This study first reported enzyme-free impedimetric biosensor-based molecularly imprinted polymers for selective and sensitive determination of L-hydroxyproline (L-hyp), a biomarker for the early diagnosis of bone diseases. In recent study, utilizing a single 3-aminophenylboronic acid (3-APBA) to create imprinted surfaces could result in a strong interaction and difficulty in removal of a template molecule. Hence, a mixture of monomer solution containing 3-APBA and o-phenylenediamine (OPD) in the presence of the L-hyp molecule was co-electropolymerized onto the screen-printed electrode using cyclic voltammetry (CV) to eradicate this mentioned limitation. The detection principle of this sensor is relied on alteration of mediator's charge transfer resistance (Rct) that could be obstructed by L-hyp occupied in imprinted surface. The successfully fabricated biosensor was explored by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and confocal scanning microscopy. Furthermore, the effect of polymer composition on the Rct response was systematically investigated. The result exhibited that the mixture of monomers could provide the highest change of Rct due to high selectivity from esterification of 3-APBA and from hydrogen bond of OPD surrounding the template. The sensor showed a significant increase in Rct in the presence of L-hyp, whereas no observable resistance change was detected in the absence thereof. The calibration curve was obtained in the range from 0.4 to 25 μg mL−1 with limits of detection (3SDblank/Slope) and quantification (10SDblank/Slope) of 0.13 and 0.42 μg mL−1, respectively. This biosensor exhibited high selectivity and sensitivity and was successfully applied to determine L-hyp in human serum samples with satisfactory results.
