 69, 544A. (5) Hayes, F. J.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1994, 66, 1860. (6) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikenbukuro, K.; Karube, I. Anal. Chem. 2003, 75, 1116. (7) Wang, J.; Liu, G.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214. Anal. Chem. 2004, 76, 7126-7130 7126 Analytical Chemistry, Vol. 76, No. 23, December 1, 2004 10.1021/ac049107l CCC: $27.50 © 2004 American Chemical Society Published on Web 11/02/2004