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In clinical studies, high serum levels (>;196 ng/ml) of tissue inhibitor of metalloproteinases-1 (TIMP-1) have been shown to be associated with shorter survival time for breast cancer patients. We investigated TIMP-1 assays based on surface acoustic wave biosensors. Biotinylated antibody, anti-TIMP-1, was immobilized via streptavidin allowing site-directed orientation of the antibody on the biosensor surface. Streptavidin was coupled via carbodiimide chemistry to an intermediate hydrogel layer. We found that reagent incubation times and concentrations used for streptavidin coupling had a strong effect on the signal response in a subsequent TIMP-1 assay. An optimized coupling procedure based on a lower streptavidin concentration allowed the detection of 2 ng/ml TIMP-1.