Determination of glucose using a coupled-enzymatic reaction with new fluoride selective optical sensing polymeric film coated in microtiter plate wells

Abstract

The determination of glucose in beverages is demonstrated using newly developed fluoride selective optical sensing polymeric film that contains aluminum (III) octaethylporphyrin (Al[OEP]) ionophore and the chromoionophore ETH7075 cast at the bottom of wells of a 96-well polypropylene microtiter plate. The method uses a dual enzymatic reaction involving glucose oxidase enzyme (GOD) and horseradish peroxidase (HRP), along with an organofluoro-substrate (4-fluorophenol) as the source of fluoride ions. The concentration of fluoride ions after enzymatic reaction is directly proportional to the glucose level in the sample. The method has a detection limit of 0.8 mmol L(-1), a linear range of 0.9- 40 mmol L(-1) and a sensitivity of 0.125 absorbance unit/decade of glucose concentration. Glucose levels in several beverage samples determined using the proposed method correlate well with a reference spectrophotometric enzyme method based on detection of hydrogen peroxide using bromopyrogallol red dye (BPR). The new method can also be used to determine H(2)O(2) concentrations in the 0.1 - 50 mmol L(-1) range using a single enzymatic reaction involving H(2)O(2) oxidation of 4-fluorophenol catalyzed by HRP. The methodology could potentially be used to detect a wide range of substrates for which selective oxidase enzymes exist (to generate H(2)O(2)), with the high throughput of simple microtiter plate detection scheme.

Keywords: Al(III)-porphyrin; Fluoride optical sensor; Glucose determination; Glucose oxidase; (GOD); Horseradish peroxidase (HRP).

Figure 1

Bienzymatic pathway, fluoride optical-membrane response mechanism, and a picture of the U-shaped 96-well microtiter plate with the optical membranes.

Figure 2

Optical film responses to fluoride at different pH's: pH 3 (■), pH 5.5 (▲), and pH 6 (●). Data points represent average ±s.d. of n=3 wells for each concentration tested.

Figure 3

Optical H₂O₂ calibration curve. Data points represent average ±s.d. for n=3 wells containing the same concentration of hydrogen peroxide.

Figure 4

Glucose calibration curves performed with external (■) and internal (▲) sample/enzymes/substrate incubation. Data points represent average ± s.d. of n=3 wells for each concentration tested.