Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted by cells. EVs contain biological information related to parental cells and provide biomarkers for disease diagnosis. We have previously shown that the levels of podocin and nephrin expression on urinary EVs may be used to diagnose renal injury associated with preeclampsia. This paper describes a nanoparticle-enabled immunoassay integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy entailed capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox-active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a novel microtiter plate that contained 16 wells with working electrodes connected to onboard counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, a microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin to nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs 1.69) suggesting that this ratio may be used for disease diagnosis.
Keywords: biomarker; electrochemical biosensors; extracellular vesicles; immunoassays; microtiter plate; nephrin; podocin; preeclampsia.