Analytical Sciences

Multiplexed Assay for Proteins Based on Sequestration Electrochemistry Using the Protein Binding Electroactive Magnetic Microbeads

Hideki KURAMITZ,* Yoko MAWATARI,* Mariwo IKEUCHI,* Osamu KUTOMI,* Noriko HATA,* Shigeru TAGUCHI,* and Kazuharu SUGAWARA**

*Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Gofuku 3190, Toyama 930–8555, Japan
**Faculty of Engineering, Maebashi Institute of Technology, Maebashi, Gunma 371–0816, Japan

The paramagnetic microbead-based electrochemical binding assay was demonstrated for detecting two kinds of protein simultaneously. The principle of this assay is based on the sequestration electrochemistry. The protein binding electroactive magnetic microbeads which are conjugated with an electroactive compound and a ligand to bind specifically with a target protein were prepared. The avidin-biotin and soybean agglutinin (SBA)-galactosamine were chosen as model protein-ligand systems. The avidin binding electroactive magnetic microbead (ABEMMb) and SBA binding electroactive magnetic microbead (SBEMMb) are constructed by biotin/thionine and galactosamine/ferrocene modified on paramagnetic microbeads. The voltammetric response for these functionalized microbeads was measured by the Nd-Fe-B magnet-incorporating carbon paste rotating disk electrode. The measurements were performed in a microliter droplet using a rotating disk electrode system. Avidin and SBA were simultaneously detected by the decrease in the current responses from the reduction of ABEMMb and SBEMMb that was caused by the binding with target proteins. The limits of detection for avidin and SBA were 4 × 10⁻¹⁰ and 2 × 10⁻¹⁰ M, respectively.

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