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Nanosized polymer films can be constructed on various surfaces via alternative electrostatic adsorption of ionic polymers. The adsorption is to a great extent determined by the state and structure of the initial surface and/or the pre-adsorbed layers. Apparently, the same rule is valid for electrostatic adsorption of enzymes, which is used for their immobilization onto surfaces. This is specifically important for design and fabrication of bioanalytical devices, e.g., biosensors, where the amount of an enzyme incorporated and the strength of its binding have an influence on basic characteristics, such as activity and operation stability, of these bioanalytical surfaces. To demonstrate this, different fabrication regimes and properties of thin polymer/enzyme films deposited onto conductive substrates (e.g., graphite, gold) were examined. The films were formed via two-steps, sequential adsorption of a polymeric component onto a conductive surface, followed by the enzyme adsorption, under different conditions (pH, salt composition, and temperature). Strong/weak linear homopolyelectrolytes, ionic amphiphilic diblock copolymers as well as dual-stimuli responsive (i.e., pH- and temperature sensitive) microgels were taken as polymeric components of the thin polymer/enzyme films. Monoenzymatic systems with uniformly distributed biomaterial or bienzymatic systems with spatially separated enzymes are reported. Specific applications in the field of biosensors are discussed. (p. 7)