Amperometric urea biosensor based on immobilized urease on polypyrrole and macroporous polypyrrole modified Pt electrode

Biosensing of urea by a biosensor as a direct detection method at ambient temperature and pressure instead of chromatography leads to a significant reduction in processing costs. Amperometric biosensors based on urease immobilization on macroporous polypyrrole (MPPy) and pyrrole on the surface of a Pt electrode were developed. Applying cyclic voltammetry (CV), we demonstrated the synthesis of MPPy using monodispersed polystyrene spheres (460 nm) as a template. CV and chronoamperometric studies were conducted to evaluate the electrochemical current of the modified electrodes. For the electrode with polypyrrole (PPy), the biosensor response was linear in the range of 1.67–8.32 mM (R = 0.99). Sensitivity, detection limit, and response time of this biosensor were 0.0035 mA mM−1 , 2.57 mM, and ∼7 s, respectively. For the electrode with MPPy, the linear range was 0.5–10.82 mM (R = 0.99). For this biosensor, sensitivity, detection limit (S/N = 3), and response time were 0.0432 mA mM−1 , 0.208 mM, and ∼5 s, respectively. The modified biosensor with MPPy showed high stability and desirable selectivity for urea.


Introduction
To improve the quality of human life, control diseases, and evaluate pollution of surface water, it is very crucial to have a low-cost, stable, and selective tool (biosensor) for real-time monitoring of urea. Excess of fertilizers, which contain urea, remains in the soil and contaminates surface water during precipitation and irrigation. The normal range of urea in human blood serum is 2.5-7.5 mM [1,2]. High levels of urea in blood serum can be due to renal failure, shock, dehydration, burns, urinary tract obstruction, and gastrointestinal bleeding. Nephritic syndrome, hepatic failure, low-protein diets, and high-carbohydrate diets cause low concentrations of urea [3][4][5].
The concentration of the urea in a sample is measured by monitoring the liberated ions using a transducer. * Correspondence: najafpour@nit.ac.ir This work is licensed under a Creative Commons Attribution 4.0 International License.
These ions are not electroactive; thus, they should be oxidized to other species. Some catalytically active supports can interact with these ions [6,7,13,16,17]. A suitable urease immobilization support must have high conductivity, high surface-to-volume ratio, and low enzyme leaching. Nanoscale conductive polymers with high surface-to-volume ratio and good conductivity are a suitable option as support. Polypyrrole (PPy) is a conductive polymer that can be synthesized using chemical and electrochemical methods in aqueous or a nonaqueous media in a wide range of pH [18][19][20]. Due to biocompatibility [21], conductivity [22], the possibility of synthesis at relatively low potentials (which means less energy consumption) [23], and, most importantly, the ability to interact (reversible deprotonation) with ammonium [24][25][26], PPy is a suitable option for urea biosensing applications. PPy electropolymerization with cyclic voltammetry (CV) creates a uniform PPy film on an electrode surface with a positive charge [27]. Since at pH values higher than the isoelectric point, urease has a negative charge, it can be adsorbed by electrostatic forces [28]. The enzyme loading on the PPy can be increased by synthesis of the nanoscale PPy on the surface of the electrode as a matrix. Nanoscale PPy (macroporous PPy) provides more sites for enzyme immobilization, preventing loss of activity and leaching of the enzyme. In order to improve the efficiency and stability of the immobilized enzyme on the surface of the electrode, a cross-linking agent (glutaraldehyde (GA)) can be used. Free amino groups of enzymes react with GA and can form a stable and very strong bonding between enzymes.
This work focuses on the development of an amperometric biosensor based on macroporous polypyrrole (MPPy) and PPy. The enzyme was immobilized by GA as a cross-linking agent (GA-Urs) and the electrostatic force on the MPPy (Pt/MPPy/GA-Urs) and PPy (Pt/PPy/GA-Urs). The results of the Pt/MPPy/GA-Urs electrode were compared against those of the biosensor with PPy as an immobilization matrix. The MPPy and PPy film morphology was evaluated using a field emission scanning electron microscope (FESEM). The electrochemical responses of the electrodes were studied by CV and chronoamperometry. The purpose of the present work is to develop urea biosensors based on MPPy and PPy, and to compare their performances. The Pt/MPPy/GA-Urs electrode showed good performance compared to the Pt/PPy/GA-Urs electrode in terms of selectivity for urea, stability, and repeatability.

Reagents and chemicals
Pyrrole monomer, urease (EC 3.5. M Ω cm −1 ) . The enzyme solution was freshly prepared in phosphate buffer (0.1 M, pH 7.0). Urea solution (1 mg/mL) was prepared by dissolving 1 g of urea in 1 L of deionized water. Other concentrations were obtained by serial dilutions.

Instrumental
All electrochemical measurements were performed in a standard three-electrode cell. A platinum wire electrode as a counter electrode, Ag/AgCl/Cl (sat) as a reference electrode, and a platinum plate electrode (4 mm in diameter) as a working electrode were used both in the chronoamperometry and CV. CV tests, urea sensing, and electropolymerization of pyrrole monomer were conducted with equipment from Bio-Logic Science Instruments Co. (Paris, France), which was connected to a computer for data acquisition. A FESEM (RIRA3, TESCAN, Czech Republic) was employed to study the morphology of the surface of the modified electrodes.
The apparent enzyme activity of the Pt/MPPy/GA-Urs bioelectrode was studied using Nessler's reagent and a UV-Vis SPEKOL 1500 spectrophotometer (Analytik Jena AG, Germany) at a wavelength of 425 nm.

PPy polymerization
Electropolymerization of pyrrole monomers was carried out by CV in a cell that contained 10 mL of the buffer consisting of 0.1 M NaCl, 50 mM monomers, and 25 mM sodium dodecyl benzenesulfonate [29].
Prior to electropolymerization, the electrodes were polished and rinsed thoroughly with deionized water to obtain a mirror surface. Then the electrodes were conditioned with CV in 0.5 M H 2 SO 4 (-0.25 to 1.2 V, 30 cycles, 100 mV s −1 ).

Preparation of MPPy
Polystyrene spheres were assembled over the surface of the working electrode by adding 10 µ L of aqueous polystyrene suspension (0.5 wt.% aqueous suspension of polystyrene containing 1.0 × 10 −6 M Triton X-100).
Triton X-100 is a nonionic surfactant that improves the stability of monodisperse polystyrene particles [30].
At high concentrations of surfactant, coagulation rather than stability is induced. Therefore, according to the literature, 10 −6 M Triton X-100 was used [31]. The aqueous suspension was placed in an ultrasound bath for 5 min before use. For water evaporation, the electrode was left in a saturated-humidity chamber. Then the same volume of the polystyrene suspension was deposited over the electrode. The electrode was then left in the saturated-humidity chamber and was finally placed in an oven at 100 • C for 4 h [31]. Afterward, electropolymerization of pyrrole monomers was performed by CV (20 cycles, 100 mV s −1 , -1.0 to 1.0 V) in 10 mL of the buffer containing 0.1 M NaCl, 50 mM monomers, and 25 mM sodium dodecyl benzenesulfonate. After electropolymerization, the polystyrene beads were removed by soaking the electrode in toluene for 24 h under stirring [31]. Then the electrode was rinsed with distilled water, followed by drying at room temperature under flowing nitrogen.

Enzyme attachment
GA contains an aldehyde functional group; when it is exposed to urease that has an amine functional group, and the aldehyde group will covalently bind to the amine group of urease [32]. The electrodes with the polymer (PPy or MPPy) were placed in a 1% GA solution and kept under stirring for 1 h. Next, the electrodes were rinsed with deionized water and dipped into a 50 mg/dL urease solution for 4 hours at 4 • C. The electrodes were then left at room temperature until dry. The amount of the enzyme on the surface of the electrode was controlled by the exposure time of the electrodes to the urease solution and also by the enzyme concentration in the solution. After preparing the electrodes, they were kept at 4 • C in PBS until use.

The activity of immobilized enzyme
The activity of the immobilized enzymes was evaluated by using Nessler's reagent and a UV-Vis spectrophotometer [16,33]. The modified electrodes were dipped for 20 min in 10 mL of urea solution (urea in PBS (0.1 M, pH 7.0)) at a certain concentration of urea, which contained 400 µ L of Nessler's solution. Ammonia reacts with Nessler's reagent ( Hg II I 2 4 ) and forms a colored product according to the following equation [13]: Absorbance of the colored species ( N H 2 Hg 2 I 3 ) was measured at λ max 425 nm for every 1 min of incubation for a total of 20 min. The activity of the immobilized urease was obtained by evaluating the rate of urea hydrolysis and ammonia formation using Eq. (3) [16,33,34]: Here, A is the difference in absorbance before and after incubation, V is the total volume of solution (10.04 cm 3 ) , ε is the millimolar absorption coefficient of Nessler's reagent at 425 nm, t is the reaction time (1 min), and s is the surface area of the working electrode (0.1256 cm 2 ) .

Morphological features
The morphological features of the electrodes were investigated using a high-resolution FESEM.

Electrochemical measurement
Chronoamperometric measurements were conducted to measure the amount of urea in the sample. The urea solution was prepared in 0.1 M phosphate buffer (pH 7.0). To do so, we used the developed electrodes, Pt/MPPy/GA-Urs or Pt/PPy/GA-Urs, as the working electrodes at fixed potential of +0.3 V vs. reference electrode (RE) for chronoamperometry [31]. Polymerization of the pyrrole was carried out with CV within the potential range of -1.0 to 1.0 V vs. RE at the scan rate of 100 mV s −1 [15]. The recorded current was directly related to the urea hydrolysis by urease. All experiments were performed at room temperature.

Preparation of Pt/MPPy/GA-Urs biosensor
Pyrrole was electropolymerized by CV (20 cycles, at 100 mV s −1 ) on the surface of a Pt electrode (4 mm in diameter). This electropolymerization was carried out in phosphate buffer containing 0.1 M NaCl, 50 mM monomers, and 25 mM sodium dodecyl benzenesulfonate. The cyclic voltammograms of the electrode are presented in Figure 1. This figure shows that PPy is growing on the surface of the electrode by increasing the number of potential cycles.
The second step of the biosensor fabrication was immobilizing the enzyme. The electrode from the previous step was placed in the GA solution, then in the urease solution, and finally allowed to dry. Figure 2 shows a schematic diagram of the fabrication procedure of the Pt/MPPy/GA-Urs electrode.

Morphological analysis
A FESEM image of the enzyme is shown in Figure 3. This figure shows nonuniform porous structures. This porosity allows ammonium to diffuse through the surface of the support matrix and then to react with PPy. This reaction transfers electrons to the Pt electrode and results in an increase in electrochemical current response. Figure 4A depicts a FESEM image of MPPy as a suitable substrate for the immobilized enzyme. For the synthesis of PPy, polystyrene beads (460 nm) were used as a template. Since MPPy has a nanosize structure, the available surface for enzyme immobilization in MPPy is greater than that of PPy as a matrix (compare the surface of the PPy in Figure 4A with 4B). The pore size of PPy in MPPy was directly determined by the diameter of the template particles (460 nm). After immobilizing the enzyme, the pore size decreased to 230 nm (see Figure 4A). Increasing the enzyme loading increased the rate of the urea hydrolysis, which in turn resulted in larger current. Moreover, due to the nanostructure matrix that provided more redox active sites (the electroactive area increased), the response time deceased.

Activity of the immobilized urease on Pt/MPPy/GA-Urs
The enzyme activity was evaluated using photometric analysis. In this method, the liberated ammonium in the sample was determined by the reaction of ammonium with Nessler's reagent. Figure 5 shows the liberated ammonium and the urea hydrolysis for 20 min. The obtained enzyme activities for the immobilized enzymes on Pt/PPy/GA-Urs and Pt/MPPy/GA-Urs electrodes were 0.64 relative and 0.78 respectively. Figure   5 demonstrates that the enzyme activity decreased after being immobilized on the substrate. The results show a 40% activity loss due to immobilization (on the Pt/MPPy/GA-Urs electrode) compared to the free enzyme. This decrease in activity can be attributed to the toxicity effect of GA. Moreover, covalent binding immobilization can influence the enzyme activity via the active conformation during immobilization [35]. Decrease in activity is lower for the Pt/MPPy/GA-Urs electrode since the MPPy electrode has a nanoscale porosity that provides a larger surface area. Due to the high surface to volume ratio of the MPPy, more enzyme was immobilized; consequently, more enzyme per unit surface area of the electrode was active.  To obtain the background current, a chronoamperometry test was performed in the buffer solution. After 120 s, the current reached steady state, and then a certain amount of the urea stock solution was added and the current was recorded versus time. PPy at 0.3 V acts as a transducer for the electrochemical production of nitric oxide from ammonia, which is the product of urea hydrolysis by urease. Therefore, the amperometric response current was obtained at 0.3 V using the Pt/PPy/GA-Urs or Pt/MPPy/GA-Urs electrodes. All experiments were conducted at room temperature at applied potential of +0.3 V vs. RE and 150 rpm agitation.

Biosensor performance
As illustrated in Figures 7 and 8  The effect of pH on the behavior of the bioelectrode was studied with 0.1 M PBS buffer. Figure 9 shows that the maximum current occurs at pH 7.0 and 7.5 for PPy and MPPy, respectively. Since the maximum enzyme activity for MPPy happens at pH of 7.5, which is closer to the pH of human blood (7.3-7.4), this clearly demonstrates the suitability of MPPy for clinical applications.

Shelf time, reproducibility, and operational stability studies
The Pt/MPPy/GA-Urs biosensor showed a 4% loss in current response after being used 6 times. Its loss in the current became 7% after a month (at 4 • C in 0.1 M PBS, pH 7.0).
To examine the biosensor for reproducibility, the electrode was modified under identical conditions several times. The modified electrodes were tested in 5 mM urea solution. The achieved results with 4.3% relative standard deviation showed that the biosensor had acceptable reproducibility.

Interference study
The effect of interferents (glucose, uric acid, ascorbic acid, and triglyceride) was studied to evaluate the behavior of the biosensor in clinical applications. According to the results in the Table, adding the interfering species to 1.67 mM urea solution did not significantly change the response current. The difference in response current of the biosensor in the presence of the interfering species, which is very small, is also provided in the Table.

Conclusions
In this study, MPPy was electropolymerized on the surface of a Pt electrode, which increased the enzyme loading. The behaviors of the developed biosensors were investigated by CV and chronoamperometry. The biosensor with MPPy and PPy showed linear behavior in the urea concentration ranging from 0. short response time, long-term stability, low detection limit, and insignificant changes in the currents after adding interfering factors. Therefore, these are effective biosensors for urea detection in physiological samples.