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An immunoassay has been developed for the detection of bacterium Escherichia coli (E. coli) through β-Galactosidase (β-Gal). For developing an immunoassay on piezoelectric quartz crystal a polystyrene nanoparticle (PSNP) surface was developed by anchoring synthesized PSNPs (60 ± 5 nm) via self-assembled monolayer of p-aminothiophenol patterned on gold-coated quartz crystal. In order to make immunoassay specific to β-Gal, antibody specific to β-Gal (anti-β-Bal) was captured through physical attraction. On exposing this immunoassay to the β-Gal antigen (3.03 ng/ml), a strong detecting signal was observed, with simultaneous detachment of antibody from immunoassay as antigen–antibody complex. No appreciable change in the frequency shift, Δf, of quartz crystal was observed upon exposure to nonspecific Brucella antigen, confirming the specific behavior of immunoassay.