Development of a ratiometric fluorescent urea biosensor based on the urease immobilized onto the oxazine 170 perchlorate-ethyl cellulose membrane

• Fabrication of a urea-sensing membrane based on the O17-EC membrane.
• Immobilization of the enzyme urease onto the O17-EC membrane.
• Detection of urea with the ratiometric fluorescence method.
• Application of the urea sensor to the determination of urea in urine solutions.

In this work, the oxazine 170 perchlorate (O17)-ethyl cellulose (EC) membrane was successfully applied in the fabrication of a urea-sensing membrane. The urea-sensing membrane was a double layer consisting of the O17-EC membrane and a layer of the enzyme urease entrapped into EC matrix. The sensing principle of urea was based on the hydrolysis reaction of urea under the catalysis of the urease to produce ammonia in water and also on the binding of ammonia with the dye O17 to create the shift in the emission wavelength from λem=630 nm to λem=565 nm. The data collected from the ratio of the fluorescence intensities at λem=630 nm and λem=565 nm was proportional to urea concentration. The urea-sensing membrane with the ratiometric method was used to measure the concentrations of urea in the range of 0.01–0.1 M with a limit of detection (LOD) of 0.027 mM and 0.1–1.0 M with LOD of 0.224 mM. It showed fast response time, high reversibility and long-term stability in this concentration range. The recovery percentage of urea concentrations of the urea-sensing membrane for two kinds of biological urine solutions (BU1, BU2) was around 85–118%. The measured results were in good agreement with standard urea concentrations in the range of 0.06 M to 1.0 M.

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