Sistema em fluxo multicomutado explorando a imobilização de enzima e reagente sólido na determinação espectrofotométrica de ureia em leite

Abstract

Detection of urea in excess of the permitted concentrations is possible with the possibility of milk fraud, which is not possible through the official method. In this work, we propose a multicommuted flow analysis system for the spectrophotometric determination of urea in milk using the urease enzyme immobilized in bacterial cellulose and the solid reagent, MOF (metal-organic framework) as colorimetric reagent. In the characterization of the MOF, the FTIR results showed the presence of the characteristic groups of MOF-Cu+2 containing carboxylate ions as ligand. The proposed analytical procedure was based on the urea hydrolysis reaction catalyzed by urease, producing NH4+, which reacts with MOF-Cu+2 forming tetramino copper (II), monitored at 450nm. The proposed system consists of two solenoid mini-pumps, a three-way solenoid valve, a column made of polymethyl methacrylate (PMMA) composed of two equal parts, separated by silica discs, where the enzymatic reaction occurs and, later, the colorimetric reaction and a multichannel (UV / VIS) spectrophotometer, model USB 4000 (Ocean Optics®). System control was developed using C language programming through an arduino® UNO board. After system optimization, linear responses were obtained between 1.0 and 50.0 mg dL-1 urea (R = 0.997, n = 11) with estimated detection and quantitation limits of 0.082 mg dL-1 and 0.272 mg dL-1 respectively, analytical frequency of 7 determinations per hour, 0.8 mL sample solution consumption, totaling 1.4 mL of residue, respectively. Potential interfering studies have shown the selectivity of the proposed method. Subsequently, samples of UHT milk, milk powder and milk compost were purchased from local markets in the metropolitan region of Recife and analyzed using the proposed system. Addition and recovery tests were performed to validate the procedure, obtaining variation between 90 and 103%, demonstrating the efficiency of the proposed method in detecting urea in milk samples. Comparing the results obtained by the proposed method and the reference method (KIT Urea CE) and applying the F test (precision) and the t test (accuracy), the results showed no significant difference at the 95% confidence level. The proposed system was robust with a structure of easy implementation and operation enabling its application in routine analysis.