CONSTRUCTION AND CHARACTERIZATION OF A NANOPLATFORM FOR ELECTROCHEMICAL DETECTION OF ANTI-PARACOCCIOIDES IMMUNOGLOBULINS

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Brazil and Latin America, being an occupational and neglected disease. PCM diagnosis and evaluation of treatment response include the detection of anti-Paracoccidioides immunoglobulins in patient sera, but conventional serology tests lack standardization and reproducibility. Immunosensors, which rely on the detection of antigen-antibody specific interaction, have been studied as a new approach for point-of-care tests, allowing fast, cheap and accessible diagnosis. Thus, our aim is the construction, characterization and evaluation of a reproductible nanoplatform for development of an electrochemical immunosensor for PCM detection. A graphene-based platform was constructed with mechanically assisted chemically exfoliated graphite, which was functionalized with a conducting polymer for nanocomposite production. rPb27 antigen from P. brasiliensis, expressed in Escherichia coli heterologous system, was used as the sensor bioreceptor, and was adsorbed to the nanocomposite for nanoconjugate production. The platform was characterized by UV-vis spectrophotometry, dynamic light scattering (DLS), zeta potential measurements and microscopy (TEM and AFM). Graphene nanoplatelets had 8 layers and 95 nm of lateral size on average, and the micrographs showed the polymeric material on nanocomposite surface. UV-vis spectra presented a blue shift on the aromatic π→π* transition after functionalization with the polymer, and a second peak from polymer conjugated chain, which had a blue shift after antigen adsorption. Zeta potential was more negative after polymer adsorption, which is consistent with the improvement of graphene dispersity in the nanocomposite. After antigen adsorption there was a reduction of zeta potential negativity, consistent with biomolecule adsorption by charge. The nanoconjugate was immobilized in the surface of screen-printed carbon electrodes for immunosensor construction, and a preliminary assessment of electrochemical response during construction and IgG detection was carried by impedance spectroscopy (EIS). Nanoconjugate adsorption led to an increase in the charge transfer resistance (Rtc) on the electrode (~370%). Rtc decreased after IgG incubation, suggesting an indirect response mediated by the electrolyte. ΔRtc after specific anti-Pb27 IgG incubation was different from the control IgG at 0,2 and 2 μg/mL (p<0,01). These results suggest that the platform is promising for anti-Paracoccidioides IgG detection. The work perspectives are the optimization of the immunosensor response, evaluation of its analytical parameters and tests with patient samples.

This work was supported by MCTI/FINEP (Ref. 0109/21 and 0513/22), CNEN, CAPES (Finance Code 001), CNPq and Fapemig.