| Summary: | <span style="font-variant: small-caps;">l</span>-Kynurenine (<span style="font-variant: small-caps;">l-</span>Kyn) is an endogenous metabolite produced in the catabolic route of <span style="font-variant: small-caps;">l</span>-Tryptophan (<span style="font-variant: small-caps;">l</span>-Trp), and it is a potential biomarker of several immunological disorders. Thus, the development of a fast and cheap technology for the specific detection of <span style="font-variant: small-caps;">l-</span>Kyn in biological fluids is of great relevance, especially considering its recent correlation with SARS-CoV-2 disease progression. Herein, a disposable screen-printed electrode based on a molecularly imprinted polymer (MIP) has been constructed: the <i>o</i>-Phenylenediamine monomer, in the presence of <span style="font-variant: small-caps;">l-</span>Kyn as a template with a molar ratio of monomer/template of 1/4, has been electropolymerized on the surface of a screen-printed carbon electrode (SPCE). The optimized kyn-MIP-SPCE has been characterized via cyclic voltammetry (CV), using [Fe(CN)<sub>6</sub>)]<sup>3−/4−</sup> as a redox probe and a scanning electron microscopy (SEM) technique. After the optimization of various experimental parameters, such as the number of CV electropolymerization cycles, urine pretreatment, electrochemical measurement method and incubation period, <span style="font-variant: small-caps;">l</span>-Kyn has been detected in standard solutions via square wave voltammetry (SWV) with a linear range between 10 and 100 μM (R<sup>2</sup> = 0.9924). The MIP-SPCE device allowed <span style="font-variant: small-caps;">l</span>-Kyn detection in human urine in a linear range of 10–1000 μM (R<sup>2</sup> = 0.9902) with LOD and LOQ values of 1.5 and 5 µM, respectively. Finally, a high selectivity factor α (5.1) was calculated for <span style="font-variant: small-caps;">l-</span>Kyn toward <span style="font-variant: small-caps;">l</span>-Trp. Moreover, the Imprinting Factor obtained for <span style="font-variant: small-caps;">l</span>-Kyn was about seventeen times higher than the IF calculated for <span style="font-variant: small-caps;">l</span>-Trp. The developed disposable sensing system demonstrated its potential application in the biomedical field.
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