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Uniform micrometer-sized polystyrene/poly(styrene-divinylbenzene) composite particles were produced by a single-step swelling of polystyrene template microspheres with styrene, divinylbenzene and benzoyl peroxide, followed by polymerization of the monomers within the swollen particles at 70 1C. Uniform crosslinked poly(styrene-divinylbenzene) microspheres of high surface area were then produced by dissolution of the polystyrene template part of the former composite particles. Hydroperoxide-conjugated microspheres were produced by ozonolysis of the crosslinked poly(styrene-divinylbenzene) particles. Redox graft polymerization of acrylonitrile and chloromethylstyrene on the hydroperoxide-conjugated particles was then accomplished. The influence of various polymerization parameters on the grafting yield was elucidated. Uniform polyaldehyde microspheres were produced from the former particles in two ways: (1) LiAlH 4 reduction of the nitrile groups of the polyacrylonitrile-grafted particles, followed by reaction of the formed primary amino groups with glutaraldehyde; (2) Sommelet reaction on the polychloromethylstyrene-grafted particles. Trypsin was then covalently bound to the polyaldehyde-grafted microspheres. A comparison between the enzymatic activity of the conjugated and free trypsin was accomplished.

A practical and relatively simple method to identify molecularly imprinted polymers capable of binding proteins via the molecular tagging (epitope-like) approach has been developed. In our two-step method, we first challenge a previously obtained anti-tag molecularly imprinted polymer with a small molecule including the said tag of choice (a biotin derivative as shown here or other) connected to a linker bound to a second biotin moiety. An avidin molecule partially decorated with fluorescent labels is then allowed to bind the available biotin derivative associated with the polymer matrix. At the end of this simple process, and after washing off all the low-affinity binding molecules from the polymer matrix, only suitable molecularly imprinted polymers binding avidin through its previously acquired small molecule tag (or epitope-like probe, in a general case) will remain fluorescent. For confirmation, we tested the selective performance of the anti-biotin molecularly imprinted polymer binding it to biotinylated alkaline phosphatase. Residual chemical activity of the enzyme on the molecularly imprinted polymer solid support was observed. In all cases, the corresponding nonimprinted polymer controls were inactive.