Kinetic study of glucose oxidase on microfluidic toner-based analytical devices for clinical diagnostics with image-based detection

Abstract

This report describes for the first time a kinetic study for the glucose oxidase reaction on microfluidic toner-based analytical devices (μTADs) based on colorimetric measurements performed by a scanner. μTADs were fabricated by a laser-printing process in a format defined by eight detection zones interconnected by microfluidic channels and one central zone to sample inlet (SI). Detection zones were filled with a cellulose paste and then spotted with potassium iodide and a mixture of glucose oxidase-horseradish peroxidase enzymes. Assays were performed by adding 60 μL of glucose or artificial serum sample solutions to the SI zone, which promoted the quick distribution of the sample through microfluidic channels by capillary action towards the detection zones. The hydrogen peroxide produced in the enzymatic reaction was monitored for a period between 0 and 30 minutes for five initial glucose concentrations ranging from 2 to 10 mM. The values achieved for the Michaelis–Menten constant (K_m), the turnover number (K_cat) and the maximum reaction rate (V_max) were 8.9 ± 1.3 mM, 41.2 ± 14.0 s⁻¹ and 19.8 ± 6.7 mM min⁻¹, respectively. The kinetic parameters found on μTADs with digital images are in agreement with the data found by other detection methods already reported in the literature. The optimum reaction time achieved by the kinetic study has allowed the quantitative determination of the glucose concentration levels in serum samples with accuracy higher than 95%. Based on the proof of clinical suitability of the microfluidic platform described herein, we believe that kinetic studies of other enzymatic assays associated with clinical diagnostics can be similarly explored to provide a full knowledge of biochemical reactions in living systems.