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Abstract: Overoxidized polypyrrole films templated with L-glutamate have been utilized for enantioselective detection of L- and D- glutamic acid. Various important fabrication factors controlling the performance of the overoxidized polypyrrole films have been investigated using fluorescence spectrometry in conjugation with the electrochemical quartz crystal microbalance technique. The measured fluorescence intensity was related to the concentration of glutamate taken up into the films. It was found that L-glutamate was inserted similar to 30 times higher into the film than D-glutamate. Several key parameters such as applied potential and pH of amino acid solution were varied to achieve the optimum sensor response. The sensor templated with L-glutamic acid also exhibited excellent selectivity over several other L- and D-amino acids. Higher enantioselectivity of overoxidized polypyrrole film than that for previously reported imprinted polymers can be attributed to the potential-induced uptake/release of targeted molecules