Electrochemical Paper-Based Analytical Device (e-PAD) Using Immobilized Prussian Blue and Antibodies for the Diagnosis of Leishmania

Leishmaniasis is a neglected disease prevalent in remote and economically disadvantaged regions. Its diagnosis can be achieved through various methods, with electroanalysis emerging as an excellent alternative for antigen detection due to its simplicity, sensitivity, and cost-effectiveness. Herein, a qualitative electrochemical paper-based analytical device (e-PAD) using unmodified screen-printed electrodes for the immunoassay of Leishmania amazonensis antigens has been developed. The detection is based on a sandwich-type assembly, utilizing two biological elements, one for capture and one for detection, with the target antigen sandwiched between them. Antibodies against Leishmania amazonensis and Prussian blue, serving as a redox mediator, were immobilized on the paper substrate. A synthetic peptide was employed as the target to demonstrate the proof-of-concept performance of the device. The formation of the immunocomplex was confirmed using horseradish peroxidase (HRP)-labeled antibodies, enabling the detection of antigen/antibody complexes in the presence of hydrogen peroxide via multiple pulse amperometry (MPA). The immunoassay exhibited good reproducibility (RSD = 5.12%) and selectivity when tested with positive and negative samples. Additionally, the ease of use and low cost of e-PAD enhance its accessibility, making it a valuable tool for the rapid and reliable diagnosis of neglected diseases. This reinforces its relevance as a practical solution in public health, particularly in underserved regions.