Two different hydrogel-coated streptavidin (SAv) piezoelectric chips were investigated. One was directly prepared by immobilizing SAv molecules covalently onto a dextran-modified crystal, and the other one was indirectly prepared by physically adsorbing SAv onto a biotin-linked dextran surface. The covalent preparation yielded 80% more SAv-binding and better subsequent adsorption of biotinylated bovine serum albumin (bBSA). Both chips displayed the best binding affinity with bBSA at pH 5.0 in a flow injection analysis and exhibited reproductive real-time response during layer-by-layer assembly of a bBSA and SAv multilayer film. In the multilayer assembly, approximately 3-7 SAv molecules were captured by each immobilized bBSA, and the estimated apparent KD values of the binding of flowing bBSA with surface SAv were 0.24 and 0.11 mu M in the first two cycles of the covalently prepared chip, respectively. Two Escherichia coli cells, each flagellum-displaying Strep-tag I and Strep-tag II, respectively, were selectively detected by both kinds of SAv chips. These studies suggest the potential application of both chips in real-time screening SAv affinity ligands from a cell-display random peptide library.