Desenvolvimento de biossensor enzimático baseado em peroxidase de bocaiúva para determinação de levodopa em fármacos

Abstract

In this work, the construction of an enzymatic biosensor based on peroxidase of bocaiúva (Acrocomia aculeata Lodd) immobilized in montmorillonite (mineral nanoargila) was optimized for the determination of levodopa in pharmaceutical formulations. Levodopa is administered in patients with Parkinson's disease, progressive degenerative neurological disease, caused by an intense decrease in dopamine production. By the action of the enzyme dopadescarboxilase, levodopa is converted to dopamine, restoring cerebral neurotransmission. The enzymatic extract of peroxidase was obtained from bocaiúva, native fruit of the cerrado biome, presenting activity of 8,270, mL-1 units. For the construction of the biosensor 1000 units mL-1 of enzyme were immobilized in montimorilonite and incorporated into a carbon-based paste containing graphite powder, multi-walled carbon nanotubes (NTCPMs) and mineral oil (Nujol) with composition 36, 4 / 9.1 / 9.1 / 45.4% m / m graphite / NTCPMs / support / Nujol. To evaluate the immobilization of the enzymatic extract in the support and the homogeneity of the carbon paste, the scanning electron microscopy technique was used. Levodopa was determined by the direct reduction of biocatalytically released quinone in 0.2 V vs. Ag / AgCl (KCl 3.0 mol L-1), using the square wave voltammetry (VOQ) technique. For the optimization of the biosensor response, several parameters were investigated. The best response was obtained in 0.1 mol L-1 phosphate buffer (pH 6.5), 4.0x10-4 mol L-1 hydrogen peroxide and 1,000.0 mL-1 peroxidase units. The parameters of the VOQ were optimized, being, frequency of 50 Hz, pulse amplitude of 100.0 mV and a scanning increment of 2.0 mV. The characteristics of this biosensor include repeatability (N = 12) with DPR of 0.9% and intermediate precision (N = 5) with DPR of 0.8%, which are within the standards established by ANVISA (2013). The analytical curve presented a linear response in the range of 0.785 to 4.60 mg L-1 (r2 = 0.992). The limits of detection and quantification were, respectively, 0.056 and 0.170 mg L-1. The stability of the biosensor was evaluated for 20 weeks and remained within the limits of statistical control (99% confidence), evidencing the efficiency of the immobilization of the enzymatic extract in the mineral nanoargila. The percent recovery percentages for a sample at three levels of fortification ranged from 101.6 ± 7.0 to 105.0 ± 7.9%, showing biosensor accuracy. The biosensor was applied for the quantification of levodopa in the drug Carbidol®, and the concentration of levodopa obtained corresponds to 91.28% of the value declared in the package insert, whereas, using the titration method in non-aqueous medium, 111 , 14%. According to t-test for 95% confidence, the values obtained by the titration method showed a significant difference between the concentrations obtained and declared in the package insert.