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Electrochemical determination of urea using a
gold nanoparticle-copolymer coated-enzyme
modified gold electrode
Seyda Korkut, Sinan Uzuncar, Muhammet Samet Kilic & Baki Hazer
To cite this article: Seyda Korkut, Sinan Uzuncar, Muhammet Samet Kilic & Baki Hazer (2019) Electrochemical determination of urea using a gold nanoparticle-copolymer coated-enzyme modified gold electrode, Instrumentation Science & Technology, 47:1, 1-18, DOI: 10.1080/10739149.2018.1447486
To link to this article: https://doi.org/10.1080/10739149.2018.1447486
Published online: 15 Mar 2018.
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https://doi.org/10.1080/10739149.2018.1447486
Electrochemical determination of urea using a gold
nanoparticle-copolymer coated-enzyme modified gold
electrode
Seyda Korkuta, Sinan Uzuncara, Muhammet Samet Kilicb, and Baki Hazerb
aDepartment of Environmental Engineering, Bulent Ecevit University, Zonguldak, Turkey; bDepartment
of Chemistry, Bulent Ecevit University, Zonguldak, Turkey
ABSTRACT
A new amperometric urea biosensor based on gold nanoparticle embedded poly(propylene-co-imidazole) was developed for the determination of urea. The urease adsorbed on the polymeric film catalyzed the hydrolysis of urea to ammonium and bicarbonate ions and the ammonium was then electrooxidized on the gold electrode with the aid of gold nanoparticles at +0.2 V versus Ag/AgCl using differential pulse voltammetry. The biosensor provided a linear current response to urea concentration from 0.1 to 30 mM, a detection limit of 36 µM, a relative standard deviation of 2.43% (n = 18), and excellent storage stability, as the current decrease was only 3% after 75 days. The operation of the biosensor was evaluated by the analysis of municipal sewage wastewater collected from the inlet pipe of the treatment plant of Zonguldak City in Turkey. The effects of possible interferants were also characterized.
KEYWORDS
Differential pulse voltammetry; gold nanoparticles; poly (propylene-co-imidazole); sewage wastewater; Urea
Introduction
Urea is a nitrogen-based organic substance and widely distributed in nature. Its determination is important in clinical chemistry, agricultural chemistry, and environmental monitoring,[1] especially in sewage water containing urea
nitrogen in the concentration range of 2–16 mg- N/L.[2] Conventional
meth-ods used for the determination of urea are gas chromatography, calorimetric and flourimetric analysis, etc.[3] However, the major disadvantages of these
methods are that they are time-consuming and require pretreatment of the samples. In addition, these methods cannot be used for field monitoring. A biosensor developed for the determination of urea in the biological samples is an option to overcome these disadvantages. Detection through electro-chemical mode is highly adopted and versatile in biosensor systems.
The immobilization of urease over electrodes via suitable matrices is the key parameter which decides the sensitivity and reproducibility of the urea none defined
CONTACT Seyda Korkut s.korkut@beun.edu.tr Department of Environmental Engineering, Bulent Ecevit University, 67100 Zonguldak, Turkey.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/list.
© 2018 Taylor & Francis
electron transfer between enzyme and the electrode, thereby enhancing the elec-trochemical sensing ability.[7] Moreover, they can immobilize enzymes through
chemical adsorption and provide a stable surface for enzyme immobilization without compromising their biological activities.[8] Unfortunately, gold
nano-particles have been used in a few number of urea biosensors.[7,9]
Polypropylene is a hydrophobic polymer which has a low cost, good mechanical property, and excellent recyclability.[10–14] In our previous study,
enzymatic fuel cell electrodes were coated with a nanocomposite, with excellent characteristics, was synthesized by combining gold nanoparticles with polypropylene chain.[15] The nanocomposite showed excellent adsorption
capacity and provided fast electron transfer, yielding efficient energy pro-duction from municipal wastewater. In the present study, a gold nanoparticle embedded poly(propylene-co-imidazole)-coated gold electrode was used for the first time for the electrochemical detection of urea. The enzymatically pro-duced ammonium was electrooxidized on the electrode with the aid of gold nanoparticles in the polymer chain. The biosensor offered relatively long stor-age stability and a good operational stability. The application of the designed biosensor to the sewage water real sample was conducted. The effect of possible interferants on the urea signals was also investigated.
Experimental Reagents
Chlorinated polypropylene (PP-Cl) (Mw 150000 Da, three repeating units have one Cl in average), imidazole, toluene (99.9% HPLC grade), sodium hydride, gold(III) chloride solution (HAuCl4), urea (99.5%), urease from Canavalia ensiformis (Jack Bean, Type III powder, 15,000–50,000 units/g
solid) and ammonium chloride (NH4Cl, ≥99.5%) were obtained from
Sigma-Aldrich. Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Merck.
Apparatus and electrochemical measurements
Three-electrode system was comprised of a gold working electrode (Ø = 2 mm), platinum wire counter electrode and Ag/AgCl (3 M NaCl)
reference electrode. Electrochemical analyses were performed using a poten-tiostat (CH Instruments 1040B). Experiments were conducted by immersing three-electrode system into a glass cell including urea solution prepared with 10 mL of 100 mM pH 8 phosphate buffer. Differential pulse voltammetry technique was used to examine electrochemical signals of urea at varied potential values. The operational condition of differential pulse voltammo-grams studies was optimized with preliminary tests to observe effective signals from the biosensor. Differential pulse voltammogram condition was set at applied potential range of 0 V and +0.8 V, voltage increment of 0.002 V, amplitude of 0.1 V, pulse width of 0.06 s, sample width of 0.02 s, and pulse period of 0.75 s.
Synthesis of gold nanoparticle-embedded poly(propylene-co-imidazole)
Gold nanoparticle-embedded poly(propylene-co-imidazole) was synthesized according to a procedure in our previous report as follows.[15] In total,
0.25 g of imidazole and 1.43 g of chlorinated polypropylene were separately dissolved in 30 mL of purified tetrahydrafuran. In total, 0.2 g of NaH in oil (60 wt%) was added to the imidazole solution. The reaction mixture was stir-red at room temperature under argon for 3 h. The sodium salt of imidazole solution was added to the chlorinated polypropylene solution and stirred for 30 min. The reaction mixture was stirred for one day and poured into 500 mL of 1 M HCl.
The precipitated polymer was filtered, washed with distilled water, and dried under vacuum at 50°C for 24 h. The final precipitate was dissolved in chloroform, reprecipitated in 200 mL of methanol, and dried under vacuum overnight at room temperature for the purification. In total, 10 mg/mL of synthesized poly(propylene-co-imidazole) were prepared in tetrahydrafuran. In total, 0.05 mL of 0.2 M aqueous HAuCl4 was added into the solution and
stirred for 30 min. The color of gold nanoparticles was observed within 2–3 min. The final solution was stirred for 1 h and poured into a 7 cm diameter Petri dish to form a film through solvent casting, then the poly(propylene-co-imidazole)/gold nanoparticles film was removed from the Petri dish. The polymeric film was washed with methanol and dried under vacuum at room temperature for 24 h. In total, 1 mg/mL of the poly (propylene-co-imidazole)/gold nanoparticle solution was prepared in toluene for readily use in electrode preparation step.
Preparation of poly(propylene-co-imidazole)/gold nanoparticles/urease electrode
The gold electrode was polished with slurries of fine alumina powders (0.3 and 0.05 mm) on a microcloth pad. The electrode was then rinsed
with double-distilled water after each cleaning step. In total, 1 µL of the poly (propylene-co-imidazole)/gold nanoparticles (1 mg/mL) solution was added to the cleaned gold surface. The electrode was then allowed to dry at room temperature for 30 min. Poly(propylene-co-imidazole)/gold nanoparticles film-coated electrode was washed with double-distilled water. In total, 5 µL of urease solution (10 mg/mL) daily prepared in 100 mM pH 8 phosphate buf-fer were added to the poly(propylene-co-imidazole)/gold nanoparticle film- coated surface. The electrode was allowed to stand for enzyme immobilization by physical adsorption for 2 h at room temperature and washed with phosphate buffer to remove unbound enzyme from the electrode surface. A schematic representation of the biosensor is shown in Scheme 1.
Results and discussion Characterization studies
Fourier-transform infrared spectra of the poly(propylene-co-imidazole)/gold nanoparticles powder were presented in our previous report.[15] Fourier-
transform infrared spectra showed that imidazole was chemically bonded to the side chains of the poly(propylene). Scanning electron microscope images of the poly(propylene-co-imidazole)/gold nanoparticles and poly(propylene-
co-imidazole)/gold nanoparticles/urease film-coated electrode surface are
shown in Figure 1. The figure shows that the surface morphology of the copolymeric film changed in presence of the immobilized urease.
Cyclic voltammetry and differential pulse voltammetry were performed to characterize electrochemical surface characteristics of poly(propylene-co- imidazole)/gold nanoparticle and poly(propylene-co-imidazole)/gold nanoparticles/urease film-coated gold electrodes. Differential pulse voltamme-try parameters were set up as presented in the “Experimental” section. Cyclic voltammetry experiments were conducted in 100 mM pH 8 phosphate buffer at a potential scan ranging between 0 and +0.8 V vs. Ag/AgCl with a scan rate of 100 mV/s.
Figure 2a shows the cyclic voltammograms of gold electrodes prepared separately with poly(propylene-co-imidazole)/gold nanoparticles and poly (propylene-co-imidazole)/gold nanoparticles/urease film in 100 mM pH 8 phosphate buffer. The current of the electrode based on the poly(propylene-co-imidazole)/gold nanoparticles/urease film was higher than
Figure 1. Scanning electron microscope image of the (a) poly(propylene-co-imidazole)/gold nanoparticles and (b) poly(propylene-co-imidazole)/gold nanoparticles/urease film-coated electrode surface at a magnification of 200 µm.
the enzymeless electrode at more positive potentials (>+0.6 V) when it was at almost same at lower potential values.
Differential pulse voltammetries were performed for both electrodes in the buffer solution and presented in Figure 2b. Differential pulse voltammetries showed that a fivefold higher current increase was observed in the wide potential scan range for the poly(propylene-co-imidazole)/gold nanoparti-cles/urease electrode in comparison to the enzymeless device. The current increase at more positive potentials can be attributed to the capacitive current which was a function of the working electrode area changed by the enzyme immobilization. Also, the current difference between the poly(propylene-co- imidazole)/gold nanoparticles/urease and the poly(propylene-co-imidazole)/ gold nanoparticles electrode was more pronounced than the current variance observed in cyclic voltammetry graphs.
Differential pulse voltammetry technique uses a series of potential pulses of increasing amplitude while the potential is also scanned with a series of pulses,
Figure 2. (a) Cyclic voltammograms: (1) poly(propylene-co-imidazole)/gold nanoparticle/urease and (2) poly(propylene-co-imidazole)/gold nanoparticles electrodes in 100 mM pH 8 phosphate buffer at a potential scan ranging between 0 and +0.8 V versus Ag/AgCl with a scan rate of 100 mV/s, (b) differential pulse voltammograms: (1) poly(propylene-co-imidazole)/gold nanoparti-cles/urease and (2) poly(propylene-co-imidazole)/gold nanoparticles electrodes in 100 mM pH 8 phosphate buffer at a potential range of 0 V and +0.8 V, voltage increment of 0.002 V, amplitude of 0.1 V, pulse width of 0.06 s, sample width of 0.02 s and pulse period of 0.75 s.
which allow time for the nonfaradaic (charging) current to decay.[16] As a
result, differential pulse voltammetry leads to significant increase in sensitivity of a voltammogram for monitoring small quantities of surface-attached molecules.[17] Therefore, differential pulse voltammetry technique was used
for further experiments because it is more sensitive for amperometric urea determination.
Detection principles for urea
Urea is enzymatically hydrolyzed by urease into HCO3 and NHþ
4 which can
be detected using various transducers such as amperometric and potentiometric.[18] This detection is directly related to urea concentration in
aqueous solution:
NH2CONH2þ2H2O þ Hþurease!HCO3 þ2NHþ4 ð1Þ
In amperometric biosensors, the response/signal strength is directly asso-ciated with urea hydrolysis rate, rapid production and electrooxidation of NHþ
4 ions on electrode surface.[19] In a conventional electrooxidation,
ammonia is dehydrogenated to adsorbed amine (–NH2), then the adsorbed
amine is dimerized to form hydrazine, which is subsequently oxidized to molecular nitrogen (N2).[20] However, the biosensor systems suffer from the
poor electroactivity of the NHþ
4 ions. To overcome this drawback, the
generated NHþ
4 ions can be catalytically oxidized by either a second enzyme
or electrooxidized on electrode surface modified with various metal catalysts.[18] The use of the second enzyme makes the sensing process difficult
since the operational conditions should suit for both enzyme to conduct bio-catalytical reactions. As far as we know, when working with metal catalysts up to now, it has not been clarified that which nitrogen species are formed at the operated potential values in biosensor reports. Therefore, it is important to investigate and clarify the effect of potential on urea biosensor signal to get accurate and reliable response.
Some experiments were conducted to identify the response of the ammonia on the designed electrode surface using differential pulse voltammetry technique. The electrooxidation ability of the enzymeless poly(propylene-co- imidazole)/gold nanoparticle film-coated electrode was investigated in 100 mM pH 8 phosphate buffer including NHþ
4 ions at various concentrations
(0–30 mM), and the differential pulse voltammogram is shown in Figure 3a. The potential was scanned between 0 and +0.8 V versus Ag/AgCl. An oxidation wave was observed at exactly +0.2 V in the graph, and the oxidation current increased by the increasing concentrations of NHþ
4 ions in the
solution at this potential value.
The same differential pulse voltammetry analysis was performed for the poly(propylene-co-imidazole) film-coated electrode without gold
nanoparticles, and no oxidation current was observed at the scanned poten-tials. The electrooxidation was achieved with the aid of gold nanoparticles which served as metal catalyst embedded into the polymeric film. Similarly, urea was detected at similar potentials in previous reports based on urea bio-sensors prepared with metal catalysts such as zirconia (ZrO2)[1] and zinc oxide
(ZnO).[21] In addition, both FeðCNÞ
6 (as the electron mediator) and
glutamate dehydrogenase (as the second enzyme) were used in these reports. Unfortunately, at the +0.2 V reported in those studies, it was not clear that whether the mediator, the second enzyme or metal catalyst was more effective in detecting urea. The same shortcoming was encountered in another amperometric urea biosensor based on TiO2 metal catalyst and FeðCNÞ6
electron mediator.[22]
The urease immobilized poly(propylene-co-imidazole)/gold nanoparticles film-coated electrode was immersed into the 100 mM pH 8 phosphate buffer,
Figure 3. (a) Differential pulse voltammograms of the poly(propylene-co-imidazole)/gold nanoparticle film electrode recorded in pH 8, 100 mM phosphate buffer containing various NHþ
4 concentrations ranging between 5 and 30 mM, (b) differential pulse voltammograms of
the poly(propylene-co-imidazole)/gold nanoparticle/urease film electrode recorded in pH 8, 100 mM phosphate buffer containing 20 mM NHþ
20 mM NHþ
4 and 20 mM urea solution, respectively. Urea and NHþ4 solutions
were prepared with the same buffer. The response characteristic of the biosensor electrode was investigated with differential pulse voltammetry at a potential scan between 0 and +0.8 V vs. Ag/AgCl. The biosensor response is presented in Figure 3b. Depending on the presence of enzyme molecules on the electrode surface, high currents (not oxidation waves) at >+0.6 V were observed in all three solutions (similar to Figure 2). As seen in Figure 3b, the urease immobilized poly(propylene-co-imidazole)/gold nanoparticles elec-trode showed the same differential pulse voltammetry characteristic with the enzymeless electrode (Figure 3a) in the NHþ
4 solution, and the oxidation
cur-rent was observed at +0.2 V. The typical NHþ
4 oxidation curve was obtained
from the biosensor electrode immersed into the urea solution, and the maximum current increase was observed at +0.2 V in comparison to the buffer signal. This can be attributed to the oxidation of NHþ
4 ions produced by the
enzymatic urea reaction on the working electrode surface.
The widely accepted mechanism for ammonia electrooxidation was pro-posed by Gerischer and Mauerer.[23] For the proposed mechanism, ammonia
molecule is adsorbed and then dehydrogenated to different adsorbed intermediate species of the type NHx, where 0 ≤x ≤ 2. The partially dehydro-genated species of adsorbed NHx are considered as active intermediates for N2 formation (final oxidation product).[24] The oxidation peak of ammonia to N
2
occurs at +0.6 V.[19,25–27] It is known that, the adsorbate is composed of NH
or NH2 rather than N at lower potentials than +0.6 V and is not oxidized up
to N2 at low potentials such as +0.2 V.[25] In addition, it was stated above,
among various noble metal and coinage surfaces, only platinum and iridium gave steady-state activity toward N2 production[26] due to the different
sorp-tion properties of adsorbed N on the different metal surfaces. At ruthenium, rhodium and palladium, N2 was formed only transiently, or not at all, because
the adsorbed NHx intermediates underwent further dehydrogenation to tightly bound atomic nitrogen. The coinage metals copper, silver and gold did not form any ammonia adsorbates,[28] and the formation of N
2 was never
observed on those.[26]
According to de Vooys et al.[29] gold has low dehydrogenation capacity or
weak affinity for adsorbed N and so do not have the ability to produce N2.
The oxidation of ammonia using gold electrodes showed (considering differential electrochemical mass spectroscopy experiments) that there is the formation of N2O.[26] It was understood that especially gold electrode played
an active role in the NO and N2O production[26,30] instead of N2 production
during the oxidation process. From this point of view, it can be concluded that dehydogenated species of NH/NH2 were probably generated on the gold
surface and oxidized to NO/N2O at +0.2 V with the aid of gold nanoparticles
Figure 4a depicts the variation of the oxidation currents (at +0.2 V) of the biosensor in increasing urea concentrations ranging between 5 and 30 mM pre-pared with 100 mM phosphate buffer at different pH values (7–8–8.4–9–10). The optimum pH of the biosensor was obtained at pH 8 which was very close
Figure 4. (a) iosensor response at +0.2 V to increasing urea concentrations ranging between 5 and 30 mM in 100 mM phosphate buffer at various pH (7–10) and (b) biosensor response at +0.2 V to polymer mass between 0.5–4 µg in the presence of 5 mM of urea.
to optimum pH of the free urease.[31] As seen in Figure 4a, the response
decrease in lower and higher pH values can be attributed to denaturation of the urease and hence limited or blocked enzymatic activity. The oxidation current of ammonia decreased at more alkaline pH values contrary as expected. In conclusion, the pH experiment revealed that the enzymatic hydrolysis step for urea was more crucial than the electrooxidation of ammonia.
Enzyme immobilization on the electrode is enabled via polymeric film layer which also provides a microenvironment for controllable access of analyte. It is expected that more dense active sites of polymer for enzyme binding per unit area are formed with increasing amount of polymer loaded on the elec-trode surface. In such a case, the enzymatic reaction rate is accelerated and results in signal improvement of bioanalytical device. However, the dense polymeric layer may cause the formation of diffusion barrier against to the analyte transport. Moreover, it can hinder the electron transfer on the elec-trode surface, especially if it is a chemically synthesized semi/nonconductive polymer layer.[28,32] Therefore, it is necessary to optimize the polymer
quantity to be coated on the electrode.
For this purpose, biosensor electrodes were prepared in a series by coating their surfaces with different amounts of poly(propylene-co-imidazole)/gold nanoparticles (0.5–0.75–1–1.5–2 and 4 µg). Each electrode was tested in 5 mM urea solution prepared with 100 mM pH 8 phosphate buffer. The generated oxidation currents of the biosensors are presented in Figure 4b. The current increased with the increasing polymer amount up to 1 µg, then decreased at polymer quantities higher than 1 µg. This decrease can be attributed to steric limitations associated with the substrate diffusion and the fact that the nonconducting polymer is likely to block the electron flow when it is coated as a thicker layer.
Analytical figures of merit for urea detection
The differential pulse voltammetry experiments (according to the setup given in the “Apparatus and electrochemical measurements” section) were conduc-ted at urea concentrations in the range of 0.1–50 mM. The peak current was plotted against the logarithm of urea concentration as a linear curve as shown
in Figure 5. The biosensor current presented a linearity to urea concentrations
between 0.1 and 30 mM (y = 23.62 log[urea] + 35.7, r2 = 0.99 and standard deviation of the slope = 1.88). The linear relationship was degraded above 30 mM. The analytical parameters obtained from the previously published urea detection reports are listed in Table 1.
The table shows that the linear range of this biosensor was wider than the other reports. Extensive efforts were made to utilize nanomaterials/metallic nanoparticles to improve the signal strength of these biosensors. It is known
that the main disadvantage of these types of electrodes is saturation and fast poisoning of the electrode surface[19,22] which probably results in narrow
measurement range. The detection limit calculated from the urea calibration plot was found to be 36 µM with a signal-to-noise ratio of 3 which was lower in comparison to other reports (Table 1).
The accuracy of the biosensor calculated according to the formula, given as (measured concentration from the calibration/actual concentration) × 100,[33]
was found to be 88.3%. The reproducibility of successive tests using the same biosensor was investigated. Eighteen successive measurements of the biosen-sor were performed in 5 mM urea solution. The relative standard deviation of the currents of these eighteen measurements was 2.43%.
The long-term stability was studied over a 75-day period by monitoring the amperometric response of the biosensor to 5 mM urea solution with everyday usage. The biosensor response retained 97% of its initial response, implicating good long-term storage stability after the usage in this time period. The bio-sensor electrode was stored in 100 mM pH 8 phosphate buffer in refrigerator. It is obvious that the biosensor displayed good functionality with regard to linear range, operational stability and storage stability.
Our preliminary experiments showed that no response was observed from the poly(propylene-co-imidazole) (without gold nanoparticles)-coated electro-des in urea solutions. This can be attributed to favorable morphology of the gold nanoparticles embedded polymeric film layer. The combination of gold nanoparticles brings together their unique properties generating a new nano-composite with excellent characteristics such as providing a stable surface for
Figure 5. Calibration curve constructed from the amperometric response of the biosensor to urea concentration ranging between 0.1 and 30 mM by differential pulse voltammetry. Conditions: applied potential range of 0 V and +0.8 V, voltage increment of 0.002 V, amplitude of 0.1 V, pulse width of 0.06 s, sample width of 0.02 s and pulse period of 0.75 s.
Table 1. Analytical characteristics of previously reported urea biosensors. Matrices Electrode Linear range (mM) Detection limit (µM) Accuracy% Relative standard deviation % Storage stability (activity retained) Voltage Reference Multiwalled carbon nanotubes/ Pt-Rh/urease/polyion complexes Indium tin oxide glass 0.05–20 50 – – Retained 50% in 20 days 0.45 Rhodinized polymer membrane Pt 0.1–2.6 50 – 5.1 Retained 86.8% in 27 days 0.60 Acrylonitrile/chitosan/Rhodium/ urease Pt 1.6–23 500 – 5.9 Retained 93% in 50 days 0.8 Ferrocene-poly(amidoamine)/ multiwalled carbon nanotubes/ urease Pencil graphite 0.2–1.8 50 95–106 1.95–2.54 Retained 62% in 3 days 0.35 Polyaniline/multiwalled carbon nanotubes/urease Pencil graphite disk 0.07–10 40 – 2.6 Retained 50% in 15 days 0.30 Polyamidoamine grafted multiwalled carbon nanotube dendrimers/urease Au 1–20 400 108.8– 111.5 2.79–3.87 Retained 83% in 15 days 0.45 Sulfonated graphene/polyaniline/ urease Indium tin oxide glass 0.12–12.3 50 96–116 – Retained 81% in 15 days between −0.2 and +0.4 Poly(propylene-co -imidazole)/gold nanoparticles/urease Au 0.1–30 36 88.3 2.43 Retained 97% in 75 days 0.2 Present work 13
The other report was based on the urea biosensor fabricated with mercap-topropionic acid capped gold nanoparticles/polypyrrole nanocomposite indium tinoxide electrode. The linear range and relative standard deviation were 0.01–10 mM and 1.4–3.9%, respectively, in this paper. The authors stated that no significant changes in response of the biosensor were observed for 45 days and a gradual decrease in response was seen thereafter for 20 days, which subsequently further decreased rapidly.[9]
Real sample analysis and interference study
The determination of urea in real samples with the biosensor was conducted using municipal sewage wastewater which was collected from the inlet pipe of the activated sludge reactor in the Wastewater Treatment Plant of Zonguldak City. The samples were filtered to remove bacteria and other particulate metabolites (0.2 µm-diameter cellulose acetate Whatman membrane filter) prior to experiments. The sample was diluted with 100 mM pH 8 phosphate buffer by 10 times to fit the urea calibration curve and remove the interference effect of other many metabolites probably found in a wastewater. We were informed that the urea concentration of the raw municipal sewage wastewater was approximately ∼25 mg/L (0.425 mM), which corresponded to the concen-tration range in the sewage water (0.067–0.56 mM).[2]
The biosensor electrode was immersed into the diluted wastewater sample, and amperometric current at +0.2 V was recorded. These experiments were repeated three times. The urea concentration of the sample was 27 mg/L (0.45 mM) by substituting the generated current value in the calibration curve equation. The biosensor analyzed urea concentrations higher than 0.425 mM with a recovery value of 106% for three measurements. This result can be attributed to the interferants such as glucose, ascorbic acid, and some ionic species (Na+ and Cl−), which are already found in municipal wastewater.
Similar interferences were observed in previously reported biosensor studies.[18,36,39]
For this reason, an interference study was conducted using the possible inter-ferants mentioned above. The concentrations of interfering species were adjusted to match a diluted urine sample,[18] and each interferant solution (0.038 mM
separately prepared in 5 mM urea solution. The poly(propylene-co-imidazole)/ gold nanoparticles/urease electrode was immersed in interferant solutions suc-cessively, and differential pulse voltammetry graphs were recorded (Figure 6). A 5 mM urea solution (without interferant) and interferant currents were com-pared with each other. The results showed that no interferences were observed from the ascorbic acid and glucose while the current response was 3.2% higher for both Na+ and Cl− ion than for the response from 5 mM urea. The biosensor
was also operated in 5 mM urea solution containing all interferants (0.038 mM ascorbic acid +0.0167 mM glucose +1.76 mM NaCl). The response obtained from this solution was 6.8% higher than for a 5 mM urea solution without the coexisting species.
Conclusion
A new urea biosensor based on the urease immobilized poly(propylene-co- imidazole)/gold nanoparticles electrode was fabricated. The liberated nonelectroactive ammonium ions were oxidized on the gold electrode with differential pulse voltammetry technique at exactly +0.2 V. A linear response of the current to the logarithm of urea concentration between 0.1 and 30 mM was observed. The biosensor provided a detection limit of 36 µM, a relative standard deviation of 2.43% (n = 18), and a measurement accuracy of 88.3%. The maximum response was obtained at the polymer quantity of 1 µg and at pH 8. The working electrode detected the urea concentration in the municipal wastewater with a recovery value of 106%. No interference effect was observed from ascorbic acid and glucose, whereas 3.2% higher cur-rent was observed from 5 mM urea solution in the presence of 1.76 mM Na+ Figure 6. Interferences due to 0.038 mM ascorbic acid, 0.0167 mM glucose, 1.76 mM Na+, and 1.76 mM Cl− in 5 mM of urea solution. Conditions: applied potential range of 0 V and +0.8 V, voltage increment of 0.002 V, amplitude of 0.1 V, pulse width of 0.06 s, sample width of 0.02 s, and pulse period of 0.75 s.
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