Desenvolvimento de um magneto-imunoensaio microfluídico com detecção eletroquímica para diagnóstico de hanseníase

Abstract

Leprosy is an infectious disease caused by the bacteria Mycobacterium leprae and its diagnosis is mostly performed by clinical examination. Thus, in this work, two magnetoimmunoassays were developed for the serological diagnosis of leprosy using amperometry and a disposable microfluidic device. The electrodes produced proved to be accurate, robust and stable for up to 70 days after screen printing. The peptides were conjugated to the surface of magnetic particles (pep/MP), being added to the serum sample to capture the anti-M. leprae. The first immunoassay used a peptide sequence derived from the 85B protein. The final bioconjugate was added to the electrode manually using a micropipette. Electrochemical detection used horseradish peroxidase (HRP) with H2O2 and hydroquinone (HQ) detection solution. The second immunoassay used a peptide sequence derived from the recombinant protein ML0405. The final bioconjugate was added to the electrode using an injection pad. Detection was performed with alkaline phosphatase (ALP) and 1-naphthyl-phosphate (1-NPP) detection solution. Precision was evaluated by the responses obtained in different electrodes of the same device, in different devices and on different days of analysis, obtaining values of relative standard deviation (RSD%) between 4.8% and 13.6%. Analyzes of healthy, PB and MB samples were performed. Both immunoassays showed excellent selectivity and specificity to identify positive patients, including PB. The first trial distinguished PB from MB patients, which makes it also applicable to disease classification. The magneto-immunoassays showed an excellent discriminatory capacity between positive and negative patients, being able to even identify PB patients with high sensitivity, specificity and precision. Thus, they have the potential to guide the appropriate treatment, improving the quality of life of patients.