Hemoglobin-modified nanoparticles for electrochemical determination of haptoglobin: Application in bovine mastitis diagnosis

Mastitis is the most common inflammatory disease in cattle, with an important economic impact on dairy farms and on the animals' health. Haptoglobin (Hp) has been proposed as a novel and effective biomarker for this disease. This work reports on the fabrication and characterization of novel hemoglobin (Hb)-modified magnetic nanoparticles (MNPs) and their application in bovine Hp detection. Firstly, chitosan-modified MNPs (MNPs-Chi) were synthesized and coated with polydopamine (pDA). MNPs-Chi@pDA were then modified with Hb because of its high affinity to Hp. The different stages during the MNPs synthesis and their modifications were evaluated with UV-Vis spectroscopy, XRD, FT-IR, thermogravimetric, electrical impedance and BET analysis. Finally, the MNPs were incorporated as solid support for Hp immobilization, and its further quantification was performed by means of an alkaline phosphatase-based assay with electrochemical detection. To do this, an electroactive substrate (1-naphthyl phosphate) and differential pulse voltammetry (DPV) were used for the indirect Hp determination. After the optimization of the main assay parameters such as incubation steps, antibody concentrations, enzymatic reaction time, among others; the Hp biosensing capacity was demonstrated in milk. The strategy presented in this work shows a wide range of detection (25-800 mu g mL(-1)), achieving a limit of detection of 43 ng mL(-1). The accuracy of the strategy was determined by recovery assays at four Hp concentrations in spiked milk samples, showing excellent results. The performance of the electrochemical sensing strategy offers an excellent and real alternative as on farm screening method for sub-clinical bovine mastitis detection.