Nanoporous Anodic Alumina As a Three-dimensional Nanostructured material for the Remote Optical Sensing of Urea
Authors: Tabrizi Mahmoud Amouzadeh , Ferré-Borrull Josep , Marsal Lluis F. 
Published: May 2020

Millions of people in the world suffer from some form of kidney disease [1]. Due to reduce the economic losses caused by this disease and also to improve the public health, the fabrication of a device to diagnose kidney disease is important for every government. Up to now, several biosensors have been reported for the determination of urea [2,3]. However, no of them is remote biosensor. The most advantage of the remote sensor is, analyzing the real samples without getting any fungal/virus/bacterial infection that can be in it. Infection. The interferometric reflectance spectroscopy (IRS) is one of the remote sensing devices. IRS is a physical method based on the interference of white light at porous thin substrates, which is used to investigate molecular interaction. This investigation is detected by the charge-coupled device (CCD) detector. Macroporous silicon (MPS) and nanoporous anodic alumina (NAA) are two of the interesting porous thin substrates for the fabrication of biosensor based on IRS [4-6]. However, NAA has become gradually influential in the IRS biosensors due to its high surface area, biocompatibility, easy functional ability, versatile, durable and low-cost substrate.
Herein, we fabricated a remote optical biosensor for the determination of urea. For this purpose first, the self-orderednanoporous anodic alumina (NAA) was fabricated by electrochemical anodization of aluminum substrates in a two-step anodization process [7, 8]. The morphological characterization of the NAA was examined by SEM. The surface of NAA has a hexagonal multi-pore structure. The average diameter pore size of NAA was 41 nm. Then, the NAA pore walls were functionalized with 3-aminopropyl tri-methoxy silane (NAA-NH2). After that, the urease enzyme was immobilized to the pore walls of NAA-NH2 by the use of glutaraldehyde as cross-linker. After that, fluorescein 5(6)-isothiocyanate (FLITC) as the pH-sensitive optical probe was attached to urease enzyme to fabricate a novel urea and trypsin biosensor. The operation principle of the proposed biosensor is based on a change in the pH of the solution during the reaction of urease and urea and then change in the absorption properties of the FLITC. The reaction of urease with urea lets to increase the pH of its environment because of producing ammonia, which is basic. So, the absorption property of FLITC that is a pH-sensitive opt-probe will increase by increasing the urea in solution due to the increase of pH solution during urease/urea reaction. Consequently, the residence of light intensity that will be detected by the CCD detector in the presence of urea is less than in the absence of it. The proposed biosensor exhibited a good response to the concentration of urea in the range of 0.12 to 3.0 mM. The limit of detection (LOD) for urea was 0.06 mM (Fig.1).
To examine its applicability in real sample analysis, the urease biosensor was used for the determination of urea in normal human urine with the standard addition method. Briefly, 1.0 mL of the filtered urine solution added to 9.0 mL of NaAC solution (0.1 M, pH 6.4) and then the solution was pumped into the analytical cell to be analyzed by NAA-NH-GLA-urease-FLITC. The concentration of urea was determined to be 192 mM. The recovery of the analysis was 92 %, considering the value determined by the fluorescent-based sensor [9].
The NAA-NH-GLA-urease-FLITC exhibited good reproducibility in the detection of urea. The effect interfering compounds were also studied on the response of biosensor. No interference was observed with 0.1 mM of L-cysteine, dopamine, glucose, and nicotinamide adenine dinucleotide and common cations and anions (0.1 mM of Na+, Al+3, K+, Cl-, NO3
-, SO4
-2).
The proposed biosensor exhibited good selectivity, linear range responsibility, and stability.
References
(1) https://www.worldkidneyday.org/2019-campaign/2019-wkd-theme/.
(2) Mishra, G.K., Mishra, R.K., Bhand, S., 2010. Biosens Bioelectron.26, 1560-1564.
(3) Parashar, U.K., Nirala, N.R., Upadhyay, C., Saxena, P.S., Srivastava, A., 2015. Appl Biochem Biotechnol. 176, 480-492.
(4) Amouzadeh Tabrizi, M., Ferré-Borrull, J, Marsal, L. F., 2019. Biosens Bioelectron 137, 279-286.
(5) Amouzadeh Tabrizi, M., Ferré-Borrull, J, Marsal, L. F., 2020. Biosens Bioelectron 137, 111828.
(6) Ferré-Borrull, J, Pallarès, J, Macías, G, Marsal, L. F., 2014 Materials 7, 5225-5253.
(7) Santos, A., Vojkuvka, L., Alba, M., Balderrama, V. S., Ferré‐Borrull, J, Pallarès, J., Marsal, L.F., Physica Status Solidi (a) 209, 2045-2048.
(8) Santos, A., Macías, G., Ferré-Borrull, J., Pallarès, J., Marsal, L.F., 2012. Appl. Mater. Interfaces 4, 3584-3588.
(9) Deng, H.-H., Li, K.-L., Zhuang, Q.-Q., Peng, H.-P., Zhuang, Q.-Q., Liu, A.-L., Xia, X.-H., Chen, W., 2018. Nanoscale 10, 6467-6473.

Figure 1