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. 2024 Feb 27;14(10):6972-6984.
doi: 10.1039/d3ra07606g. eCollection 2024 Feb 21.

Ammonium release in synthetic and human urine by a urease immobilized nanoconstruct

Manab Diasi  1 , Rinki Singh  1 , Amarjyoti Das Mahapatra  1 , Renuka L  1 , Hitarth Patel  1 , Hasit Ganatra  2 , Bhaskar Datta  1   3
Affiliations

Ammonium release in synthetic and human urine by a urease immobilized nanoconstruct

Manab Diasi et al. RSC Adv. .

Abstract

In this work, we have studied the ability of urease immobilized on glutaraldehyde crosslinked chitosan coated magnetic iron oxide nanoparticles (Urease/GA/CS/MIONPs), for the hitherto unreported comparative hydrolysis of urea in synthetic (SUr) and real human urine (HUr). The prepared Urease/GA/CS/MIONPs were characterized by a combination of Fourier transform infrared spectroscopy (FTIR), field emission-scanning-electron-microscopy (FESEM), energy dispersive X-ray spectroscopy (EDX) and dynamic light scattering (DLS). The nanoconstructs display the highest ammonium ion liberation post-urea hydrolysis in 1/20 or 1/24-fold dilutions of SUr and HUr, respectively. The optimum activity of immobilized urease is observed at pH 7, and the nanoconstructs facilitate efficient urea-hydrolysis till at least 45 °C. Kinetic analysis of the immobilized urease shows km and vmax of 14.81 mM, 12.36 mM, and 18.55 μM min-1 and 10.10 μM min-1, towards SUr and HUr, respectively. The magnetization of the immobilized urease is suitable for reuse across multiple cycles of urea hydrolysis in SUr and HUr. The robust performance of Urease/GA/CS/MIONPs in SUr and HUr is promising for generating ammonium as a useable source of nitrogen from human urine, and underscores the suitability of SUr as a urine mimic for such interventions.

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Conflict of interest statement

There are no conflicts of interest to declare.

Figures

Fig. 1
Fig. 1. Schematic depicting the synthesis of (a) magnetic iron-oxide nanoparticles (MIONPs), (b) glutaraldehyde crosslinked chitosan-modified magnetic iron-oxide nanoparticles (GA/CS/MIONPs), and (c) urease-immobilized glutaraldehyde crosslinked chitosan-modified magnetic iron-oxide nanoparticles (Urease/GA/CS/MIONPs). The representations of the constructs and intermediates are not proportional in scale.
Fig. 2
Fig. 2. ATR-FTIR spectra of (a) MIONPs, (b) CS/MNIOPs, (c) GA/CS/MIONPs and (d) Urease/GA/CS/MIONPs.
Fig. 3
Fig. 3. SEM images of (a) MIONPs, (b) GA/CS/MIONPs and (c) Urease/GA/CS/MIONPs.
Fig. 4
Fig. 4. X-ray diffraction patterns of (a) synthesized MIONPs nanoparticles, (b) chitosan coated iron oxide nanoparticles (CS/MIONPS) and (c) glutaraldehyde crosslinked GA/CS/MIONPs.
Fig. 5
Fig. 5. Plot of magnetization (M) as a function of applied magnetic field (H) for MIONPs nanoparticles and the Urease/GA/CS/MIONPs, at room temperature.
Fig. 6
Fig. 6. Effect of dilution on the monitoring of ammonium ions at 25 °C, (a) in synthetic urine and (b) in human urine, without the application of urease nanoconstruct treatment. The Berthelot reaction is performed in DI water. The standard error of measurement was in the range of 1–3%.
Fig. 7
Fig. 7. Effect of dilution on urea hydrolysis and ammonium ion monitoring following treatment with urease nanoconstruct at 25 °C, (a) in synthetic urine (SUr), and (b) in real fresh human urine (HUr). The Berthelot reaction is conducted in deionized water. The Berthelot reaction is performed in DI water. The standard error of measurement was in the range of 1–8%.
Fig. 8
Fig. 8. Effect of pH on urea hydrolysis and ammonium ion monitoring, (a) in synthetic urine (SUr), and (b) in real fresh human urine (HUr), following treatment with urease nanoconstruct. Both SUr and HUr are diluted at a ratio of 1/20 in PBS solution, with pH ranging from 3 to 10 at a temperature of 25 °C. The Berthelot reaction is performed in the PBS buffer solution.
Fig. 9
Fig. 9. Effect of temperature on urea hydrolysis and ammonium ion monitoring in both human urine (HUr) and synthetic urine (SUr) following treatment with urease nanoconstruct. Both SUr and HUr are diluted at a ratio of 1/20. The Berthelot reaction is carried out in DI water. The standard error of measurement was in the range of 1–10%.
Fig. 10
Fig. 10. Evaluation of urease nanoconstruct and ammonium ion monitoring over six cycles of application in both human urine (HUr) and synthetic urine (SUr). Both SUr and HUr are diluted at a ratio of 1/20. The Berthelot reaction is performed in DI water. The standard error of measurement was in the range of 1–10%.
Fig. 11
Fig. 11. Urease–urea reaction kinetic plots (a), (b) at different time intervals for several concentrations of urea, (c) and (d) Lineweaver–Burk plot of urease immobilized GA/CS/MIONPs in synthetic urine (SUr) and real fresh human urine (HUr).
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