RESUMO
In this work, a genosensor for the electrochemical detection of genomic DNA from Mycobacterium tuberculosis was developed. The biosensor is based on self-assembled monolayers of mercaptobenzoic acid (MBA) and magnetite nanoparticles (Fe3O4Nps) on bare gold electrode for immobilization of DNA probe. The aim of this work was the development of a platform based on cysteine-coated magnetic Fe3O4Nps linked via the carboxylate group from MBA to the work electrode surface and subsequently to the DNA probe. The probe-genome interaction was evaluated using a [Fe(CN)6](4-)/[Fe(CN)6](3-) redox pair. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to evaluate the bioelectrochemical behavior of the sensor. Atomic force microscopy images showed Fe3O4Nps immobilized across the electrode surface. The interaction of the sensor with different genome DNA concentrations resulted in changes in the charge transfer resistance, indicating a possible use for tuberculosis detection at low concentrations (detection limit of 6ngµL(-1)).
Assuntos
Benzoatos/química , DNA Bacteriano , Nanopartículas de Magnetita/química , Mycobacterium tuberculosis , Sondas de Oligonucleotídeos , Compostos de Sulfidrila/química , Tuberculose , Cisteína/química , DNA Bacteriano/química , DNA Bacteriano/genética , Espectroscopia Dielétrica , Técnicas Eletroquímicas , Nanopartículas de Magnetita/ultraestrutura , Microscopia de Força Atômica , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Tuberculose/diagnóstico , Tuberculose/genéticaRESUMO
Primers targeting the Plasmodium small-subunit (SSU) rDNA were designed to amplify DNA from P. vivax, P. falciparum, P. malariae, and P. ovale, using conventional PCR, two-step nested PCR (HNPCR), and single tube hemi-nested PCR (STHNPCR). The limit of detection of parasite DNA for the conventional PCR, HNPCR, and STHNPCR were 10 pg, 0.01 pg, and 0.1 pg, respectively, indicating that the STHNPCR is 100-fold more sensitive than conventional PCR, and only 10 times less sensitive than HNPCR. In addition, the detection limit was also defined using blood from a patient infected with P. falciparum. Using the saponin method, the detection limit of the conventional PCR, HNPCR, and STHNPCR were 70, 0.7, and 0.07 parasites/microl, respectively. Finally, the three techniques were evaluated using blood from 30 patients receiving antimalarial treatment, and negative by microscopy and conventional PCR. The HNPCR could still detect specific DNA in 16/30 patients, whereas STHNPCR detected parasite DNA in 10/30 patients, but the difference was not statistically significant. No significant correlation was found between presence of clinical manifestations and presence of parasite DNA, detected by either HNPCR or STHNPCR. We conclude that these sensitive molecular diagnostic systems can be used for the diagnosis of asymptomatic oligoparasitemic patients.
