G.A. Messina, P.R. Aranda, S.V. Pereira, F.A. Bertolino, J. Raba
INQUISAL, National University of San Luis, CONICET,
Keywords: Toxocara, microfluidic immunosensor, laser-induced fluorescence, quantum dot, silica nanoparticles
Summary:Toxocariasis, one of the most common zoonotic infection worldwide, is caused by Toxocara canis (T. canis), or less commonly, Toxocara cati [1,2]. In humans, the infection is acquired by oral route through accidental ingestion of infective eggs from soil-contaminated hands, consumption of poorly sanitized vegetables and raw or undercooked meats [3,4]. Toxocara infective eggs hatch into the first portion of the intestine. Subsequently, the juvenile stages are distributed throughout the body, generating symptoms from mild to severe manifestations. The possibility of early diagnosis is of great importance, allowing proper management and treatment of patients suffering from toxocariasis. In last years, nanotechnology has contributed to the development of miniaturized immunosensor-based devices with high-throughput analytical properties [5,6]. Different nanomaterials such as quantum dots (QDs), silica nanoparticles (SNs), and other nanoparticles have emerged as promising alternatives for a wide range of immunosensors applications. The objective of this work was to develop a microfluidic immunosensor that include the use of nanomaterials for the quantitative determination of IgG antibodies to IgG anti-T.canis. For the development of the microfluidic immunosensor, excretory-secretory antigens from T. canis second-stage larvae (TES) were obtained according to the technique described by Gillespie (1995) . The IgG anti-T.canis antibodies detection in serum samples were carried out using a non-competitive format immunoassay. TES immobilized on 3-aminopropyl-functionalized silica-nanoparticles (AP-SNs) covalently incorporated in the central channel of the device are recognized specifically by the anti-T. canis antibodies in the sample. The subsequent detection was achieved by adding a second antibody labeled with cadmium selenide zinc sulfide quantum dots (CdSe-ZnS QDs) specific to human IgG. The concentration of IgG anti-T. canis antibodies present in the serum sample was measured by LIF detector, using excitation lambda at 491 nm and emission at 540 nm. Relevant studies of experimental variables that affect the performance of microfluidic immunosensor for IgG anti-T. canis antibodies determination were done. Between them, the optimal flow rate, incubation time, concentration of TES to be immobilized, enzymatic activity and the amplification effect resulting from the incorporation of the AP-SNs, were studied The combination of two different nanomaterials; AP-SNs as bioaffinity supports and QDs as fluorescent labels, enabled us to achieve a useful alternative tool for T. canis diagnostic. SNs proved to be an excellent choice for optical sensing, increasing the active area and consequently the sensitivity. The total assay time was 30 minutes, having made LIF detection in less than 1 minute. The detection limit calculated for the proposed methodology was 0.12 ng mL-1 and the coefficients of intra- and inter-assay variation were less than 6%. The results show the usefulness of the developed immunosensor for the fast determination of IgG antibodies anti T. canis.