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In this study, a more efficient, convenient, and cost-effective way was proposed to design and fabricate a polydimethylsiloxane (PDMS) microfluidic device using 3D printing and transfer techniques. Typically, sensor measurements can only be conducted by immersing the sensor in a solution under static conditions. However, in this study, a newly designed microfluidic device combined with a microfluidic system enables dynamic measurements with sensors. By controlling the flow and temperature of the test liquid, the physiological environment of the human body under various conditions can be simulated, resulting in more accurate and realistic data. During the dynamic condition, an average sensitivity of 6.396 mV/(mg/dL) and linearity of 0.999 were obtained at a flow rate of $75.0 \mu \text{L}$ /min. When the temperature was at 37°C, the biosensor achieved an average sensitivity of 4.381 mV/(mg/dL) and a linearity of 0.999. The dynamic response time was 7 seconds, and the drift rate was 1.47 mV/hr. The experimental results obtained under dynamic conditions showed a significant improvement in the sensing characteristics of the urea biosensor compared with the results obtained under static conditions.

This study proposes a power management circuit integrated with a readout circuit for measuring urea biosensors. The proposed power management circuit replaces the bulky power supply traditionally used in measurement systems. It operates with only a single 3V button cell for voltage reduction and stabilization. We have designed and manufactured a printed circuit board (PCB) that integrates passive components and power management readout circuit chip for measuring a urea biosensor. A RuO2 thin film was prepared on a flexible printed circuit board (FPCB) through radio frequency (RF) sputtering. To fabricate the urea sensor, we used 3-Aminopropyltriethoxysilane ( $\gamma $ -APTES), glutaraldehyde, and urease. The average sensitivity of the urea sensor was evaluated to be 3.121 (mV/(mg/dL)), with a linearity of 0.994 and a reproducibility of 2.59 %. These results confirm the stability and reliability of the sensor architecture.

Present study is specially designed for the recent advances in biosensors to detect and quantify urea concentration. Urea (carbamide) is an organic compound made up of the carbonyl (C=O) functional group with two −NH2 groups having chemical formula CO (NH2)2. In nature, urea is found everywhere as the result of various processes, and in the human body, urea is an end product of nitrogen metabolism. An excessive concentration of urea in the human body is responsible for different critical diseases such as indigestion, acidity, ulcers, cancer, malfunctioning of kidneys, renal failure, urinary tract obstruction, dehydration, shock, burns, gastrointestinal bleeding, and so on. Moreover, below the normal level may cause hepatic failure, nephritic syndrome, cachexia, and so on. As well as in various fields such as fishery, dairy, food preservation, agriculture, and so on, urea is normally found and its detection is necessary. In urea biosensors, enzyme urease (Urs) is used as a bioreceptor element and retains its long last activity is the critical issue in front of the researcher. During recent decades, different nanoparticles (zinc oxide, nickel oxide, iron oxide, titanium dioxide, tin(IV) oxide, etc.), conducting polymer (polyaniline, polypyrrole, etc.), conducting polymer–nanoparticles composites, carbon materials (carbon nanotubes, graphene oxide, reduced graphene oxide graphene), and so on are used in urea biosensors. The main emphasis of the present study is to provide cumulative and comprehensive information about the sensing parameters of urea biosensors based on the materials used for enzyme immobilization. Besides this special task, this review provides a fruitful discussion on the basics of biosensors briefly for new and upcoming researchers. Thus, the present study may act as a gift for a large audience that come from different fields and are working in biosensors research. [ABSTRACT FROM AUTHOR]

From our other works, we have developed urea biosensor based on graphene oxide/nickel oxide sensing film modified either by Au nanoparticles or ${\gamma }$ -Fe2O3 nanoparticles. In this study, we have further developed back-end calibration circuit to reduce the drift effect and hysteresis effect of the two types of the urea biosensors. The back-end calibration circuit is composed of non-inverting amplifiers, error amplifiers, P-MOSFET transmission transistor, feedback networks, output voltage capacitors and resistor dividers. After applying the back-end calibration circuit, the drift rate of urea biosensor modified by Au NPs is reduced from 3.06 mV/hr to 0.28 mV/hr, which is 90.85% reduction. The drift rate of urea biosensor modified by ${\gamma }$ -Fe2O3 NPs is reduced from 3.92 mV/hr to 0.57 mV/hr, which is 85.46% reduction. Through the back-end calibration circuit to reduce the hysteresis effect, the hysteresis voltage for the forward cycle and reverse cycle of the urea biosensor modified by Au NPs are reduced by 26% and 30%, respectively. The hysteresis voltage for the forward cycle and reverse cycle of the urea biosensor modified by ${\gamma }$ -Fe2O3 NPs are reduced by 23% and 28%, respectively.

In this work, we used radio frequency (RF) system and screen printing technology to fabricate urea biosensor based on urease-maghemite nanoparticles (γ-Fe2O3 NPs)/graphene oxide (GO)/nickel oxide (NiO) sensing membrane on silver conductive wire printed polyethylene terephthalate (PET) substrate. The V – T measurement system was used to investigate the sensing characteristics of urea biosensors based on GO/NiO sensing membrane modified by γ-Fe2O3 NPs. The sensing membrane was modified by adding different contents of γ-Fe2O3 NPs. The concentration of γ-Fe2O3 NPs was 1 mg/mL. When the volume ratio of urease/γ-Fe2O3 NPs was 1/1, it had the best sensitivity. The average sensitivity, the linearity, and the limit of detection of (LOD) of the best urea biosensor in this work were 52.36 mV/decade ± 0.22 mV/decade, 0.996, and $7.91~μM, respectively. The effect of temperature on the urea biosensor was investigated from 25 °C to 65 °C. The stability of urea biosensors was evaluated by the effect of hysteresis and long-term storage stability. After 29 days, the average sensitivity remained 80.62% of the initial value. Compared with other urea biosensors, our urea biosensor based on urease-γ-Fe2O3 NPs/GO/NiO sensing membrane has better average sensitivity. [ABSTRACT FROM AUTHOR]

Prussian blue (PB) is an electrochromic material, which can be used as a signal transducer in the formation of optical urea biosensors. The previous researches in electrochromic properties of PB demonstrated the optical PB response to ammonium ions, which occurs when ammonium ions are interacting with PB layer at a constant 0.2 V vs Ag|AgCl|KClsat potential. In this work PB optical dependence on ammonium ions concentration was applied in the formation of electrochromic urea biosensor. Biosensor was formed by modifying the optically transparent indium tin oxide (ITO) coated glass electrode (glass/ITO) with Prussian blue layer and immobilizing urease (glass/ITO/PB‐urease). Calibration curve showed the linear dependency (R2=0.995) between the change of maximal absorbance (ΔA) and urea concentration in concentration range varying from 3 mM to 30 mM. The highest sensitivity (4 ΔA M−1) of glass/ITO/PB‐urease biosensor is in the concentration range from 7 mM to 30 mM. It was determined that working principle of the glass/ITO/PB‐urease biosensor is not related to pH changes occurring during enzymatic hydrolysis of urea. [ABSTRACT FROM AUTHOR]

Nickel oxide nanoparticle (NiO−NP) and polypyrrole (PPy) composite were deposited on a Pt electrode for fabrication of a urea biosensor. To develop the sensor, a thin film of PPy−NiO composite was deposited on a Pt substrate that serves as a matrix for the immobilization of enzyme. Urease was immobilized on the surface of Pt/PPy−NiO by a physical adsorption. The response of the fabricated electrode (Pt/PPy−NiO/Urs) towards urea was analyzed by chronoamperometry and cyclic voltammetry (CV) techniques. Electrochemical response of the bio‐electrode was significantly enhanced. This is due to electron transfer between Ni2+ and Ni3+ as the electro‐catalytic group and the reaction between polypyrrole and the urease‐liberated ammonium. The fabricated electrode showed reliable and demonstrated perfectly linear response (0.7–26.7 mM of urea concentration, R2= 0.993), with high sensitivity (0.153 mA mM−1 cm−2), low detection of limit (1.6 μM), long stability (10 weeks), and low response time (∼5 s). The developed biosensor was highly selective and obtained data were repeatable and reproduced using PPy‐NiO composite loaded with immobilized urease as urea biosensors. [ABSTRACT FROM AUTHOR]

Non-covalent complexes of urease/polyethylene glycol (PEG)-aldehyde were synthesized using regular molar ratios of urease and PEG-aldehyde at room temperature. The physical properties of the non-covalent complexes were analyzed in order to investigate the impact of coupling ratio, temperature, pH, storage stability, and thermal stability. Urease activity was analyzed by UV–Vis spectrophotometer at 630 nm. The results showed that the strongest thermal resistance was obtained using nU/nPEG:1/1 (mg/mL) complex within all molar ratios tested. The enzymatic activity of nU/nPEG:1/1 complex doubled the activity of the free enzyme. Therefore, this complex was chosen to be used in the analyses. When coupled with PEG-aldehyde, urease exhibited improved activity between pH 4.0–9.0 and the optimum pH was found to be 7.0. The thermal inactivation results of the complex demonstrated that higher activity remained (40%) when compared with the free enzyme (10%) at 60 °C. The storage stability of the non-covalent complex was 4 weeks which was greater than the storage stability of the free enzyme. A kinetic model was suggested in order to reveal the mechanism of enzymatic conversion. Potentiometric urea biosensor was prepared using two different membranes: carboxylated poly vinyl chloride (PVC) and palmitic acid containing PVC. The potentiometric responses of both sensors were tested against pH and temperature and the best results were obtained at pH 7.0 and 20–30 °C. Also, selectivity of the suggested biosensors toward Na+, Li+ Ca2+, and K+ ions was evaluated and the reproducibility responses of the urea biosensors were measured with acceptable results. [ABSTRACT FROM AUTHOR]

Abstract As a unique strategy, a sensitive urea biosensor media involving dip coated ZnO and Fe 3 O 4 nanocomposite, at the optimized instrumental deposition conditions, on the F-doped SnO 2 conducting glass was designed (Fe 3 O 4 -ZnO/SnO 2 :F) to achieve an excellent platform for urease enzyme (Urs) immobilization (Urs/Fe 3 O 4 -ZnO/SnO 2 :F). The Fe 3 O 4 -ZnO nanocomposite selection principles were based on urea biosensing improvement by Urs immobilization on ZnO nanoparticles by electrostatic adsorption, while Fe 3 O 4 selection criteria were dependent upon giving high electronic density to the biosensor surface as an electrostatic repulsion layer at the biological media for the anionic interferents. Surface morphology characterization of the nanocomposite film by field emission-scanning electron microscopy (FE-SEM) displays nanocomposite film as an operative biosensing area for Urs enzyme immobilization. Step by step monitoring of Urs/Fe 3 O 4 -ZnO/SnO 2 :F biosensor fabrication was done using electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Urs/Fe 3 O 4 -ZnO/SnO 2 :F biosensor was exploited for urea quantification by I-V assessment. The I-V consequences show high sensitivity for urea detection within 5–150 mg dL−1 and limit of detection as 5 mg dL−1. Consequently, the proposed method presented a potential application for producing the disposable sensor with good repeatability. Additionally, fast response of fabricated sensor can usually allow real-time analysis of real samples. Graphical abstract Unlabelled Image Highlights • Unique disposable urea biosensor construction based on composite transducer. • Employing dip coating technique as a novel strategy for composite sensing film fabrication. • Urea biosensing based on I-V assessment due to its sensitivity, rapidity, and repeatability. • Fabricated sensor fast response can usually allow a real-time analysis of real samples. [ABSTRACT FROM AUTHOR]

Numerous studies have addressed the utilization of glutaraldehyde (GA) as a homobifunctional cross-linker. However, its applicability has been impeded due to several issues, including the tendency of GA molecules to undergo polymerization. Herein, a portable urea biosensor was developed for the real-time monitoring of the flow of physiological fluids; this was achieved by using disuccinimidyl cross-linker-based urease immobilization. Urease was immobilized on a porous polytetrafluoroethylene (PTFE) solid support using different disuccinimidyl cross-linkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and bis-N-succinimidyl-(pentaethylene glycol) ester (BS(PEG)5). A urease activity test revealed that DSS exhibited the highest urease immobilizing efficiency, whereas FT-IR analysis confirmed that urease was immobilized on the PTFE membrane via DSS cross-linking. The membrane was inserted in a polydimethylsiloxane (PDMS) fluidic chamber that generated an electrochemical signal in the presence of a flowing fluid containing urea. Urea samples were allowed to flow into the urea biosensor (1.0 mL/min) and the signal was measured using chronoamperometry. The sensitivity of the DSS urea biosensor was the highest of all the trialed biosensors and was found to be superior to the more commonly used GA cross-linker. To simulate real-time monitoring in a human patient, flowing urea-spiked human serum was measured and the effective urease immobilization of the DSS urea biosensor was confirmed. The repeatability and interference of the urea biosensor were suitable for monitoring urea concentrations typically found in human patients.

The fabrication, characterization, and application of a nanostructured zinc oxide/graphene (ZnO/Gr) transducer were described for the determination of urea concentration. For this purpose, thin films of sol–gel‐synthesized ZnO nanostructure and Gr were prepared using the spin coating technique on the conductive fluorinated–tin oxide (FTO) substrate. Appropriate correlations between the spinning parameters and thin film features were established so that an efficient ZnO–Gr thin film was obtained. Field emission scanning electron microscopy (FE‐SEM) images prove thin film pores to be an operative biosensing zone for urease (Urs) enzyme immobilization. Step‐by‐step monitoring of FTO/ZnO‐Gr/Urs biosensor fabrication was achieved using electrochemical methods. The impedimetric results of a fabricated FTO/ZnO‐Gr/Urs biosensor show high sensitivity for urea detection within 5.0–125.0 mg/dL and a limit of detection of 3.36 mg/dL. In our research group in the thin film and nanotechnology laboratory, we are working on the development of semiconductor‐based transducers for enzymatic biosensors and impedimetric assessment for biosensing. This work reports a novel transducer using spin‐coated ZnO–graphene nanostructure thin film on FTO surface and its effects on the improvement of enzymatic urea biosensing. [ABSTRACT FROM AUTHOR]

Spin coated zinc oxide (ZnO)-carbon active porous media on the Fluorine-doped Tin Oxide (FTO) coated glass is presented as an engaged nomination for novel application in enzymatic urea biosensor. Some correlations between the processing parameters spinning speed, spinning duration, volume of solution and ZnO:carbon active ratio and respective thin film characteristics were determined, then uniform ZnO-carbon active the uniform film was achieved at the optimum deposition conditions. FE-SEM was employed to investigate the morphology and hardness of ZnO-carbon active thin film. The FE-SEM image illustrates cavities of a thin film as an efficient transducer area for immobilization of urease enzyme (Urs). Stepwise study of FTO/ZnO-carbon active/Urs biosensor manufacturing was performed by voltammetric and impedimetric techniques. The results revealed a good sensitivity for impedimetric urea retrieval between 8.0-110.0 mg dL-1 with detection limit of 5.4 mg dL-1. [ABSTRACT FROM AUTHOR]

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.

The goal of this study was to reduce the drift effect of RuO2 urea biosensors. A new calibration circuit (NCC) based on the voltage regulation technique with the advantage of having a simple structure was presented. To keep its simplicity, the proposed NCC was composed of a non-inverting amplifier and a voltage calibrating circuit. A ruthenium oxide (RuO2) urea biosensor was fabricated to test the calibrating characteristics of the drift rate of the proposed NCC. The experiment performed in this study was divided into two main stages. For the first stage, a sound RuO2 urea biosensor testing environment was set-up. The RuO2 urea sensing film was immersed in the urea solution for 12 h and the response voltage was measured using the voltage-time (V−T) measurement system and the proposed NCC. The results of the first stage showed that the RuO2 urea biosensor has an average sensitivity of 1.860 mV/(mg/dL) and has a linearity of 0.999 which means that the RuO2 urea biosensor had been well fabricated. The second stage of the experiment verified the proposed NCC’s functions, and the results indicated that the proposed NCC reduced the drift rate of RuO2 urea biosensor to 0.02 mV/hr (98.77% reduction).

Urea analysis is of considerable interest in clinical, agricultural and environmental chemistry. Urea biosensor helps in on-site monitoring of urea. The aim of this study was to evaluate the potential use of nanocellulose reinforced composite film as substrate for urea biosensor. Nanocellulose was prepared from non-spinnable short staple cotton fibres by controlled microbial hydrolysis process. This was used as a substrate to immobilize urease enzyme and, subsequently used to reinforce the starch/polyvinyl alcohol biocomposite film. Nanocellulose acted as both carrier for the enzyme and reinforcing agent in the film. Use of 1.5% nanocellulose increased the tensile strength of the resultant film to the tune of 2.5-fold. About 68% immobilization efficiency of urease onto nanocellulose was observed in the reported process. The biosensor could detect the presence of urea linearly in the range of 10 to 1000 ppm concentration in water with a response time between 30 sec and 1 min. The storage lifetime was 2 months when stored in 4°C with the activity more than 90%. The study revealed that the nanocomposite film could significantly improve the performance and storability of urea biosensor. [ABSTRACT FROM AUTHOR]

Herein, we demonstrate synthesis and application of two-dimensional (2D) rectangular ytterbium oxide (Yb 2 O 3 ) nanodisks via a facile hydrothermal method. The structural, morphological, compositional, crystallinity, and phase properties of as-synthesized nanodisks were carried out using several analytical techniques that showed well defined 2D rectangular nanodisks/sheet like morphologies. The average thickness and edge length of the nanosheet structures were 20 ± 5 nm and 600 ± 50 nm, respectively. To develop urea biosensor, glassy carbon electrodes (GCE) were modified with Yb 2 O 3 nanodisks, followed by urease immobilization and Nafion membrane covering (GCE/Yb 2 O 3 /Urease/Nafion). The fabricated biosensor showed sensitivity of 124.84 μA mM −1 cm −2 , wide linear range of 0.05–19 mM, detection limit down to ~ 2 μM, and fast response time of ~ 3 s. The developed biosensor was also used for the urea detection in water samples through spike-recovery experiments, which illustrates satisfactory recoveries. In addition, the obtained desirable selectivity towards specific interfering species, long-term stability, reproducibility, and repeatability further confirm the potency of as-fabricated urea biosensor. [ABSTRACT FROM AUTHOR]

The urease membrane was immobilized on the surface of indium gallium zinc oxide (IGZO) based on polyethylene terephthalate substrate, which was used to develop an arrayed flexible urea biosensor in this study. In order to improve the properties of arrayed flexible IGZO/Al urea biosensor, graphene oxide (GO) was employed to modify the properties of arrayed flexible IGZO urea biosensor. Compared with the average sensitivity of 1.53 mV/(mg/dl) and linearity of 0.931 for arrayed flexible IGZO/Al urea biosensor, the average sensitivity and linearity of arrayed flexible IGZO urea biosensor modified by GO achieved 3.06 mV/(mg/dl) and 0.970, respectively. As the result, 5 mM pH 7.0 PBS buffer solution was the optimal measurement environment for Urease/IGZO/GO/Al urea biosensor, and which the average sensitivity was 3.65 [mV/(mg/dl)]. Anti-interference testing showed the arrayed flexible IGZO/Al urea biosensor modified by GO was not easily interfered from the other materials. [ABSTRACT FROM PUBLISHER]

A new bioanalytical method for urea determination by using the immobilized urease on the highly luminescent ZnS quantum dots (QDs), which functioned as the pH fluorescent label has been developed. Bioconjugation of ZnS QDs to urease via amide bond was performed to obtain the ZnS QDs-urease bioconjugate. Upon enzymatic hydrolysis of urea by the immobilized urease, a pH change to a more alkaline condition has led to the deprotonation of ZnS QDs, and an increase in the fluorescence intensity can be observed. The fluorescence intensity of the urease conjugated ZnS QDs changed linearly with the urea concentrations between 4 × 10 −9 M and 4 × 10 −3 M (R 2 = 0.992) at pH 6 with a calibration sensitivity of 179.46 intensity/decade. No noticeable influence by the Ca 2+ , Mg 2+ , K + and Na + ions on the response of the fluorescent pH bioprobe. Urea determination in soil sample by using the urea bioprobe was in good agreement with the standard DMAB (p-dimethylaminobenzaldehyde) UV–vis spectrophotometric method. By employing the strategy of ZnS QDs-urease bioconjugation, the stable covalent link between the two substances has appeared to widen the dynamic range and lower the detection limit for urea compared to free enzyme and QDs in solution for bioassay of urea concentration. [ABSTRACT FROM AUTHOR]

A novel method for the rapid modification of fullerene for subsequent enzyme attachment to create a potentiometric biosensor is presented. Urease was immobilized onto the modified fullerene nanomaterial. The modified fullerene-immobilized urease (C60-urease) bioconjugate has been confirmed to catalyze the hydrolysis of urea in solution. The biomaterial was then deposited on a screen-printed electrode containing a non-plasticized poly(n-butyl acrylate) (PnBA) membrane entrapped with a hydrogen ionophore. This pH-selective membrane is intended to function as a potentiometric urea biosensor with the deposition of C60-urease on the PnBA membrane. Various parameters for fullerene modification and urease immobilization were investigated. The optimal pH and concentration of the phosphate buffer for the urea biosensor were 7.0 and 0.5 mM, respectively. The linear response range of the biosensor was from 2.31 × 10−3 M to 8.28 × 10−5 M. The biosensor’s sensitivity was 59.67 ± 0.91 mV/decade, which is close to the theoretical value. Common cations such as Na+, K+, Ca2+, Mg2+ and NH4+ showed no obvious interference with the urea biosensor’s response. The use of a fullerene-urease bio-conjugate and an acrylic membrane with good adhesion prevented the leaching of urease enzyme and thus increased the stability of the urea biosensor for up to 140 days.

In this study, a portable urea sensor that monitors the urea concentration in flow conditions was fabricated. We propose an electrochemical sensor that continually measures the urea concentration of samples flowing through it at a constant flow rate in real time. For the electrochemical sensing, a porous silk fibroin membrane with immobilized urease was mounted in a polydimethylsiloxane (PDMS) sensor housing. The fabricated urea sensor elicited linear current–concentration characteristics in the clinically significant concentration range (0.1–20 mM) based on peritoneal dialysis. The sensor maintained the linear current–concentration characteristics during operation in flow conditions.

Vanadium pentoxide nanorods (V 2 O 5 -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/V 2 O 5 -nanorods detected H + ions (pH) with sensitivity between 52–61 mV/pH (close to Nernstian theoretical value). As a proof-of-concept, a urea biosensor has been developed, upon immobilization of urease enzyme on PAH/V 2 O 5 -nanorods electrodes. The biosensor could detect urea in a 0.05–5 mM 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. [ABSTRACT FROM AUTHOR]

The sensing element of biosensor is prepared by the immobilization of urease onto nano-structured polyaniline (PANi)-Nafion ® (NSPN)/Au/Al 2 O 3 synthesized by the chronopotentiometric technique. The sensitivity of lead ion at an amperometric urease based biosensor is obtained to be 743.5 µA ppm −1 for the linear dynamic range of 0.1–1.0 ppm Pb 2+ . The irreversible inhibiting kinetic models of the mercury and lead ions at the amperometric biosensors are proposed. The values of K d (dissociation equilibrium constant of adsorbed type of enzyme and heavy metal ion) and k irr (irreversible inhibition reaction rate constant) for mercury and lead ions are obtained to be 0.45 ppm, 1.66 × 10 −3 s −1 , 8.67 ppm and 3.16 × 10 −3 s −1 , respectively. Compared with lead ion detection, the higher sensitivity to mercury ion of the biosensor is mainly caused by a higher inhibition efficiency ( k irr / K d ). [ABSTRACT FROM AUTHOR]

Biosensing nitrogenous compounds like urea is required to control the incidents of Economically Motivated Adulteration (EMA). In this study, we report the FluMag Systematic Evolution of Ligands by Exponential Enrichment (FluMag-SELEX) method to isolate a urea specific DNA aptamer with a dissociation constant ( K d ) of 232 nM. The interaction of DNA aptamer with urea has been confirmed by affinity assay, CD analysis, melting curve analysis and truncation studies. Unlike other urea sensing methods reported so far, using this urea aptamer, we demonstrate a simple, ‘non-enzymatic’ easy-to-use, dual readout aptasensor that exploits unmodified gold nanoparticles (AuNPs) to transduce the signals of aptamer binding to urea in terms of intrinsic fluorescence differences and color changes simultaneously. This method is free from complicated sample processing and labeling steps. The urea aptasensor displays high selectivity for urea and is free from interference from common milk adulterants. The developed aptasensor reliably detects urea adulteration in milk. The response signals linearly correlate with the increasing concentrations of urea in milk ranging from 20 mM to 150 mM with detection limit of 20 mM. We also show that this aptasensor can also be used as a simple fluorescence based “turn-on” sensor. The results obtained in this study are comparable to the commercial urease based detection methods. [ABSTRACT FROM AUTHOR]

Urea is one of the metabolic products of protein metabolism. The accurate determination of urea is important in glomerular filtration rate determination, renal function testing, and other biomedical applications. This research presents a simple fabrication of potentiometric urea biosensor based on a urease-immobilized polypyrrole (PPy). Thin film of PPy doped with hydroquinone monosulfonate (HQS) was electropolymerized on stainless steel supporting electrode. Urease was immobilized onto the PPy film by mean of physical adsorption. X-ray photoelectron spectroscopy (XPS) was performed to confirm the existence of urease on PPy surface. The PPy film immobilized with urease activity of 200 U/mL showed the sensitivity of 43.4 ± 3.3 mV/pUrea (r2 > 0.99) in the urea concentration ranging from 0.5 to 10 mM (pUrea 2.0-3.3). The response time was less than 60 sec. The urease-immobilized PPy film offers a great potential in the use of conducting PPy film as an effective transducer to convert physiochemical change into an electronic signal, enhancing in performance of the biosensor. [ABSTRACT FROM AUTHOR]

Uniform sputtered nanoporous zinc oxide (Nano-ZnO) thin film on the conductive fluorinated-tin oxide (FTO) layer was applied to immobilize urease enzyme (Urs) for urea detection. Highly uniform nanoporous ZnO thin film were obtained by reactive radio frequency (RF) magnetron sputtering system at the optimized instrumental deposition conditions. Characterization of the surface morphology and roughness of ZnO thin film by field emission-scanning electron microscopy (FE-SEM) exhibits cavities of nanoporous film as an effective biosensing area for enzyme immobilization. Step by step monitoring of FTO/Nano-ZnO/Urs biosensor fabrication were performed using electrochemical methods such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Fabricated FTO/Nano-ZnO/Urs biosensor was used for urea determination using EIS experiments. The impedimetric results show high sensitivity for urea detection within 0.83–23.24 mM and limit of detection as 0.40 mM. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] LbL films containing nanostructured polyaniline were fabricated in a simple way. [•] Urea biosensor was constructed using urease immobilized on nanostructured polyaniline. [•] The system showed an excellent sensitivity to pH and urea with a small voltage drift. [•] A large dynamic range of urea detection was achieved. [•] The proposed biosensor can be used in clinical analyses. [Copyright &y& Elsevier]

Abstract: A simple and novel potentiometric biosensor for urea detection was prepared by employing an electrosynthesized polymer with buffering capability. It was obtained by deposition of a weighed amount of urease (Ur) at a glassy carbon (GC) electrode followed by immobilization by an electrosynthesized poly-o-phenylenediamine (PPD) film. An unconventional “upside-down” (UD) geometry was employed for the electrochemical cell. The response of GC/Ur/PPD sensor is linear with urea concentration in the range 10μM to 1mM (15mV/mM, R 2 =0.9999) due to buffering capability of PPD film, which represents a novel role of electrosynthesized polymers in their application to biosensors. At higher concentrations, the more common Nernstian response (28mV/decade, R 2 =0.9987) is observed. The sensor exhibits a sufficient sensitivity for practical determinations, rapid response and long term stability. [Copyright &y& Elsevier]

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μμL of cell-free extract containing 7.47 mg protein mL−−1, 2 μμL of glutaraldehyde (5%, v/v) and 10 μμL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0--10.0 μμM urea, a detection limit of 2.0 μμM for urea, a response time of 20 s, a sensitivity of 58.245 % per μμM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition. [ABSTRACT FROM AUTHOR]

Abstract: A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10−6 to 10−1 molL−1 urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting. [Copyright &y& Elsevier]

Abstract: A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pHopt, T opt, thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927μAmM−1 cm−2. The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%. [Copyright &y& Elsevier]

Simple conducting polymer–polyaniline-based sensors/biosensors, working either in potentiometric or UV/Vis spectrophotometric mode, are proposed. Disposable sensors were produced by coating polyaniline layers, cast from aqueous dispersion of the polymer nanoparticles, on a transparent plastic polyacetate foil. In the potentiometric mode, polyaniline layers are sensitive to a number of metal cations, while in UV/Vis mode, changes of absorbance were recorded only in case of a chemical reaction of cations with the polymer. Pronounced sensitivity of tested sensors to ammonia was used for potentiometric/spectrophotometric biosensing purposes studied on a model example of urease-based sensors of urea. The highest sensitivity and reproducibility of such sensors were observed in urea concentration ranging from 1 to 10 mM. [ABSTRACT FROM AUTHOR]

Abstract: This investigation evaluated an EGFET biosensor in which the enzyme was immobilized on a nitrocellulose film. A porous nitrocellulose layer, formed by an inverse phase process, was deposited directly on a SnO2/ITO gate by spin-coating. To ensure optimal surface chemistry conditions under which the EGFET had appropriate pH sensitivity and protein-immobilizing capacity, the nitrocellulose film manufacturing conditions were investigated, and were found to be determined by the inverse phase process, the rate of the spin coater, and the concentration of nitrocellulose. To determine the protein-immobilizing capacity of the nitrocellulose-modified EGFET, urease was immobilized on the nitrocellulose film, and was adopted as a urea biosensor. The device exhibited a response of 136mV for urea at a concentration of 64mg/dl. The porous surface of nitrocellulose constitutes a low diffusion barrier, and has a fast response time of 20s. In a stability test, the sensor maintained its excellent performance in detecting urea in 27 measurements performed over 60 days. The developed nitrocellulose-modified EGFET was therefore proved to have potential application as a biosensor for determining enzymes or antigen-antibody reactions. [Copyright &y& Elsevier]

Abstract: A novel potentiometric urea biosensor has been developed for selective and quantitative recognition of urea by immobilizing urease onto Ti/urease-imprinted TiO2 film and monitoring the potentiometric response caused by the immobilized urease/urea reaction system. Urease immobilization on TiO2 film was investigated using a potentiometer, and factors affecting its immobilization such as concentration of urease, pH and ionic strength were discussed in detail. The performance of the developed urea biosensor was evaluated, and the obtained urea biosensor exhibited shorter response time (25s), wider linear range (8μM–3mM), lower detection limit (5.0μM) and good stability (with about 93.3% of the original response signal retained after 1month). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples. [Copyright &y& Elsevier]

Abstract: An amperometric biosensor was fabricated for the quantitative determination of urea in aqueous medium using hematein, a pH-sensitive natural dye. The urease (Urs) was covalently immobilized onto an electrode made of gold nanoparticles functionalized with hyperbranched polyester-Boltron® H40 (H40–Au) coated onto an indium–tin oxide (ITO) covered glass substrate. The covalent linkage between the Urs enzyme and H40–Au nanoparticles provided the resulting enzyme electrode (Urs/H40–Au/ITO) with a high level of enzyme immobilization and excellent lifetime stability. The response studies were carried out as a function of urea concentration with amperometric and photometric measurements. The biosensor based on Urs/H40–Au/ITO as the working electrode showed a linear current response to the urea concentration ranging from 0.01 to 35mM. The urea biosensor exhibited a sensitivity of 7.48nA/mM with a response time of 3s. The Michaelis–Menten constant for the Urs/H40–Au/ITO biosensor was calculated to be 0.96mM, indicating the Urs enzyme immobilized on the electrode surface had a high affinity to urea. [Copyright &y& Elsevier]

Abstract: Urease (Ur) and glutamate dehydrogenase (GLDH) have been co-immobilized onto superparamegnatic iron oxide (Fe3O4) nanoparticles-chitosan (CH) based nanobiocomposite film deposited onto indium-tin-oxide (ITO) coated glass plate via physical adsorption for urea detection. The magnitude of magnetization (60.9emu/g) of Fe3O4 nanoparticles (∼22nm) estimated using vibrating sample magnetometer (VSM) indicates superparamagnetic behaviour. It is shown that presence of Fe3O4 nanoparticles results in increased active surface area of CH-Fe3O4 nanobiocomposite for immobilization of enzymes (Ur and GLDH), enhanced electron transfer and increased shelf-life of nanobiocomposite electrode. Differential pulse voltammetry (DPV) studies show that Ur-GLDH/CH-Fe3O4/ITO bioelectrode is found to be sensitive in the 5–100mg/dL urea concentration range and can detect as low as 0.5mg/dL. A relatively low value of Michaelis–Menten constant (K m, 0.56mM) indicates high affinity of enzymes (Ur and GLDH) for urea detection. [Copyright &y& Elsevier]

Abstract: The lyophilized biomass of bacterium Brevibacterium ammoniagenes was immobilized in polystyrene sulphonate–polyaniline (PSS–PANI) conducting polymer on a Pt twin wire electrode by potentiostatic electropolymerization. The bacterial cells retained their viability as well as urease activity under entrapped state, as confirmed with bacterial live–dead fluorescent assay and enzymatic assays. The entrapped cells were visualized using scanning electron microscope. The immobilized cells were used as a source of unpurified urease to develop a conductometric urea biosensor. The catalytic action of urease in the sensor released ammonia, thereby causing an increase in the pH of the microenvironment. The pH dependant change in the resistivity of the polymer was used as the basis of sensing mechanism. The sensor response was linear over a range of 0–75mM urea with a sensitivity of 0.125mM−1. The sensor could be reused for 12–15 independent measurements and was quite stable in dry as well as buffered storage condition at 4°C for at least 7 days. [Copyright &y& Elsevier]

Abstract: A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity. [Copyright &y& Elsevier]