Journal of Biotechnology 122 (2006) 16–27 Effect of mixing on reaction–diffusion kinetics for protein hydrogel-based microchips D.A. Zubtsov, S.M. Ivanov, A.Yu. Rubina, E.I. Dementieva ∗ , V.R. Chechetkin, A.S. Zasedatelev Engelhardt Institute of Molecular Biology of Russian Academy of Sciences, Vavilova Str. 32, Moscow 119991, Russia Received 29 March 2005; received in revised form 11 July 2005; accepted 4 August 2005 Abstract Protein hydrogel-based microchips are being developed for high-throughput evaluation of the concentrations and activities of various proteins. To shorten the time of analysis, the reaction–diffusion kinetics on gel microchips should be accelerated. Here we present the results of the experimental and theoretical analysis of the reaction–diffusion kinetics enforced by mixing with peristaltic pump. The experiments were carried out on gel-based protein microchips with immobilized antibodies under the conditions utilized for on-chip immunoassay. The dependence of fluorescence signals at saturation and corresponding saturation times on the concentrations of immobilized antibodies and antigen in solution proved to be in good agreement with theoretical predictions. It is shown that the enhancement of transport with peristaltic pump results in more than five-fold acceleration of binding kinetics. Our results suggest useful criteria for the optimal conditions for assays on gel microchips to balance high sensitivity and rapid fluorescence saturation kinetics. © 2005 Elsevier B.V. All rights reserved. Keywords: Protein hydrogel microchips; Reaction–diffusion kinetics; Peristaltic pump; Antibody–antigen interaction; Fluorescence measure- ment 1. Introduction Biological microchips, or arrays of individual ele- ments containing various probes (DNA, proteins, oligosaccharides, cells, etc.), serve as miniature tools for different research and practical purposes (for ∗ Corresponding author. Tel.: +7 095 135 99 80; fax: +7 095 135 14 05. E-mail address: kdem@biochip.ru (E.I. Dementieva). review see, e.g. Nature Genetics, 2002; Kolchinsky and Mirzabekov, 2002; Cutler, 2003; Seong and Choi, 2003; Stoll et al., 2004). Recently, three-dimensional hydrogel-based microchips with immobilized DNA and proteins were developed at the Moscow Institute of Molecular Biology (Rubina et al., 2003, 2004). Immo- bilization of probes within three-dimensional hydro- gels offers many advantages over two-dimensional sur- face immobilization used by most microchip manu- facturers. In particular, the increase in immobilization 0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.08.010