Advanced DNA–Gold Biointerface for PCR‐Free Molecular Detection of <i>Leishmania infantum</i>

Abstract PCR‐free approaches are the most promising technologies for molecular point‐of‐care (PoC). In this context, the detection of not amplified genetic targets through electro‐optical transduction is successfully investigated. While PCR‐free approaches are widely studied, there are only a few studies investigating the factors that modulate both the kinetics and the effectiveness of target capture. Among these, the probes grafting density and the isoelectric properties of the biointerface are crucial since they conditionate the charge field around biomolecules during and after the target recognition. In this work, an experimental and theoretical study of a gold biointerface functionalized with oligonucleotide probes is presented for the direct detection by cooperative hybridization of the kinetoplast (k)DNA of Leishmania infantum (LI). The biointerface is characterized by surface free energy (SFE) analysis and contact angle (CA) to investigate the grafting of probes and the surface isoelectric properties upon the duplex formation with the genetic target. Experimental data are compared with a theoretical model, based on the prediction of adsorption energies, which effectively reflects the charge profile of the functionalized surface. Lastly, the biointerface is characterized by electrochemical impedance spectroscopy (EIS) and the sensing performances assess in the frame of its suitability for PoC applications.