A novel potentiometric sensor based on urease/ bovine serum albumin-poly(3,4-ethylenedioxythiophene)/Pt for urea detection

1 Introduction

Urea, CO(NH₂)₂, is a by-product of protein metabolism and is produced at around 10 kg/year in the liver as a part of the urea cycle in the human body. Urea can be dissolved in the blood and is excreted by the kidney as a component of urine. To assess kidney function, tests that determine the urea level in the blood and urine are commonly used. Conventional methods of urea detection involve the use of high-performance liquid chromatography [1] and calorimetric [2] and fluorometric [3] analysis; however, these are not portable because such tests can only be carried out at centralized locations and require sample dilution to eliminate the matrix effect. Thus, it is essential to develop portable urea biosensors.

Urease, an enzyme that catalyzes urea hydrolysis, has been used to detect urea via enzyme immobilization on different substrates to form potentiometric [4–6], amperometric [7, 8], conductometric [9], optical [10], and piezoelectric [11] urea sensors. Among these sensors, the potentiometric biosensors are easily fabricated and have been used to detect different bio-materials through the immobilization of enzymes on the electrodes. In the development of electrochemical urea biosensors, this step is the key parameter that decides the sensitivity and reproducibility of the sensor. A variety of polymers have been employed to immobilize urease [12–20]; this can be done by introducing urease directly into the monomer solution to trap the urease in the polymer matrix, by binding urease molecules to the polymer chains through electrostatic interactions, or by forming covalent bonds between the enzyme and the functional groups of the polymer.

Hamilton and Breslin [21] developed a highly sensitive urea sensor incorporating urease into a polypyrrole film with sulfonated-β-cyclodextrin dopant. The formation of an inclusion complex between urea and a sulfonated-β-cyclodextrin host in an aqueous solution led to a good sensitivity of 5.79 μC/μm. Osaka and colleagues [15, 16] fabricated urea biosensors based on a composite film of electroinactive polypyrrole with the polyion complex. The polyion enzyme complex was cast over polypyrrole by the successive deposition of polyacrylate or polystyrenesulfonate, urease, and polylysine solutions. The addition of the polyion resulted in urea sensors with a rapid, sensitive, and stable response signal. Lakard and colleagues [22–24] developed urea sensors based on conducting polymers and polyelectrolytes films. They found that adsorption of a urease-chitosan multilayer onto an electrodeposited polyaniline film was efficient for urea sensing. However, the short lifespan, delayed response time, and other limitations of the prepared membranes are some aspects that require further investigation.

In this work, we employed an electrically conductive polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), to immobilize bovine serum albumin (BSA) on the Pt electrode and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) as a coupling agent to bind BSA with the enzyme urease. We studied the effect of PEDOT thickness, pH value, and the temperature of the urea solution on the detection sensitivity. The long-term stability of the sensor was also examined.

2 Experimental section

3 Results and discussion

Figure 1 shows the time-dependent potentiometric responses of the urea biosensors with the BSA-PEDOT/Pt electrode with (Fig. 1a) and without (Fig. 1b) the immobilized urease enzyme on the electrode to solutions with different urea concentrations. The immobilized enzyme on the BSA-PEDOT-based Pt electrode catalyzed the hydrolysis of urea, leading to the formation of ammonium (NH₄⁺), bicarbonate (HCO₃⁻), and hydroxide (OH⁻) ions. These ionic products changed the potential difference between the fully functionalized Pt (enzyme/BSA-PEDOT/Pt) and the AgCl/Ag reference electrodes. The electrode was subsequently exposed to solutions with urea concentrations ranging from 10⁻⁵ to 10⁻²m after the potential reached steady state. The steady-state potential was defined as the value with the rate of potential changes at <1 mV/min. The urea detection sensitivity was defined as the potential difference over a decade (order of magnitude) of urea concentration difference, which was derived from the slope of the linear plot of potential difference between the fully functionalized Pt electrod, and the AgCl/Ag reference electrode versus the logarithmic urea concentration, as the insert shows in Fig. 1. A sensitivity of 15.2 mV/decade of urea concentration was achieved for the fully functionalized enzyme/BSA-PEDOT/Pt sensor. The sensitivity of the same sensor without the immobilized enzyme was only 3.2 mV/decade. To confirm the effect of BSA and PEDOT on the sensing sensitivity, two reference sensors without BSA were also fabricated. The first reference was fabricated with the same procedure as for the preparation of the enzyme/BSA/PEDOT/Pt sensor with the presence of BSA in the EDOT monomer solution during the PEDOT polymerization. The second sensor was prepared by adding the urease to the EDOT monomer solution during the PEDOT polymerization. The responses of these two sensors are illustrated in Fig. 2. Both of these reference sensors exhibited a very low urea sensing sensitivity similar to the response of the BSA-PEDOT/Pt sensor without urease. This confirmed that there were no direct physical or chemical bonds between the urease molecules and the PEDOT; PEDOT does not immobilize the urease molecules without the presence of BSA.

Fig. 1:

Time-dependent potentiometric responses of the BSA/PEDOT/Pt sensor, (a) with and (b) without the immobilized urease enzyme on the electrode, to solutions with different urea concentrations. The insert is the potential difference of the sensor exposed to 10⁻⁵ to 10⁻¹m urea in 0.01 m phosphate buffer solution (pH = 7.0).

Fig. 2:

Potential difference of the different sensors exposed to 10⁻⁵ to 10⁻¹m urea in 0.01 m phosphate buffer solution (pH = 7.0).

Figure 3 shows the effect of EDOT monomer concentration during the polymerization on urea detection sensitivity. Different concentrations of EDOT monomers ranging from 0.08 to 0.72 m were used in this study, and the thickness of the PEDOT film on the Pt electrode was linearly proportional to the EDOT monomer concentration. As described above, the PEDOT was polymerized on the Pt electrode from the EDOT monomers dispersed in the electrode solution through electropolymerization at a fixed voltage of 2 V for 20 min in the presence of 10⁻⁴m BSA solution. An optimal EDOT monomer concentration of 0.24 m was found to achieve a maximal urea detection sensitivity of 15.2 mV/decade. A 1-μm-thick composite film of BSA-PEDOT was formed on the Pt electrode using the 0.24-m EDOT monomer solution. These conditions resulted in trapping an optimal number of BSA molecules in the PEDOT matrix while maintaining good electrical conductivity of the PEDOT.

Fig. 3:

Urea detection sensitivity as a function of EDOT monomer concentration during PEDOT polymerization.

For the PEDOT film polymerized from the EDOT monomer solution with a concentration lower than 0.24 m, the number of BSA molecules trapped in the PEDOT film was limited; thus, the composite BSA-PEDOT film provided fewer BSA molecules capable of bonding with enzyme molecules. Therefore, the detection sensitivity decreased to 8.3 mV/decade. For the sensor prepared with an EDOT monomer concentration higher than 0.24 m, a thicker BSA-PEDOT film was formed, and the urea detection sensitivity was even further reduced to 3.7 mV/decade. Although more BSA molecules were embedded in the PEDOT film, the increased thickness of the film prevented the BSA molecules from interacting with the enzyme. The additional inactive BSA actually reduced the electrical conductivity of the PEDOT and blocked the charge transfer. Figure 4 shows the scanning electron microscope (SEM) pictures of the Pt electrode surface before and after BSA/PEDOT film coating. The fresh Pt surface consisted of many oriented cracks owing to the sanding process for the Pt wire, as illustrated in Fig. 4a. Figure 4b shows the Pt surface coated with the BSA-PEDOT composite film. As a result of the PEDOT film coating, the cracks on the Pt surface were not as obvious relative to the bare Pt wire. The particles on the BSA-PEDOT/Pt surface might be PEDOT precipitates formed during polymerization.

Fig. 4:

SEM pictures of Pt electrode surfaces (a) before and (b) after BSA/PEDOT film coating.

The effect of enzyme concentration on the urea sensing sensitivity was also studied. In this experiment, the enzyme concentrations tested ranged from 40 to 200 mg/mL. As shown in Fig. 5, the urea detection sensitivity initially increased with the enzyme concentration and then leveled off. For the condition with lower enzyme concentrations, there were not enough enzyme molecules to chemically bond to the BSA molecules immobilized in the PEDOT; therefore, the detection sensitivity was proportional to the enzyme concentration. Once the enzyme concentration reached 100 mg/mL, the urea detection sensitivity stayed constant because the available BSA molecules immobilized in the PEDOT are already bonded to the enzyme molecules.

Fig. 5:

Urea detection sensitivity as a function of the enzyme concentration.

The effect of the temperature of the buffer solution on urease activity was also studied. As shown in Fig. 6a, 25 °C was the ambient temperature that resulted in the highest detection sensitivity. At temperature higher than 25 °C, the detection sensitivity was reduced to about 70 %, whereas lower temperatures also resulted in lower detection sensitivity. This enzyme activity is strongly dependent on the particular three-dimensional (3D) structure of the enzyme and its ability to interact with its substrate; increased or decreased temperature greatly compromises the 3D structure of an enzyme and affects its ability to catalyze the reaction. This prevents the proper interaction between the enzyme and the substrate molecule and results in a decreased sensitivity. Similarly, lower temperatures decrease the enzyme’s ability to adjust its active site to obtain the optimal configuration for the urea molecule to access, leading to a lower detection sensitivity.

Fig. 6:

Urea detection sensitivity as a function of (a) solution temperature and (b) pH of the buffer solution.

The effect of the pH on the enzyme activity is shown in Fig. 6b. Optimal enzyme activity was observed at a pH value of 5.8, which is close to the isoelectrical potential (5.5) of urease. A similar result was reported by Howell and Sumner [25]. There was a slight decrease in urease activity when the pH of the solution was lower than 5.8 but a much sharper reduction in the enzyme activity for conditions where the pH value was higher than 7. The carboxyl and amine functional groups of urease are ionized in acidic and basic solutions, respectively; thus, the ionized urease exhibits a different 3D shape and the original active site of the enzyme is no longer able to form the urease-urea complex. This contributes to the observed reduction in urease activity.

After the first detection, the sensor was thoroughly rinsed with a PBS buffer solution (pH = 7, 10 mm) to wash off the urea solution. The procedures for urea detection and sensor surface reactivation were repeated five times. The sensor showed good detection repeatability, as shown in Fig. 1a. Then, the sensor was rinsed thoroughly with the PBS buffer solution (pH = 7, 10 mm) to wash off the residual urea before it was immersed in PBS buffer solution (pH = 7.4, 10 mm) and stored at room temperature. Sensors were re-tested for urea detection every 7 days. For these tests, the procedures for urea detection and sensor surface reactivation were also repeated five times for a period of 10 weeks. Figure 7 shows the sensitivities of the sensors tested after different storage times at a fixed urea concentration of 10⁻⁵m against the storage time. The sensor after 1 week of storage showed around a 10 % reduction in the sensitivity. The average degradation rate was roughly 9 % per week for the period of 2 months. As there is degradation of the detection sensitivity, the sensor would need to be re-calibrated for urea concentration determination after storage; however, there would be no need to re-calibrate for the detection of the presence or absence of urea. The decrease in detection sensitivity after 10 weeks of storage was not caused by the Pt electrode, but rather due to the decrease in enzyme activity [5]. Another important finding was that the response time of the sensor stored for 10 weeks was 10 s compared to 5 s for the fresh sensor. The decreased number of active enzymes after long-term storage causes a longer response time.

Fig. 7:

Urea detection sensitivity of the sensors tested after different storage times at a fixed urea concentration of 10⁻⁵m.

Glucose, KCl, and NaCl were tested as potential interfering species. A comparison of sensitivities on various interfering analytes is shown in Fig. 8. Clearly, the highest sensitivity was observed for urea, which was almost seven times larger than that for NaCl, as shown in Fig. 8. No obvious response currents were obtained for glucose. Thus, it is concluded that this newly developed potentiometric sensor is specific for urea.

Fig. 8:

The selectivity of the newly developed urea sensor.

4 Conclusions

A potentiometric urea sensor employing a urease/BSA-PEDOT/Pt electrode has been demonstrated to have a sensitivity of 15.2 mV/decade. BSA trapped in the PEDOT matrix successfully immobilizes urease molecules on the surface of the BSA-PEDOT/Pt electrode through strong amide covalent bonding between the carboxyl functional groups on the enzyme and the amines on the BSA using EDC and NHS as coupling agents. A 1-μm-thick BSA-PEDOT composite film has the optimal thickness to trap the maximal amount of BSA molecules in the PEDOT matrix, while maintaining a good electrical conductivity of the PEDOT for urea detection. Optimal enzyme activity was observed at a pH value of 5.8, which is close to the isoelectrical potential (5.5) of urease. Once the enzyme concentration reached 100 mg/mL, all the available BSA molecules immobilized in the PEDOT are bonded to the enzyme molecules. The enzyme activity is strongly dependent on the 3D structure of the enzyme and its ability to interact with its substrate; 25 °C was the optimal ambient temperature to achieve the highest detection sensitivity. The average degradation rate of this urea sensor was roughly 9 % per week for a period of 2 months.