Immobilization Of Urease Enzyme Research Articles

Development and Characterization of a Paper-Based Sensor for Colorimetric Urea Detection in Artificial Urine

Toxic nitrogen, which is formed as a result of the breakdown of proteins, is converted into nontoxic urea by the enzymes in the "urea cycle". While a small part of the produced urea is eliminated with sweat, a large part is removed from the body by the kidneys with urine. Therefore, urea determination in blood and urine provides important information about kidney functions. In this study, a colorimetric paper based sensor was developed and characterized for use in urea detection. Basically, urea measurement was carried out by immobilization of urease enzyme on pH sensitive papers. The determination of urea was made by observing the changes in pH caused by ammonia with basic properties, which is formed as a result of the destruction of urea by urease. In the presence of different concentrations of urea, the colorimetric changes on the sensor were visualized with a camera and analyzed with the ImageJ program in the computer environment. In the first stage, a calibration curve showing the relationship between urea concentration and color intensity was obtained, and then the detection limit and sensitivity of the system were determined from this curve. Finally, the selectivity of the sensor was demonstrated by testing the different molecules involved.

Immobilization of urease enzyme on chitosan/polyvinyl alcohol electrospun nanofibers

Electrospun nanofibers have gained much attention for enzyme immobilization due to their high surface-to-volume ratio. In this study, urease was immobilized on chitosan/poly(vinyl alcohol) (PVA) nanofibers by both adsorption and crosslinking methods. In order to obtain nanofibers with more desirable properties, solutions with different ratios of chitosan and PVA were electrospun and crosslinked using glutaraldehyde. Comparing SEM images of the nanofibers, before and after immersing them in phosphate buffer, it was shown that higher chitosan content leads to more stable fibers. So, the solution with the chitosan to PVA ratio of 40:60 was used for enzyme immobilization. Then, the effects of initial protein concentration, temperature, incubation time, and method of immobilization were investigated to reach the highest enzyme activity. Under similar immobilization conditions, covalently immobilized urease showed higher activity, compared to uncrosslinked immobilized enzyme. Besides, it retained 30% of its initial activity after 10 times usage. So, this method was chosen for further investigation. Not only the activity of the immobilized enzyme was much higher than the free enzyme in a wide range of pH and temperature, but also stability of the immobilized enzyme was improved. Immobilized urease was then used to remove thiourea which is a toxic compound. Findings indicated 60% hydrolysis of initial thiourea in 12 h. In conclusion, the findings showed that chitosan/PVA nanofibers are suitable candidates for the immobilization of urease.

Urease-Immobilized PEI Cryogels for the Enzymatic Hydrolysis of Urea and Carbon Dioxide Uptake

Here, the immobilization of the urease enzyme within polyethyleneimine (PEI) cryogels (Urease@PEI cryogel) is reported during the synthesis via the entrapment method. The immobilization yield of the enzymes within PEI cryogels was determined as >85%, and the activity% of the prepared Urease@PEI cryogel systems was calculated as >90% with respect to equal amounts of native urease enzyme. The working stability of Urease@PEI cryogel systems was observed to be better than native urease enzymes in the pH range of 5.0–9.0 and the temperature range of 15–45 °C. Furthermore, the immobilization of urease enzyme within PEI cryogels afforded great performance for use in continuous systems with more than 70% activity. Moreover, Urease@PEI cryogel systems preserved more than 50% activity even after 20 consecutive uses and showed superior storage stability, e.g., storage at 25 °C for 10 days resulted in >50% activity. Most importantly, the possible utilization of Urease@PEI cryogel systems for CO2 adsorption, which is the byproduct of the enzymatic reaction of urea, from the reaction media was investigated. The CO2 capture capability was calculated as 0.42, 0.27, and 0.11 cm3/g at 1 bar and 25 °C for Urease@PEI-1, Urease@PEI-2, and Urease@PEI-3 cryogels, respectively.

Characterization of PVA-Enzyme Coated Indicator Electrodes GA coated again with PVC-KTpClPB-o-NPOE SEM-EDS, FTIR and XRD analysis

This study aims to characterize the tungsten-urea analyte indicator electrode. The method used is biosensor potentiometry with urease enzyme immobilization technique. This indicator electrode was coated with PVA-enzyme coated with glutaraldehyde (GA) 2.9% coated with PVC-KTpClPB- o-NPOE with o-NPOE variation of 61% and 66%. Characterization of coated indicator electrodes using SEM-EDS, FTIR and XRD analysis. A1-4 61% indicator electrode sample coated PVA-enzyme 1x coated with glutaraldehyde (GA) 2.9% 1x coated PVC-KTpClPB- o-NPOE 1x, with o-NPOE 61%. A3-4 61% indicator electrode sample coated PVA-enzyme 3x coated with glutaraldehyde (GA) 2.9% 1x coated PVC-KTpClPB- o-NPOE 1x, with o-NPOE 61%. Likewise, the reasoning of samples A1-4 66% and A3-4 66%. There are four indicator electrodes made with the notation A1-4 61%, A1-4 66%, A3-4 61% and A3-4 66%. The best results were obtained at the indicator electrode sample A1-4 61%, contributing to the urea sensor of the potentiometer cell

Solution Blowing Spinning Technology towards Green Development of Urea Sensor Nanofibers Immobilized with Hydrazone Probe.

Cellulose has been one of the most widespread materials due to its renewability, excellent mechanical properties, biodegradability, high absorption ability, biocompatibility and cheapness. Novel, simple and green colorimetric fibrous film sensor was developed by immobilization of urease enzyme (U) and tricyanofuran hydrazone (TCFH) molecular probe onto cellulose nanofibers (CNF). Cellulose acetate nanofibers (CANF) were firstly prepared from cellulose acetate using the simple, green and low cost solution blowing spinning technology. The produced CANF was exposed to deacetylation to introduce CNF, which was then treated with a mixture of TCFH and urease enzyme to introduce CNF-TCFH-U nanofibrous biosensor. CNF were reinforced with tricyanofuran hyrazone molecular probe and urease enzyme was encapsulated into calcium alginate biopolymer to establish a biocomposite film. This CNF-TCFH-U naked-eye sensor can be applied as a disposable urea detector. CNF demonstrated a large surface area and was utilized as a carrier for TCFH, which is the spectroscopic probe and urease is a catalyst. The biochromic CNF-TCFH-U nanofibrous biosensor responds to an aqueous medium of urea via a visible color signal changing from off-white to dark pink. The morphology of the generated CNF and CNF-TCFH-U nanofibrous films were characterized by different analytical tools, including energy-dispersive X-ray patterns (EDX), polarizing optical microscope (POM), Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM). SEM images of CNF-TCFH-U nanofibers demonstrated diameters between 800 nm and 2.5 μm forming a nonwoven fabric with a homogeneous distribution of TCFH/urease-containing calcium alginate nanoparticles on the surface of CNF. The morphology of the cross-linked calcium alginate nanoparticles was also explored using transmission electron microscopy (TEM) to indicate an average diameter of 56–66 nm. The photophysical performance of the prepared CNF-TCFH-U was also studied by CIE Lab coloration parameters. The nanofibrous film biosensor displayed a relatively rapid response time (5–10 min) and a limit of detection as low as 200 ppm and as high as 1400 ppm. Tricyanofuran hydrazone is a pH-responsive disperse dye comprising a hydrazone detection group. Determination of urea occurs through a proton transfer from the hydrazone group to the generated ammonia from the reaction of urea with urease.

Smartphone based optical biosensor for the detection of urea in saliva

In the present study, we have developed a smartphone based handheld optical biosensor for determination of urea in saliva. A simple strategy was adopted by immobilization of urease enzyme along with a pH indicator on a filter paper based strip. The strip changed color upon the reaction with urea present in saliva and the color change can be estimated using our smartphone based application based on RGB profiling. Calibration of the biosensor was carried out using spiked saliva samples and an exponentially decreasing calibration curve has been obtained for green pixel intensity in the broad range (10–1000 mgdL−1) with a linear detection range of 10–260 mgdL−1 and a response time of 20 s. The sensitivity reported for the biosensor in the clinically significant range was −0.005 average pixels sec−1/mgdL−1 with a LOD of 10.4 mgdL−1. Studies carried out on spiked saliva samples showed a good correlation between salivary urea estimated using our biosensor against phenol-hypochlorite based spectroscopic procedure. Development of a smartphone based biosensor for urea estimation eliminates the need for procuring a dedicated instrument as well as trained technician for daily monitoring and saves time as compared to traditional laboratory methods of analysis.

Design of amperometric urea biosensor based on self-assembled monolayer of cystamine/PAMAM-grafted MWCNT/Urease

In this work, we report novel amperometric urea biosensor based on immobilization of Urease enzyme onto self-assembled polyamidoamine grafted multiwalled carbon nanotube (MWCNT-PAMAM) dendrimers (from G1, first generation up to G5) on Au electrode. Bio-sensing electrode’s performance parameters such as optimum pH, temperature, and applied potential together with reproducibility were investigated. The electrode based on fifth generation of MWCNT-PAMAM and urease enzyme showed excellent performance in urea detection at an applied potential of 0.45V with short response time of 3s, wide linear range of 1–20mM, detection limit of 0.4mM, and sensitivity of 6.6nA/mM. No significant response was detected in the presence of possible interferon molecules. Non-diluted human blood plasma has been used for real sample application where electrode showed excellent performance in urea detection.

Immobilization of urease enzyme on nanoceria modifies secondary and tertiary protein structures

Abstract Urease catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. The increase in pH from the urease reaction causes a broad range of deleterious effects. Nanoceria (cerium oxide) possesses unique chemical properties under a redox reaction. This study investigated the synthesis of nanoceria via a hydrothermal method and determined its interaction with urease enzyme. Transmission electron microscopy results showed a cubic-figured nanoceria with a size of ~15 nm. Urease was immobilized on nanoceria through adsorption. The maximum velocity (V max) and Michaelis constant (K m) of the free urease and urease immobilized on nanoceria decreased after interaction with nanoceria, and the Lineweaver-Burk plot showed an uncompetitive inhibition. The thermodynamic study of the adsorption process showed an endothermic reaction. The interaction changed the secondary and tertiary structures of urease as demonstrated by the circular dichroism study (the decrease in both α- and β-structure percentages). The fluorescence study revealed a change in the tertiary structure. The FTIR for the nanoceria—urease complex showed no changes in the covalent bonds, which indicated the involvement of physical forces in the interaction between urease and nanoceria.

Potentiometric detection of chemical species by spin-assisted assembly of vanadium pentoxide nanorods

Vanadium pentoxide nanorods (V2O5-nanorods) and poly(allylamine hydrochloride) (PAH) were assembled onto gold-coated substrates via spin-assisted assembly technique and used as a chemically sensitive electrodes. PAH/V2O5-nanorods detected H+ ions (pH) with sensitivity between 52–61mV/pH (close to Nernstian theoretical value). As a proof-of-concept, a urea biosensor has been developed, upon immobilization of urease enzyme on PAH/V2O5-nanorods electrodes. The biosensor could detect urea in a 0.05–5mM dynamic range. The spin-assisted assembly technique enables the combination of different materials in a simple way and offers advantages for the construction of functional electrodes.

Cobalt oxide magnetic nanoparticles–chitosan nanocomposite based electrochemical urea biosensor

In this study, a potentiometric urea biosensor has been fabricated on glass filter paper through the immobilization of urease enzyme onto chitosan/cobalt oxide (CS/Co3O4) nanocomposite. A copper wire with diameter of 500 µm is attached with nanoparticles to extract the voltage output signal. The shape and dimensions of Co3O4 magnetic nanoparticles are investigated by scanning electron microscopy and the average diameter is approximately 80–100 nm. Structural quality of Co3O4 nanoparticles is confirmed from X-ray powder diffraction measurements, while the Raman spectroscopy has been used to understand the chemical bonding between different atoms. The magnetic measurement has confirmed that Co3O4 nanoparticles show ferromagnetic behavior, which could be attributed to the uncompensated surface spins and/or finite size effects. The ferromagnetic order of Co3O4 nanoparticles is raised with increasing the decomposition temperature. A physical adsorption method is adopted to immobilize the surface of CS/Co3O4 nanocomposite. Potentiometric sensitivity curve has been measured over the concentration range between 1 × 10−4 and 8 × 10−2 M of urea electrolyte solution revealing that the fabricated biosensor holds good sensing ability with a linear slope curve of ~45 mV/decade. In addition, the presented biosensor shows good reusability, selectivity, reproducibility and resistance against interferers along with the stable output response of ~12 s.