A large response range reflectometric urea biosensor made from silica-gel nanoparticles

Abstract

A new silica-gel nanospheres (SiO2NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO2NPs were basically synthesized based on sol-gel chemistry using a modified Stober method. The SiO2NPs surfaces were modified with amine (-NH2) functional groups for urease immobilization in the presence of glutaric acid (GA) cross-linker. The chromoionophore pH-sensitive dye ETH 5294 was physically adsorbed on the functionalized SiO2NPs as pH transducer. The immobilized urease determined urea concentration reflectometrically based on the colour change of the immobilized chromoionophore as a result of the enzymatic hydrolysis of urea. The pH changes on the biosensor due to the catalytic enzyme reaction of immobilized urease were found to correlate with the urea concentrations over a linear response range of 50-500 mM (R2 = 0.96) with a detection limit of 10 mM urea. The biosensor response time was 9 min with reproducibility of less than 10% relative standard deviation (RSD). This optical urea biosensor did not show interferences by Na+, K+, Mg2+ and NH4+ ions. The biosensor performance has been validated using urine samples in comparison with a non-enzymatic method based on the use of p-dimethylaminobenzaldehyde (DMAB) reagent and demonstrated a good correlation between the two different methods (R2 = 0.996 and regression slope of 1.0307). The SiO2NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors.

Figure 1.

Schematic illustration of the SiO₂NPs-based optical urea biosensor.

Figure 2.

hydrolysis and condensation of TEOS in the Stober method.

Figure 3.

SEM images of SiO₂NPs for (a) t1 and (b) t2 compositions.

Figure 4.

The FTIR spectra for SiO₂NPs (t2 composition), SiO₂NPs-NH₂, SiO₂NPs-NH₂-GA and SiO₂NPs-NH₂-GA-urease.

Figure 5.

Schematic illustration for SiO₂NPs biochemical surface modification.

Figure 6.

The XRD spectra for (a) SiO₂NPs (t2 composition); (b) SiO₂NPs-NH₂ and (c) SiO₂NPs-NH₂-GA.

Figure 7.

Urease incubation period versus percentage of immobilized urease (n = 3).

Figure 8.

Effect of buffer concentration on urea biosensor response at 650 nm using 300 mM urea at pH 7 (n = 3).

Figure 9.

Reflectance spectra and calibration curve of urea biosensor obtained in the urea concentration range of 50–500 mM at 0.05 M phosphate buffer (pH 7) (n = 6).

Figure 10.

Response time of the biosensor towards 500 mM urea in 50 mM phosphate buffer at pH 7 (n = 3).

Figure 11.

The correlation between biosensor and DMAB standard method for urea determination in urine samples (n = 3).

Figure 12.

The optical urea biosensor response over 55 days experimental period using 600 mM urea in 50 mM phosphate buffer (pH 7) (n = 3).

Figure 13.

The maximum response changes of the biosensor towards various cations from 10⁻⁵ to 10⁻¹ M and urea at the wavelength of 650 nm (n = 3).