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Urea impedimetric biosensor based on reactive RF magnetron sputtered zinc oxide nanoporous transducer
Electrochimica Acta 146 (2014) 538–547
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Urea impedimetric biosensor based on reactive RF magnetron sputtered zinc oxide nanoporous transducer Sayed Ahmad Mozaffari ∗ , Reza Rahmanian, Mohammad Abedi, Hossein Salar Amoli Thin Layer and Nanotechnology Laboratory, Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), P.O. Box 33535-111, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 19 May 2014 Received in revised form 18 August 2014 Accepted 22 August 2014 Available online 18 September 2014 Keywords: Nanoporous ZnO thin film Urea biosensor Reactive RF magnetron sputtering Electrochemical impedance spectroscopy
a b s t r a c t 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. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Urea [(NH2 )2 CO] serves an important role in the metabolism of nitrogen-containing compounds and is the main nitrogencontaining substance in the urine of mammals. Urea is widely distributed in nature, and its analysis is considerable interested in clinical and agricultural chemistry. The normal level of urea in blood serum is from 2.5 to 7.1 mM and it is used as a marker of renal function, though it is inferior to other markers such as creatinine because blood urea levels are influenced by other factors such as diet and dehydration [1]. Increasing in urea level in blood and urine can cause urinary tract obstruction, dehydration, shock, burns, and gastrointestinal bleeding, whereas reducing in urea level may be seen in nephritic syndrome, cachexia and hepatic failure [2]. The assay of urea can be followed by many different techniques as spectrometry [3–5], potentiometry [6–9], conductometry [10–12], coulometry [13] and amperometry [14]. Nowadays several attempts have been widely done to develop urea electrochemical biosensor, due to their inherent advantages such as robustness, easy miniaturization, excellent detection limits, and requirement for very small amounts of analyte [15–20].
∗ Corresponding author. Tel.: +98 21 56276637; fax: +98 21 56276265. E-mail addresses: [email protected], [email protected] (S.A. Mozaffari). http://dx.doi.org/10.1016/j.electacta.2014.08.105 0013-4686/© 2014 Elsevier Ltd. All rights reserved.
Numerous types of electrochemical biosensors have been used for the detection and estimation of urea concentration based on Urs enzyme which utilize the catalyzed hydrolysis of urea [21–25], following by potentiometric assessments. Potentiometric biosensors based on Urs have been the most preferred choice due to simple mode of detection based on either detection of ammonium ions or pH change. The high potassium ion interference is the major disadvantage of these electrodes [26–28]. Besides, several attempts have been done for development of urea determination based on amperometric methods due to its effectiveness, simplicity, ease of calibration, etc [29–31]. The electrochemical impedance spectroscopy (EIS) is a powerful, non-destructive and informative technique to study electrical and electrochemical properties of the interfaces which has been proved to be a promising method for impedimetric biosensing assessment due to its reliability via electrochemical signal transduction and capability to monitor electrode surface modifications just by looking impedance curves [32–37]. In addition to using impedimetric assessment as an efficient signal transduction method, the construction of transducer surface has significant effect on the analytical performance of biosensor. Nanostructured metal oxides such as zinc oxide (ZnO) have been recently used for fabrication of transducer surface of biosensors because of their unique ability to promote electron transfer between electrode and active site of desired enzyme.
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Nanostructured ZnO can be prepared using sol-gel [38], electrophoresis [39], RF sputtering [40], hydrothermal [41], and molecular beam epitaxy [42] techniques. In this study an easy regenerable FTO/Nano-ZnO/Urs biosensor was fabricated by utilizing cavities of nanoporous ZnO thin film as an excellent platform for enzyme immobilization in order to retaining maximum specificity of biocatalyst and analyte interaction, and having minimum loss of enzyme activity. Zinc oxide nanoporous thin films were prepared by reactive RF magnetron sputtering technique onto conductive FTO coated glass (FTO/Nano-ZnO), and urea biosensor were prepared by gentle treatment of FTO/NanoZnO matrix with Urs (FTO/Nano-ZnO/Urs). Effect of urea/urine on the electrical properties of Urs immobilized ZnO thin films were studied by varying potential across the film and measuring the corresponding current. Urea measurements were carried out by an impedimetric method utilizing FTO/Nano-ZnO/Urs electrode served as sensing electrode. The proposed FTO/Nano-ZnO/Urs biosensor showed a fast response time of less than 4 seconds (s) and retained good enzymatic activity for more than three weeks when kept at 4 ◦ C temperature when not in use. 2. Experimental 2.1. Materials and apparatus A fluorine-doped SnO2 conductive glass purchased from Dyesol Company. Urs (urease enzyme, E.C.3.5.1.5 from Jack Bean 100 U mg-1 ), Urea (ACS reagent 99.9%), and all chemicals used in this project were of analytical reagent grade and purchased from Sigma Aldrich. Phosphate buffer solution (PBS) was prepared from K2 HPO4 and KH2 PO4 and the pH was adjusted to 7.4. Urs solution was prepared in PBS, 0.1 M, pH 7.4 containing 100 units for 3 h. A stock solution of urea was prepared in 0.1 M PBS, and stored at 4 ◦ C. The low concentration standard solutions of urea were freshly prepared before the measurements. The deposition of ZnO thin films was carried out in a reactive RF magnetron sputtering system (Nano-structured Coatings Co., Tehran, Iran) using 99.999% pure Zn target under different conditions. The surface analysis of ZnO thin film was carried out using a Tescan Mira II FE-SEM. Voltammetric and impedimetric experiments were performed with a potentiostat/galvanostat (PGSTAT. 302N, Autolab, Eco-Chemie, The Netherlands). All electrochemical experiments were carried out in a conventional three electrode system at ambient temperature. FTO/Nano-ZnO/Urs electrode served as working electrode (surface area = 1 cm2 ), Pt and SCE electrodes were used as counter and reference electrodes, respectively. The electrochemical cell was placed in a Faraday cage to eliminate any environmental stray effects. For EIS measurements, 10 mV peak-topeak ac amplitude was applied at -0.45 V dc potential versus SCE, a range of frequencies from 100 kHz to 10 mHz was scanned, and the impedances were recorded. The analysis of EIS data was performed using Zview/Zplot (Scribner Associates, Inc.) on the basis of Macdonald’s algorithm (LEVM 7) using a complex non-linear least square (CNLS) approximation method [43]. 2.2. Fabrication of FTO/Nano-ZnO/Urs biosensor Immobilization of enzymes is an important feature in designing the bio recognition part of enzyme based biosensors. Enzyme immobilization appears as a key factor to develop efficient biosensors with appropriate performances such as good operational and storage stability, high sensitivity, high selectivity, short response time and high reproducibility. Immobilized biomolecules have to maintain their structure, their function, to retain their biological activity after immobilization, to remain tightly bound to the surface
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and not to be desorbed during the use of biosensor. Having maximum retaining specificity of biocatalyst and analyte interaction and minimum loss of enzyme activity are determinative keys in the selection of enzyme immobilization protocol. Moreover, an ideal biosensor has to be stable for long term application. Various immobilization strategies can be envisioned as: adsorption, entrapment and encapsulation, covalence and cross-linking. Covalent attachment of the biomolecule to the substrate is one of the most stylish immobilization methods accessible, although others as adsorption, entrapment and cross-linking are repeatedly used [2,44]. Urs was immobilized by soaking the FTO/Nano-ZnO in a PBS, 0.1 M, pH 7.4 containing 0.2 mg mL-1 of Urs (100 units of Urs) for 3 h at ambient temperature. The FTO/Nano-ZnO/Urs was then washed and kept in PBS until use. A covalently attachment of Urs with FTO/Nano-ZnO was expected to provide FTO/Nano-ZnO/Urs biosensor [45]. Step by step monitoring of FTO/Nano-ZnO/Urs biosensor fabrication were performed using electrochemical methods such as current vs. potential (I-V curve), CV and EIS techniques. After completing these steps, the biosensors were applied as working electrode for urea determination using impedimetric measurements. 2.3. Determination of enzyme activity after immobilization After Urs immobilization, the electrical property of FTO/ Nano-ZnO/Urs was studied to determine the enzyme activity. For this measurement, a cell was made, which consists of gold wire ( 0.3 mm, 5 cm length) as an electrode and FTO/Nano-ZnO/Urs as another electrode. Potential from 0.0 to 1.0 V was applied to the film and the corresponding dc current was measured. The ratio of voltage to current is used as a measure of enzyme activity. For each sample, five sets of measurement were carried out at the ambient temperature. Urea solutions with various concentrations of urea such as 0.0, 0.83, 11.62 mM and 23.24 mM were prepared in PBS and used as an electrolyte. Amount of electrolyte was kept constant as 10 ml for all measurements. Since the amount of urea with respect to PBS has changed with increasing molar concentration, this effect would reflect in the enzyme activity curves indicating the variation due to urea, as presented in Fig. 1. The slope of I-V curve (I/V) was used as a measure of enzyme activity of the biosensor [45]. Finally, human urine sample diluted with buffer solution from 1:9 to 5:5 (i.e. a total 10 mL of solution was prepared where urine concentration was varied from 1 mL to 5 mL). Enhancement in the sample current is obvious after immobilization i.e. the film conductivity increases after Urs immobilization. As can be seen in Fig. 1, it is the clear evidence that the change in response is owing to the Urs immobilization. Although urea can react directly with Nano-ZnO, the Urs–urea reaction kinetics (catalytic reaction) are different than that of Nano-ZnO–urea, therefore, the extent of reaction may not be same as that after Urs immobilization. This increase is attributed to the catalytic reaction of Urs–urea as below; Urs
NH2 CONH2 + 2H2 O−→2NH4+ + CO32−
(1)
This reaction results two NH4 + and CO3 2− from uncharged urea which raises the conductivity of the FTO/Nano-ZnO/Urs by providing excess electron to the conduction band. The effect of urea concentration is clearly apparent from the enzyme activity curves, so increasing enzyme activity is observed, with increasing urea concentration. The high activity FTO/Nano-ZnO/Urs is due to high surface area of nanoporous ZnO thin film as a family of the richest nanostructure. Analogous to that of urea sensing, FTO/Nano-ZnO electrode was tested for urine samples before and after enzyme immobilization. Urine was diluted with buffer solution from 1:9 to 5:5. Increase in
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Fig. 1. Typical I–V curves showing the effect of urease immobilization on ZnO films with increasing urea concentration, (A) before and (B) after Urs immobilization. The inset of A shows a higher magnificent I-V curve from 0.4 to 0.8 V.
sensitivity is noticed with increasing urine concentration (data not shown). Immobilization results in an increase in the current with applied voltage which supports the abovementioned reaction in Eq. 1.
mentioned parameters was varied while keeping other parameters constant. Film thickness has been shown to have great influences on many physical properties, such as mechanical properties and electrical resistivity [50]. In this project, the sputtering conditions are chosen to be optimum for ZnO thin film formation with the lowest electrical resistivity. In Fig. 2A the dependence of ZnO thin film electrical resistivity on film thickness from 100 to 400 nm is shown. Increasing of ZnO thin film thickness from 100 to 400 nm resulted in decreasing its electrical resistivity of film. Increasing film thickness following by annealing process reduces defects in the film and causes low resistivity of thin film. For this study the film thickness of about 300 nm was selected as the optimum thickness. The Ar:O2 gas flow ratio is very effective paprameter in reactive RF magnetron sputtering for obtaining the nanoporous ZnO thin film with lower electrical resistivity. By decreasing the amount of oxygen in gas flow ratio, lower electrical resistivity of thin film will be observed (Fig. 2B). This behavior depicts the increasing or decreasing effect of oxygen amount in the sputtering gases ratio on the formation of ZnO:Zn phases in the thin film, which finally leads to higher or lower electrical resistivity of thin film. Total gas pressure has a vital effect in plasma formation, and directly influences on the deposition rate and accordingly the porosity and physical homogeneity of ZnO thin film (Fig. 2C). Total gas pressure and deposition rate are very important to optimize concurrently to achieve better homogenous thin film. RF sputtering power strongly affects on the electrical resistivity of ZnO thin film (Fig. 2D). Lower RF sputtering power will decrease the amounts of Zn sputtered atoms from the target and consequently increases the possibility of reactive formation of ZnO molecules outgoing towards the substrate which leads to higher electrical resistivity of thin film. As much as the RF sputtering power increases up to 80 Watts (W), the amounts of non-reacted Zn sputtered atoms and the deficiencies of oxygen in the substrate will increase and leads to achieve lower electrical resistivity. However, the excessive supply of RF sputtering power over than 80 W may cause thin film degradation by the bombardment of highly energized particles, resulting an inhomogeneous thin film. The optimum conditions for obtaining uniform nanoporous ZnO thin film were considered in continue as 300 nm, 20:1, 3.0 × 10-2 Torr and 80 Watts for sputtered film thickness, Ar:O2 gas flow ratio, mixed gases pressure (total pressure), and sputtering power, respectively.
3. Results and discussion 3.1. Fabrication of FTO/Nano-ZnO matrix by reactive RF magnetron sputtering
3.2. Optical transmission and morphological characterization of sputtered ZnO thin film
The basic principles of reactive RF magnetron sputtering have been described elsewhere [46–48]. In reactive sputtering, the deposited thin film is formed by chemical reaction between the target materials and a reactive gas which is introduced into the vacuum chamber. Oxide and nitride films are often fabricated using reactive sputtering [49]. The composition of thin film can be controlled by varying the relative pressures of the inert and reactive gases. In this study, reactive magnetron sputtering was carried out in argon and oxygen mixed gas atmosphere by supplying RF power at a frequency of 13.56 MHz (a frequency chosen because of its noninterference with radio-transmitted signals). In order to obtain very uniform nanoporous ZnO thin film, four sets of depositions were carried out at various film thickness (100 to 400 nm), Ar:O2 gas flow ratio (5:5 to 20:1 sccm/min), mixed gases pressure (total gas pressure from 5.0 × 10-3 to 5.0 × 10-2 Torr), and sputtering power (40 to 90 Watts). In every set of samples, only one of the above
In Fig. 3 typical optical transmission spectrum recorded for ZnO thin film is shown. The sharp absorption onset and the high transmission values at wavelengths above 338 nm exhibit the optical quality and low concentration of defects such as pits and voids of the ZnO thin film. The periodic and continuous annealing of ZnO thin film at 250 ◦ C cause better aligned textured films which is required for a homogeneous matrix for enzyme immobilization in biosensor fabrication. Fig. 4 (A-C) illustrates the characterization of surface morphology of nanoporous ZnO thin film by FE-SEM at different magnifications. The revealed feature confirms the uniform pores are open and extend through the surface into the bulk which is believed to play an important role in enzyme immobilization. In addition, higher magnification image of the surface shows the nanosize distribution of ZnO particles provides high surface area nanostructured matrix which can lead to a greater amount of an immobilized enzyme on the surface (Fig. 4D).
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Fig. 2. Effect of film thickness (A), Ar:O2 sputtering gases ratio (B), total gas pressure (C), and RF magnetron sputtering power (D) on FTO/Nano-ZnO characteristics.
3.3. Impedimetric and voltammetric characterization of FTO/Nano-ZnO/Urs biosensor in the absence of urea EIS measurements were used to trace the events during the formation of FTO/Nano-ZnO/Urs biosensor. EIS is a powerful technique for studying electrical and electrochemical interfacial properties of a large variety of systems. Concerning the use of EIS to study thin films in contact with electrolyte solutions, three different contributions, bulk, interfacial and electrolytes may be determined [32–35,51]. The interfacial behavior is widely described by Randles
Fig. 3. Transmission spectrum recorded for sputtered ZnO thin film.
equivalent circuit (Fig. 5A) [52], in such model, the diffusion or Warburg impedance (Zw ) [53,54] is in series with the charge transfer resistance (Rct ) and both are generally in parallel with the double layer capacitance (Cdl ), and in series with the solution resistance (Rs ), while in Fig. 5B modified Randles model in which the doublelayer capacitance is replaced by constant phase element (CPE) is observed. In this study, modified Randles model was used for the approximation of EIS data of impedimetric urea detection, and the chai-square of 2 = 1.70 × 10−4 was obtained for data analysis. Fig. 6A shows the impedance spectra presented as Nyquist plots (-Z vs. Z ) upon the layer by layer assembly of FTO/Nano-ZnO/Urs and characterization of them in urea-free PBS containing 5 mM [Fe(CN)6 ]3-/4- , which is reflected by the appearance of a semicircle part on the EIS spectrum, corresponding to a charge transfer resistance (Rct ) of a marker in electrolyte solution. Three layers of (a) FTO, (b) FTO/Nano-ZnO, and (c) FTO/Nano-ZnO/Urs result in a change of the electron-transfer resistance from (a) Rct = 0.96 k to (b) Rct = 4.6 k and (c) Rct = 5.4 k, respectively. The Rct value of ZnO thin film (4.6 k, curve b) with respect to FTO electrode (0.96 k, curve a) is higher, which is related to the lower electrical conductivity of semiconducting ZnO nanoporous thin film. However, the Rct value further increases (from 4.6 k to 5.4 k, curve c) after the immobilization of Urs onto FTO/Nano-ZnO electrode due to insulating characteristics of Urs [17,45,55,56]. Cyclic voltammetric study of layer by layer assembly of FTO/Nano-ZnO/Urs in urea-free PBS containing 5 mM [Fe(CN)6 ]3-/4− is shown in Fig. 6B. The magnitude of anodic peak current for FTO/Nano-ZnO electrode (0.171 mA, curve b) is lower than that of bare FTO electrode (2.09 mA, curve a) reveals that ZnO thin film shows an obstruction to the electron transfer
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Fig. 4. FE-SEM images of deposited ZnO thin film at different magnifications (A-C), and the particle size distribution histogram (D).
Fig. 5. Equivalent circuits used for impedance data approximation: (A) Randles model and (B) modified Randles model in which the double-layer capacitance is replaced by CPE. The Rs , Rct , Cdl and Zw represent the solution resistance, charge transfer resistance, double-layer capacitance and Warburg impedance, respectively.
from/to the electrode surface due to its semiconducting behavior. The magnitude of peak current response for FTO/Nano-ZnO/Urs electrode shows decreases in electron transfer from/to the electrode surface (0.144 mA, curve c) which is shown the insulating characteristics of Urs [17,45,55,56]. These results are consistent with the results of studying stepwise layer by layer assembly were obtained by EIS experiments. The scan rate dependency of voltammetric studies conducted on the FTO/Nano-ZnO/Urs biosensor as a function of scan rate (0.01-0.1 V/s) reveals that the magnitude of current response is linearly dependent on the square root of the scan rate (Fig. 6B, inset). This suggests that the electrochemical reaction is controlled diffusion process with facile charge transfer kinetics. A schematic of the proposed mechanism for electron transfer at the FTO/Nano-ZnO/Urs electrode in the presence of [Fe(CN)6 ]3-/4− redox marker is shown in Fig. 7. By comparing curve b and c in Fig. 6A and 6B which represent the EIS and CV spectra of [Fe(CN)6 ]3−/4− redox reaction at FTO/Nano-ZnO and FTO/NanoZnO/Urs electrodes, it can be observed that Urs layer decreases direct electron transfer with electrode substrate compare with ZnO thin film.
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Fig. 6. (A) The Nyquist plots (-Z vs. Z ) obtained for the Faradaic impedance measurements in urea-free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3-/4− on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c). (B) Cyclic voltammogram obtained in urea-free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3-/4− on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c) at the scan rate of 0.1 V/s. The inset shows the effect of scan rate (0.01- 0.1 V/s) on the response current of the FTO/Nano-ZnO/Urs biosensor.
3.4. Impedimetric and voltammetric characterization of FTO/Nano-ZnO/Urs biosensor in the presence of urea In addition to investigation of layer by layer assembly of FTO/Nano-ZnO/Urs electrode in the presence of [Fe(CN)6 ]3-/4− as a redox probe, the electrochemical behavior of each layer and final biosensor verified in the presence of urea. EIS technique was employed to study the Rct at the interfacial properties of the FTO/Nano-ZnO/Urs biosensor via urea (Fig. 8A). The Rct value in the presence of urea at ZnO thin film (55 k, curve b) is lower than that of FTO electrode (197 k, curve a) which is due to direct urea reaction with Nano-ZnO [45]. Further decreasing in Rct value (15.8 k, curve c) was observed after immobilization of Urs onto FTO/Nano-ZnO matrix due to fast hydrolysis of urea by enzyme, which can represent faster, repeatable and reproducible response of FTO/Nano-ZnO/Urs compare with slower, unpredictable and non-reproducible response of FTO/Nano-ZnO. Whereas the rate of urea hydrolysis at FTO/Nano-ZnO is not at all up to the extent of enzymatic hydrolysis at FTO/Nano-ZnO/Urs. Cyclic voltammetric studies of layer by layer assembly of FTO/Nano-ZnO/Urs in PBS containing 0.83 mM urea are shown in Fig. 8B. By comparing curve a and b, it can be observed that an
Fig. 7. Schematic of the mechanistic model proposed for electron transfer at the (a) FTO, (b) FTO/Nano-ZnO, and (c) FTO/Nano-ZnO/Urs biosensor in urea free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3-/4− .
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Fig. 9. Schematic representation of the detection mechanism of FTO/Nano-ZnO/Urs urea biosensor.
Fig. 8. (A) The Nyquist plots (-Z vs. Z ) obtained for the impedance measurements in PBS (pH 7.4) containing 0.83 mM urea on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c); the inset shows the magnified curve c. (B) Cyclic voltammogram obtained at 0.1 V/s scan rate in PBS (pH 7.4) containing 0.83 mM urea on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c) at the scan rate of 0.1 V/s; the inset shows the magnified curve a.
anodic peak is appeared for FTO/Nano-ZnO in the presence of urea which is due to partial hydrolysis of urea at ZnO thin film [45]. The magnitude of anodic peak current response increases greatly by Urs on FTO/Nano-ZnO/Urs biosensor (2.0 × 10−2 mA, curve c). These results are consistent with the results of studying stepwise layer by layer assembly obtained by EIS experiments in the presence of urea which supports the obtained I-V curves in section 2.3. 3.5. ZnO nanostructure and expected sensing mechanism The stability and performance of the enzyme on the matrix surface strongly depends on the immobilization and surface chemistry. Among the various immobilization techniques, covalent attachment of the biomolecule to the substrate is one of the most stylish immobilization methods. The optimum buffer pH for hydrolysis of urea by Urs was considered as 7.4 at which Urs enzyme retains its maximum activity and natural structure that is required to improve detection limit and sensitivity for urea detection [57–62]. Many researchers clarify the change in the electrical conductance of a highly porous semiconducting film sensor in the presence of toxic gases due to the reactions occurring on the surface [45,63]. Fig. 9 shows the mechanism. At first, atmospheric oxygen molecules are physisorbed on the surface sites, which get ionized by extracting an electron from the conduction band while moving from one
site to another, and are thus ionosorbed on the surface as Oads − . This leads to a decrease in the conductance of the transducer, as indicated by an increase in potential barrier at the grain boundaries as illustrated in Fig. 9A. Upon exposing FTO/Nano-ZnO/Urs to urea, an enzymatic reaction takes places between Urs and urea which can be represented as Fig. 9B. Urs
NH2 CONH2 + 2H2 O−→2NH4+ + CO32− − R + Oads → RO + e−
(2) +
It results in production of NH4 ion, which reacts with the surface adsorbed oxygen (Oads − ) and thereby release the trapped electron to the conduction band of ZnO as indicated by the decrease of potential barrier at grain boundary as shown in Fig. 9C. Further, when reducing gas (R) molecules (ammonia in the present case) react with pre-adsorbed negatively charged oxygen adsorbates, the trapped electrons are given back to conduction band of the material. The energy released during the decomposition of adsorbed ammonia molecules would be sufficient for the electrons jumping up into the conduction band so as to increase conductivity of the biosensor [45,63]. The results obtained by CV (Fig. 8B) supports the above-mentioned sensing mechanism, which clearly points to an oxidation and reduction process. 3.6. Working curve for impedimetric measurements The electrochemical response of FTO/Nano-ZnO/Urs biosensor has been impedimetrically measured as a function of urea concentration. As shown in Fig. 10A, the magnitude of Rct decreases on
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Fig. 10. (A) The Nyquist plots (-Z vs. Z ) obtained on the FTO/Nano-ZnO/Urs biosensor in the presence of different concentration of urea: (a) 0.83 mM; (b) 1.66 mM; (c) 3.32 mM; (d) 4.98 mM; (e) 6.64 mM; (f) 8.30 mM; (g) 9.96 mM; (h) 11.62 mM; (i) 13.28 mM; (j) 14.94 mM; (k) 16.60 mM; (l) 18.26 mM; (m) 19.92 mM; (n) 21.58 mM; (o) 23.24 mM. (B) Variation of charge transfer resistance (Rct ) as a function of urea concentration represents calibration curve.
addition of urea concentration from 0.83 to 23.24 mM (curve a to curve o). The quick response indicates that porous structure of ZnO matrix yields a very low mass transport barrier and results in a rapid diffusion from solution to the enzyme with keeping its bioactivity. At physiological pH (pH = 7.4), ZnO thin film (isoelectric point (IEP) ∼ 9) possesses a positive charge surface which provide a friendly micro-environment for negatively charge urease (IEP∼5.9) to retain its activity and also promote fast electron transfer [61]. The impedimetric studies clearly indicate that the process is a reversible process and confirms the Urs–urea enzymatic catalysis. From impedimetric studies and step by step studies, it is confirmed that the developed sensors can be re-used. The calibration curve was obtained for urea at the FTO/Nano-ZnO/Urs biosensor by monitoring its Rct responses (|Rct | = |Rct urea -Rct buffer (blank) |). Fig. 10B displays a working curve shown in a linear scale [Rct (k) = 8.813 (±0.164) + 0.637 (±0.012) × [urea] (mM); R2 = 0.995] in a concentration range of 0.83 to 23.24 mM of urea. The FTO/Nano-ZnO/Urs biosensor exhibits detection limit of 0.40 mM for urea and sensitivity of 0.637 k per mM with a linear range within 0.83–23.24 mM. These results confirm that ZnO thin film matrix provides attractive microenvironment for the enzyme immobilization to retain its desired natural activities. Several assays were made on blood serum to test the precision of the FTO/Nano-ZnO/Urs biosensor. The urea concentration was determined by the calibration curve (Table 1). Corresponding experiments were carried out with a spectrophotometric method by a local hospital. The results exhibited good consistent and precision between the two methods and confirmed the proposed
method is useful for application in real samples with good precision and accuracy. The proposed FTO/Nano-ZnO/Urs biosensor has a fast response time of less than 4s and retained good enzymatic activity for more than three weeks when kept at 4 ◦ C temperature and not in use. In addition, the stability of the FTO/Nano-ZnO/Urs biosensor was tested by comparing the catalytic ability to the urea with different storage time at 4 ◦ C. For a fixed concentration of urea, three same biosensors lost about 0.0%, 4.0% and 8.0% of their original activity after storing time of 1 day, 2 weeks and 6 weeks, respectively. Table 2 compares response characteristics of the FTO/Nano-ZnO/Urs biosensor with the other reported urea biosensors. The biosensor of the present work clearly shows good linear range, detection limit and response time respect to results published in previous studies [64–71].
Table 1 Determination of urea level in blood serum. Method
Determined by Spectrophotometry (mM) Measured by FTO/Nano-ZnO/Urs (mM) R.S.D.a (n = 5) (%) a
Sample number 1
2
3
4
5
2.52b 2.53 1.15
4.24 4.19 2.21
5.56 5.54 1.86
6.82b 6.85 2.55
4.51 4.53 2.1
R.S.D.: Relative Standard Deviation. Represents the hyper- or hypo-level of urea in blood serum, the normal urea level in blood serum is between 2.50 and 7.10 mM. b
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Table 2 Analytical characteristics of FTO/Nano-ZnO/Urs biosensor compared with other reported values of urea biosensors in the literature. Ref.
[64] [65] [66] [67] [68] [69] [70] [71] this work
Detection method
Cyclic Voltammetry Amperometric Potentiometric Conductometric Optical Colorimetric Manometric Potentiometric Impedimetric
Response Characteristics RT
D.L. (mM)
D.R. (mM)
2 min 10 min 1–5 min 5 min 2 min 4s
2.24 0.04 0.83 3.32 0.99 1.66 1.99 1.66 0.40
1.66–13.28 0.49–4.98 0.83–23.24 3.32–19.92 1.66–66.4 1.99–109.56 1.66–16.43 0.83–23.24
Enzyme immobilization strategy
Matrix
Physisorption Covalent binding Entrapment Physical adsorption Entrapment Ionic binding Free enzyme Physical adsorption Electrostatic adsorption
ZnO nanoparticles Polyaniline Nafion/Au/ceramic composite film Gelatin Polyaniline-Poly (n-butylmethacrylate) Cellulose membrane Dead cellulose Whatman paper ZnO nanoporous thin film
Detection Range [D.R.], Detection Limit [DL], Response Time [RT].
4. Conclusions As a result, the performance of biosensor depends on (i) the amount of immobilized enzyme which is related to the morphology and surface area/volume ratio of matrix, and (ii) the porosity of matrix which is providing rapid diffusion from solution to the immobilized enzyme. In this study, reactive RF sputtered nanoporous ZnO thin film with excellent uniformity of thickness has been used for fabrication of the electrochemical urea biosensor via immobilization of Urs. Fabricated biosensor showed high sensitivity, low detection limit, broad dynamic range, fast response time, high stability and long term reproducibility by retaining enzyme activity due to the suitable immobilization of Urs in nanoporous ZnO thin film. Porous nanomaterials provide a larger surface area available for enzyme binding and decrease the diffusion distance for the substrate to access the immobilized enzyme, which may improve the performance of the enzyme electrode. Besides, the close enzyme–substrate contact through covalent bonding, may promote faster detection kinetics, once the electrochemically detectable species (ammonia) is produced closer to the transducer, reducing diffusion resistance, and thus increasing sensitivity and diminishing response times. Impedimetric analysis of the proposed biosensor indicated a fast response time of less than 4s with a wide linear range from 0.83 to 23.24 mM and limit of detection as 0.40 mM for urea. Acknowledgement S. A. Mozaffari acknowledges the support of the Iranian Research Organization for Science and Technology, Iran Nanotechnology Initiative Council (INIC) and Nano-structured Coatings Co. for this research. References [1] J. Traynor, R. Mactier, C. Geddes, How to measure renal function in clinical practice, BMJ 333 (2006) 733–737. [2] M. Singh, N. Verma, A.K. Garg, N. Redhu, Urea biosensors, Sens. Actuators B 134 (2008) 345–351. [3] G.P. Nikoleli, D.P. Nikolelis, C. Methenitis, Construction of a simple optical sensor based on air stable lipid film with incorporated urease for the rapid detection of urea in milk, Anal. Chim. Acta 675 (2010) 58–63. [4] H.C. Tsai, R.A. Doong, Simultaneous determination of pH, urea, acetylcholine and heavy metals using array-based enzymatic optical biosensor, Biosens, Bioelectron. 20 (2005) 1796–1804. [5] B. Kovacs, G. Nagy, R. Dombi, K. Toth, Optical biosensor for urea with improved response time, Biosens. Bioelectron. 18 (2003) 111–118. [6] G.P. Nikoleli, M.Q. Israr, N. Tzamtzis, D.P. Nikolelis, M. Willander, N. Psaroudakis, Structural characterization of graphene nanosheets for miniaturization of potentiometric urea lipid film based biosensors, Electroanalysis 24 (2012) 1285–1295. [7] A. Ali, M. AlSalhi, M. Atif, A.A. Ansari, M.Q. Israr, J. Sadaf, E. Ahmed, O. Nur, M. Willander, Potentiometric urea biosensor utilizing nanobiocomposite of chitosan-iron oxide magnetic nanoparticles, in: Journal of Physics: Conference Series, IOP Publishing, 2013, pp. 012024.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Urea impedimetric biosensor based on reactive RF magnetron sputtered zinc oxide nanoporous transducer Sayed Ahmad Mozaffari ∗ , Reza Rahmanian, Mohammad Abedi, Hossein Salar Amoli Thin Layer and Nanotechnology Laboratory, Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), P.O. Box 33535-111, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 19 May 2014 Received in revised form 18 August 2014 Accepted 22 August 2014 Available online 18 September 2014 Keywords: Nanoporous ZnO thin film Urea biosensor Reactive RF magnetron sputtering Electrochemical impedance spectroscopy
a b s t r a c t 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. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Urea [(NH2 )2 CO] serves an important role in the metabolism of nitrogen-containing compounds and is the main nitrogencontaining substance in the urine of mammals. Urea is widely distributed in nature, and its analysis is considerable interested in clinical and agricultural chemistry. The normal level of urea in blood serum is from 2.5 to 7.1 mM and it is used as a marker of renal function, though it is inferior to other markers such as creatinine because blood urea levels are influenced by other factors such as diet and dehydration [1]. Increasing in urea level in blood and urine can cause urinary tract obstruction, dehydration, shock, burns, and gastrointestinal bleeding, whereas reducing in urea level may be seen in nephritic syndrome, cachexia and hepatic failure [2]. The assay of urea can be followed by many different techniques as spectrometry [3–5], potentiometry [6–9], conductometry [10–12], coulometry [13] and amperometry [14]. Nowadays several attempts have been widely done to develop urea electrochemical biosensor, due to their inherent advantages such as robustness, easy miniaturization, excellent detection limits, and requirement for very small amounts of analyte [15–20].
∗ Corresponding author. Tel.: +98 21 56276637; fax: +98 21 56276265. E-mail addresses: [email protected], [email protected] (S.A. Mozaffari). http://dx.doi.org/10.1016/j.electacta.2014.08.105 0013-4686/© 2014 Elsevier Ltd. All rights reserved.
Numerous types of electrochemical biosensors have been used for the detection and estimation of urea concentration based on Urs enzyme which utilize the catalyzed hydrolysis of urea [21–25], following by potentiometric assessments. Potentiometric biosensors based on Urs have been the most preferred choice due to simple mode of detection based on either detection of ammonium ions or pH change. The high potassium ion interference is the major disadvantage of these electrodes [26–28]. Besides, several attempts have been done for development of urea determination based on amperometric methods due to its effectiveness, simplicity, ease of calibration, etc [29–31]. The electrochemical impedance spectroscopy (EIS) is a powerful, non-destructive and informative technique to study electrical and electrochemical properties of the interfaces which has been proved to be a promising method for impedimetric biosensing assessment due to its reliability via electrochemical signal transduction and capability to monitor electrode surface modifications just by looking impedance curves [32–37]. In addition to using impedimetric assessment as an efficient signal transduction method, the construction of transducer surface has significant effect on the analytical performance of biosensor. Nanostructured metal oxides such as zinc oxide (ZnO) have been recently used for fabrication of transducer surface of biosensors because of their unique ability to promote electron transfer between electrode and active site of desired enzyme.
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Nanostructured ZnO can be prepared using sol-gel [38], electrophoresis [39], RF sputtering [40], hydrothermal [41], and molecular beam epitaxy [42] techniques. In this study an easy regenerable FTO/Nano-ZnO/Urs biosensor was fabricated by utilizing cavities of nanoporous ZnO thin film as an excellent platform for enzyme immobilization in order to retaining maximum specificity of biocatalyst and analyte interaction, and having minimum loss of enzyme activity. Zinc oxide nanoporous thin films were prepared by reactive RF magnetron sputtering technique onto conductive FTO coated glass (FTO/Nano-ZnO), and urea biosensor were prepared by gentle treatment of FTO/NanoZnO matrix with Urs (FTO/Nano-ZnO/Urs). Effect of urea/urine on the electrical properties of Urs immobilized ZnO thin films were studied by varying potential across the film and measuring the corresponding current. Urea measurements were carried out by an impedimetric method utilizing FTO/Nano-ZnO/Urs electrode served as sensing electrode. The proposed FTO/Nano-ZnO/Urs biosensor showed a fast response time of less than 4 seconds (s) and retained good enzymatic activity for more than three weeks when kept at 4 ◦ C temperature when not in use. 2. Experimental 2.1. Materials and apparatus A fluorine-doped SnO2 conductive glass purchased from Dyesol Company. Urs (urease enzyme, E.C.3.5.1.5 from Jack Bean 100 U mg-1 ), Urea (ACS reagent 99.9%), and all chemicals used in this project were of analytical reagent grade and purchased from Sigma Aldrich. Phosphate buffer solution (PBS) was prepared from K2 HPO4 and KH2 PO4 and the pH was adjusted to 7.4. Urs solution was prepared in PBS, 0.1 M, pH 7.4 containing 100 units for 3 h. A stock solution of urea was prepared in 0.1 M PBS, and stored at 4 ◦ C. The low concentration standard solutions of urea were freshly prepared before the measurements. The deposition of ZnO thin films was carried out in a reactive RF magnetron sputtering system (Nano-structured Coatings Co., Tehran, Iran) using 99.999% pure Zn target under different conditions. The surface analysis of ZnO thin film was carried out using a Tescan Mira II FE-SEM. Voltammetric and impedimetric experiments were performed with a potentiostat/galvanostat (PGSTAT. 302N, Autolab, Eco-Chemie, The Netherlands). All electrochemical experiments were carried out in a conventional three electrode system at ambient temperature. FTO/Nano-ZnO/Urs electrode served as working electrode (surface area = 1 cm2 ), Pt and SCE electrodes were used as counter and reference electrodes, respectively. The electrochemical cell was placed in a Faraday cage to eliminate any environmental stray effects. For EIS measurements, 10 mV peak-topeak ac amplitude was applied at -0.45 V dc potential versus SCE, a range of frequencies from 100 kHz to 10 mHz was scanned, and the impedances were recorded. The analysis of EIS data was performed using Zview/Zplot (Scribner Associates, Inc.) on the basis of Macdonald’s algorithm (LEVM 7) using a complex non-linear least square (CNLS) approximation method [43]. 2.2. Fabrication of FTO/Nano-ZnO/Urs biosensor Immobilization of enzymes is an important feature in designing the bio recognition part of enzyme based biosensors. Enzyme immobilization appears as a key factor to develop efficient biosensors with appropriate performances such as good operational and storage stability, high sensitivity, high selectivity, short response time and high reproducibility. Immobilized biomolecules have to maintain their structure, their function, to retain their biological activity after immobilization, to remain tightly bound to the surface
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and not to be desorbed during the use of biosensor. Having maximum retaining specificity of biocatalyst and analyte interaction and minimum loss of enzyme activity are determinative keys in the selection of enzyme immobilization protocol. Moreover, an ideal biosensor has to be stable for long term application. Various immobilization strategies can be envisioned as: adsorption, entrapment and encapsulation, covalence and cross-linking. Covalent attachment of the biomolecule to the substrate is one of the most stylish immobilization methods accessible, although others as adsorption, entrapment and cross-linking are repeatedly used [2,44]. Urs was immobilized by soaking the FTO/Nano-ZnO in a PBS, 0.1 M, pH 7.4 containing 0.2 mg mL-1 of Urs (100 units of Urs) for 3 h at ambient temperature. The FTO/Nano-ZnO/Urs was then washed and kept in PBS until use. A covalently attachment of Urs with FTO/Nano-ZnO was expected to provide FTO/Nano-ZnO/Urs biosensor [45]. Step by step monitoring of FTO/Nano-ZnO/Urs biosensor fabrication were performed using electrochemical methods such as current vs. potential (I-V curve), CV and EIS techniques. After completing these steps, the biosensors were applied as working electrode for urea determination using impedimetric measurements. 2.3. Determination of enzyme activity after immobilization After Urs immobilization, the electrical property of FTO/ Nano-ZnO/Urs was studied to determine the enzyme activity. For this measurement, a cell was made, which consists of gold wire ( 0.3 mm, 5 cm length) as an electrode and FTO/Nano-ZnO/Urs as another electrode. Potential from 0.0 to 1.0 V was applied to the film and the corresponding dc current was measured. The ratio of voltage to current is used as a measure of enzyme activity. For each sample, five sets of measurement were carried out at the ambient temperature. Urea solutions with various concentrations of urea such as 0.0, 0.83, 11.62 mM and 23.24 mM were prepared in PBS and used as an electrolyte. Amount of electrolyte was kept constant as 10 ml for all measurements. Since the amount of urea with respect to PBS has changed with increasing molar concentration, this effect would reflect in the enzyme activity curves indicating the variation due to urea, as presented in Fig. 1. The slope of I-V curve (I/V) was used as a measure of enzyme activity of the biosensor [45]. Finally, human urine sample diluted with buffer solution from 1:9 to 5:5 (i.e. a total 10 mL of solution was prepared where urine concentration was varied from 1 mL to 5 mL). Enhancement in the sample current is obvious after immobilization i.e. the film conductivity increases after Urs immobilization. As can be seen in Fig. 1, it is the clear evidence that the change in response is owing to the Urs immobilization. Although urea can react directly with Nano-ZnO, the Urs–urea reaction kinetics (catalytic reaction) are different than that of Nano-ZnO–urea, therefore, the extent of reaction may not be same as that after Urs immobilization. This increase is attributed to the catalytic reaction of Urs–urea as below; Urs
NH2 CONH2 + 2H2 O−→2NH4+ + CO32−
(1)
This reaction results two NH4 + and CO3 2− from uncharged urea which raises the conductivity of the FTO/Nano-ZnO/Urs by providing excess electron to the conduction band. The effect of urea concentration is clearly apparent from the enzyme activity curves, so increasing enzyme activity is observed, with increasing urea concentration. The high activity FTO/Nano-ZnO/Urs is due to high surface area of nanoporous ZnO thin film as a family of the richest nanostructure. Analogous to that of urea sensing, FTO/Nano-ZnO electrode was tested for urine samples before and after enzyme immobilization. Urine was diluted with buffer solution from 1:9 to 5:5. Increase in
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Fig. 1. Typical I–V curves showing the effect of urease immobilization on ZnO films with increasing urea concentration, (A) before and (B) after Urs immobilization. The inset of A shows a higher magnificent I-V curve from 0.4 to 0.8 V.
sensitivity is noticed with increasing urine concentration (data not shown). Immobilization results in an increase in the current with applied voltage which supports the abovementioned reaction in Eq. 1.
mentioned parameters was varied while keeping other parameters constant. Film thickness has been shown to have great influences on many physical properties, such as mechanical properties and electrical resistivity [50]. In this project, the sputtering conditions are chosen to be optimum for ZnO thin film formation with the lowest electrical resistivity. In Fig. 2A the dependence of ZnO thin film electrical resistivity on film thickness from 100 to 400 nm is shown. Increasing of ZnO thin film thickness from 100 to 400 nm resulted in decreasing its electrical resistivity of film. Increasing film thickness following by annealing process reduces defects in the film and causes low resistivity of thin film. For this study the film thickness of about 300 nm was selected as the optimum thickness. The Ar:O2 gas flow ratio is very effective paprameter in reactive RF magnetron sputtering for obtaining the nanoporous ZnO thin film with lower electrical resistivity. By decreasing the amount of oxygen in gas flow ratio, lower electrical resistivity of thin film will be observed (Fig. 2B). This behavior depicts the increasing or decreasing effect of oxygen amount in the sputtering gases ratio on the formation of ZnO:Zn phases in the thin film, which finally leads to higher or lower electrical resistivity of thin film. Total gas pressure has a vital effect in plasma formation, and directly influences on the deposition rate and accordingly the porosity and physical homogeneity of ZnO thin film (Fig. 2C). Total gas pressure and deposition rate are very important to optimize concurrently to achieve better homogenous thin film. RF sputtering power strongly affects on the electrical resistivity of ZnO thin film (Fig. 2D). Lower RF sputtering power will decrease the amounts of Zn sputtered atoms from the target and consequently increases the possibility of reactive formation of ZnO molecules outgoing towards the substrate which leads to higher electrical resistivity of thin film. As much as the RF sputtering power increases up to 80 Watts (W), the amounts of non-reacted Zn sputtered atoms and the deficiencies of oxygen in the substrate will increase and leads to achieve lower electrical resistivity. However, the excessive supply of RF sputtering power over than 80 W may cause thin film degradation by the bombardment of highly energized particles, resulting an inhomogeneous thin film. The optimum conditions for obtaining uniform nanoporous ZnO thin film were considered in continue as 300 nm, 20:1, 3.0 × 10-2 Torr and 80 Watts for sputtered film thickness, Ar:O2 gas flow ratio, mixed gases pressure (total pressure), and sputtering power, respectively.
3. Results and discussion 3.1. Fabrication of FTO/Nano-ZnO matrix by reactive RF magnetron sputtering
3.2. Optical transmission and morphological characterization of sputtered ZnO thin film
The basic principles of reactive RF magnetron sputtering have been described elsewhere [46–48]. In reactive sputtering, the deposited thin film is formed by chemical reaction between the target materials and a reactive gas which is introduced into the vacuum chamber. Oxide and nitride films are often fabricated using reactive sputtering [49]. The composition of thin film can be controlled by varying the relative pressures of the inert and reactive gases. In this study, reactive magnetron sputtering was carried out in argon and oxygen mixed gas atmosphere by supplying RF power at a frequency of 13.56 MHz (a frequency chosen because of its noninterference with radio-transmitted signals). In order to obtain very uniform nanoporous ZnO thin film, four sets of depositions were carried out at various film thickness (100 to 400 nm), Ar:O2 gas flow ratio (5:5 to 20:1 sccm/min), mixed gases pressure (total gas pressure from 5.0 × 10-3 to 5.0 × 10-2 Torr), and sputtering power (40 to 90 Watts). In every set of samples, only one of the above
In Fig. 3 typical optical transmission spectrum recorded for ZnO thin film is shown. The sharp absorption onset and the high transmission values at wavelengths above 338 nm exhibit the optical quality and low concentration of defects such as pits and voids of the ZnO thin film. The periodic and continuous annealing of ZnO thin film at 250 ◦ C cause better aligned textured films which is required for a homogeneous matrix for enzyme immobilization in biosensor fabrication. Fig. 4 (A-C) illustrates the characterization of surface morphology of nanoporous ZnO thin film by FE-SEM at different magnifications. The revealed feature confirms the uniform pores are open and extend through the surface into the bulk which is believed to play an important role in enzyme immobilization. In addition, higher magnification image of the surface shows the nanosize distribution of ZnO particles provides high surface area nanostructured matrix which can lead to a greater amount of an immobilized enzyme on the surface (Fig. 4D).
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Fig. 2. Effect of film thickness (A), Ar:O2 sputtering gases ratio (B), total gas pressure (C), and RF magnetron sputtering power (D) on FTO/Nano-ZnO characteristics.
3.3. Impedimetric and voltammetric characterization of FTO/Nano-ZnO/Urs biosensor in the absence of urea EIS measurements were used to trace the events during the formation of FTO/Nano-ZnO/Urs biosensor. EIS is a powerful technique for studying electrical and electrochemical interfacial properties of a large variety of systems. Concerning the use of EIS to study thin films in contact with electrolyte solutions, three different contributions, bulk, interfacial and electrolytes may be determined [32–35,51]. The interfacial behavior is widely described by Randles
Fig. 3. Transmission spectrum recorded for sputtered ZnO thin film.
equivalent circuit (Fig. 5A) [52], in such model, the diffusion or Warburg impedance (Zw ) [53,54] is in series with the charge transfer resistance (Rct ) and both are generally in parallel with the double layer capacitance (Cdl ), and in series with the solution resistance (Rs ), while in Fig. 5B modified Randles model in which the doublelayer capacitance is replaced by constant phase element (CPE) is observed. In this study, modified Randles model was used for the approximation of EIS data of impedimetric urea detection, and the chai-square of 2 = 1.70 × 10−4 was obtained for data analysis. Fig. 6A shows the impedance spectra presented as Nyquist plots (-Z vs. Z ) upon the layer by layer assembly of FTO/Nano-ZnO/Urs and characterization of them in urea-free PBS containing 5 mM [Fe(CN)6 ]3-/4- , which is reflected by the appearance of a semicircle part on the EIS spectrum, corresponding to a charge transfer resistance (Rct ) of a marker in electrolyte solution. Three layers of (a) FTO, (b) FTO/Nano-ZnO, and (c) FTO/Nano-ZnO/Urs result in a change of the electron-transfer resistance from (a) Rct = 0.96 k to (b) Rct = 4.6 k and (c) Rct = 5.4 k, respectively. The Rct value of ZnO thin film (4.6 k, curve b) with respect to FTO electrode (0.96 k, curve a) is higher, which is related to the lower electrical conductivity of semiconducting ZnO nanoporous thin film. However, the Rct value further increases (from 4.6 k to 5.4 k, curve c) after the immobilization of Urs onto FTO/Nano-ZnO electrode due to insulating characteristics of Urs [17,45,55,56]. Cyclic voltammetric study of layer by layer assembly of FTO/Nano-ZnO/Urs in urea-free PBS containing 5 mM [Fe(CN)6 ]3-/4− is shown in Fig. 6B. The magnitude of anodic peak current for FTO/Nano-ZnO electrode (0.171 mA, curve b) is lower than that of bare FTO electrode (2.09 mA, curve a) reveals that ZnO thin film shows an obstruction to the electron transfer
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Fig. 4. FE-SEM images of deposited ZnO thin film at different magnifications (A-C), and the particle size distribution histogram (D).
Fig. 5. Equivalent circuits used for impedance data approximation: (A) Randles model and (B) modified Randles model in which the double-layer capacitance is replaced by CPE. The Rs , Rct , Cdl and Zw represent the solution resistance, charge transfer resistance, double-layer capacitance and Warburg impedance, respectively.
from/to the electrode surface due to its semiconducting behavior. The magnitude of peak current response for FTO/Nano-ZnO/Urs electrode shows decreases in electron transfer from/to the electrode surface (0.144 mA, curve c) which is shown the insulating characteristics of Urs [17,45,55,56]. These results are consistent with the results of studying stepwise layer by layer assembly were obtained by EIS experiments. The scan rate dependency of voltammetric studies conducted on the FTO/Nano-ZnO/Urs biosensor as a function of scan rate (0.01-0.1 V/s) reveals that the magnitude of current response is linearly dependent on the square root of the scan rate (Fig. 6B, inset). This suggests that the electrochemical reaction is controlled diffusion process with facile charge transfer kinetics. A schematic of the proposed mechanism for electron transfer at the FTO/Nano-ZnO/Urs electrode in the presence of [Fe(CN)6 ]3-/4− redox marker is shown in Fig. 7. By comparing curve b and c in Fig. 6A and 6B which represent the EIS and CV spectra of [Fe(CN)6 ]3−/4− redox reaction at FTO/Nano-ZnO and FTO/NanoZnO/Urs electrodes, it can be observed that Urs layer decreases direct electron transfer with electrode substrate compare with ZnO thin film.
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Fig. 6. (A) The Nyquist plots (-Z vs. Z ) obtained for the Faradaic impedance measurements in urea-free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3-/4− on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c). (B) Cyclic voltammogram obtained in urea-free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3-/4− on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c) at the scan rate of 0.1 V/s. The inset shows the effect of scan rate (0.01- 0.1 V/s) on the response current of the FTO/Nano-ZnO/Urs biosensor.
3.4. Impedimetric and voltammetric characterization of FTO/Nano-ZnO/Urs biosensor in the presence of urea In addition to investigation of layer by layer assembly of FTO/Nano-ZnO/Urs electrode in the presence of [Fe(CN)6 ]3-/4− as a redox probe, the electrochemical behavior of each layer and final biosensor verified in the presence of urea. EIS technique was employed to study the Rct at the interfacial properties of the FTO/Nano-ZnO/Urs biosensor via urea (Fig. 8A). The Rct value in the presence of urea at ZnO thin film (55 k, curve b) is lower than that of FTO electrode (197 k, curve a) which is due to direct urea reaction with Nano-ZnO [45]. Further decreasing in Rct value (15.8 k, curve c) was observed after immobilization of Urs onto FTO/Nano-ZnO matrix due to fast hydrolysis of urea by enzyme, which can represent faster, repeatable and reproducible response of FTO/Nano-ZnO/Urs compare with slower, unpredictable and non-reproducible response of FTO/Nano-ZnO. Whereas the rate of urea hydrolysis at FTO/Nano-ZnO is not at all up to the extent of enzymatic hydrolysis at FTO/Nano-ZnO/Urs. Cyclic voltammetric studies of layer by layer assembly of FTO/Nano-ZnO/Urs in PBS containing 0.83 mM urea are shown in Fig. 8B. By comparing curve a and b, it can be observed that an
Fig. 7. Schematic of the mechanistic model proposed for electron transfer at the (a) FTO, (b) FTO/Nano-ZnO, and (c) FTO/Nano-ZnO/Urs biosensor in urea free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3-/4− .
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Fig. 9. Schematic representation of the detection mechanism of FTO/Nano-ZnO/Urs urea biosensor.
Fig. 8. (A) The Nyquist plots (-Z vs. Z ) obtained for the impedance measurements in PBS (pH 7.4) containing 0.83 mM urea on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c); the inset shows the magnified curve c. (B) Cyclic voltammogram obtained at 0.1 V/s scan rate in PBS (pH 7.4) containing 0.83 mM urea on FTO (a), FTO/Nano-ZnO (b), and FTO/Nano-ZnO/Urs (c) at the scan rate of 0.1 V/s; the inset shows the magnified curve a.
anodic peak is appeared for FTO/Nano-ZnO in the presence of urea which is due to partial hydrolysis of urea at ZnO thin film [45]. The magnitude of anodic peak current response increases greatly by Urs on FTO/Nano-ZnO/Urs biosensor (2.0 × 10−2 mA, curve c). These results are consistent with the results of studying stepwise layer by layer assembly obtained by EIS experiments in the presence of urea which supports the obtained I-V curves in section 2.3. 3.5. ZnO nanostructure and expected sensing mechanism The stability and performance of the enzyme on the matrix surface strongly depends on the immobilization and surface chemistry. Among the various immobilization techniques, covalent attachment of the biomolecule to the substrate is one of the most stylish immobilization methods. The optimum buffer pH for hydrolysis of urea by Urs was considered as 7.4 at which Urs enzyme retains its maximum activity and natural structure that is required to improve detection limit and sensitivity for urea detection [57–62]. Many researchers clarify the change in the electrical conductance of a highly porous semiconducting film sensor in the presence of toxic gases due to the reactions occurring on the surface [45,63]. Fig. 9 shows the mechanism. At first, atmospheric oxygen molecules are physisorbed on the surface sites, which get ionized by extracting an electron from the conduction band while moving from one
site to another, and are thus ionosorbed on the surface as Oads − . This leads to a decrease in the conductance of the transducer, as indicated by an increase in potential barrier at the grain boundaries as illustrated in Fig. 9A. Upon exposing FTO/Nano-ZnO/Urs to urea, an enzymatic reaction takes places between Urs and urea which can be represented as Fig. 9B. Urs
NH2 CONH2 + 2H2 O−→2NH4+ + CO32− − R + Oads → RO + e−
(2) +
It results in production of NH4 ion, which reacts with the surface adsorbed oxygen (Oads − ) and thereby release the trapped electron to the conduction band of ZnO as indicated by the decrease of potential barrier at grain boundary as shown in Fig. 9C. Further, when reducing gas (R) molecules (ammonia in the present case) react with pre-adsorbed negatively charged oxygen adsorbates, the trapped electrons are given back to conduction band of the material. The energy released during the decomposition of adsorbed ammonia molecules would be sufficient for the electrons jumping up into the conduction band so as to increase conductivity of the biosensor [45,63]. The results obtained by CV (Fig. 8B) supports the above-mentioned sensing mechanism, which clearly points to an oxidation and reduction process. 3.6. Working curve for impedimetric measurements The electrochemical response of FTO/Nano-ZnO/Urs biosensor has been impedimetrically measured as a function of urea concentration. As shown in Fig. 10A, the magnitude of Rct decreases on
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Fig. 10. (A) The Nyquist plots (-Z vs. Z ) obtained on the FTO/Nano-ZnO/Urs biosensor in the presence of different concentration of urea: (a) 0.83 mM; (b) 1.66 mM; (c) 3.32 mM; (d) 4.98 mM; (e) 6.64 mM; (f) 8.30 mM; (g) 9.96 mM; (h) 11.62 mM; (i) 13.28 mM; (j) 14.94 mM; (k) 16.60 mM; (l) 18.26 mM; (m) 19.92 mM; (n) 21.58 mM; (o) 23.24 mM. (B) Variation of charge transfer resistance (Rct ) as a function of urea concentration represents calibration curve.
addition of urea concentration from 0.83 to 23.24 mM (curve a to curve o). The quick response indicates that porous structure of ZnO matrix yields a very low mass transport barrier and results in a rapid diffusion from solution to the enzyme with keeping its bioactivity. At physiological pH (pH = 7.4), ZnO thin film (isoelectric point (IEP) ∼ 9) possesses a positive charge surface which provide a friendly micro-environment for negatively charge urease (IEP∼5.9) to retain its activity and also promote fast electron transfer [61]. The impedimetric studies clearly indicate that the process is a reversible process and confirms the Urs–urea enzymatic catalysis. From impedimetric studies and step by step studies, it is confirmed that the developed sensors can be re-used. The calibration curve was obtained for urea at the FTO/Nano-ZnO/Urs biosensor by monitoring its Rct responses (|Rct | = |Rct urea -Rct buffer (blank) |). Fig. 10B displays a working curve shown in a linear scale [Rct (k) = 8.813 (±0.164) + 0.637 (±0.012) × [urea] (mM); R2 = 0.995] in a concentration range of 0.83 to 23.24 mM of urea. The FTO/Nano-ZnO/Urs biosensor exhibits detection limit of 0.40 mM for urea and sensitivity of 0.637 k per mM with a linear range within 0.83–23.24 mM. These results confirm that ZnO thin film matrix provides attractive microenvironment for the enzyme immobilization to retain its desired natural activities. Several assays were made on blood serum to test the precision of the FTO/Nano-ZnO/Urs biosensor. The urea concentration was determined by the calibration curve (Table 1). Corresponding experiments were carried out with a spectrophotometric method by a local hospital. The results exhibited good consistent and precision between the two methods and confirmed the proposed
method is useful for application in real samples with good precision and accuracy. The proposed FTO/Nano-ZnO/Urs biosensor has a fast response time of less than 4s and retained good enzymatic activity for more than three weeks when kept at 4 ◦ C temperature and not in use. In addition, the stability of the FTO/Nano-ZnO/Urs biosensor was tested by comparing the catalytic ability to the urea with different storage time at 4 ◦ C. For a fixed concentration of urea, three same biosensors lost about 0.0%, 4.0% and 8.0% of their original activity after storing time of 1 day, 2 weeks and 6 weeks, respectively. Table 2 compares response characteristics of the FTO/Nano-ZnO/Urs biosensor with the other reported urea biosensors. The biosensor of the present work clearly shows good linear range, detection limit and response time respect to results published in previous studies [64–71].
Table 1 Determination of urea level in blood serum. Method
Determined by Spectrophotometry (mM) Measured by FTO/Nano-ZnO/Urs (mM) R.S.D.a (n = 5) (%) a
Sample number 1
2
3
4
5
2.52b 2.53 1.15
4.24 4.19 2.21
5.56 5.54 1.86
6.82b 6.85 2.55
4.51 4.53 2.1
R.S.D.: Relative Standard Deviation. Represents the hyper- or hypo-level of urea in blood serum, the normal urea level in blood serum is between 2.50 and 7.10 mM. b
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Table 2 Analytical characteristics of FTO/Nano-ZnO/Urs biosensor compared with other reported values of urea biosensors in the literature. Ref.
[64] [65] [66] [67] [68] [69] [70] [71] this work
Detection method
Cyclic Voltammetry Amperometric Potentiometric Conductometric Optical Colorimetric Manometric Potentiometric Impedimetric
Response Characteristics RT
D.L. (mM)
D.R. (mM)
2 min 10 min 1–5 min 5 min 2 min 4s
2.24 0.04 0.83 3.32 0.99 1.66 1.99 1.66 0.40
1.66–13.28 0.49–4.98 0.83–23.24 3.32–19.92 1.66–66.4 1.99–109.56 1.66–16.43 0.83–23.24
Enzyme immobilization strategy
Matrix
Physisorption Covalent binding Entrapment Physical adsorption Entrapment Ionic binding Free enzyme Physical adsorption Electrostatic adsorption
ZnO nanoparticles Polyaniline Nafion/Au/ceramic composite film Gelatin Polyaniline-Poly (n-butylmethacrylate) Cellulose membrane Dead cellulose Whatman paper ZnO nanoporous thin film
Detection Range [D.R.], Detection Limit [DL], Response Time [RT].
4. Conclusions As a result, the performance of biosensor depends on (i) the amount of immobilized enzyme which is related to the morphology and surface area/volume ratio of matrix, and (ii) the porosity of matrix which is providing rapid diffusion from solution to the immobilized enzyme. In this study, reactive RF sputtered nanoporous ZnO thin film with excellent uniformity of thickness has been used for fabrication of the electrochemical urea biosensor via immobilization of Urs. Fabricated biosensor showed high sensitivity, low detection limit, broad dynamic range, fast response time, high stability and long term reproducibility by retaining enzyme activity due to the suitable immobilization of Urs in nanoporous ZnO thin film. Porous nanomaterials provide a larger surface area available for enzyme binding and decrease the diffusion distance for the substrate to access the immobilized enzyme, which may improve the performance of the enzyme electrode. Besides, the close enzyme–substrate contact through covalent bonding, may promote faster detection kinetics, once the electrochemically detectable species (ammonia) is produced closer to the transducer, reducing diffusion resistance, and thus increasing sensitivity and diminishing response times. Impedimetric analysis of the proposed biosensor indicated a fast response time of less than 4s with a wide linear range from 0.83 to 23.24 mM and limit of detection as 0.40 mM for urea. Acknowledgement S. A. Mozaffari acknowledges the support of the Iranian Research Organization for Science and Technology, Iran Nanotechnology Initiative Council (INIC) and Nano-structured Coatings Co. for this research. References [1] J. Traynor, R. Mactier, C. Geddes, How to measure renal function in clinical practice, BMJ 333 (2006) 733–737. [2] M. Singh, N. Verma, A.K. Garg, N. Redhu, Urea biosensors, Sens. Actuators B 134 (2008) 345–351. [3] G.P. Nikoleli, D.P. Nikolelis, C. Methenitis, Construction of a simple optical sensor based on air stable lipid film with incorporated urease for the rapid detection of urea in milk, Anal. Chim. Acta 675 (2010) 58–63. [4] H.C. Tsai, R.A. Doong, Simultaneous determination of pH, urea, acetylcholine and heavy metals using array-based enzymatic optical biosensor, Biosens, Bioelectron. 20 (2005) 1796–1804. [5] B. Kovacs, G. Nagy, R. Dombi, K. Toth, Optical biosensor for urea with improved response time, Biosens. Bioelectron. 18 (2003) 111–118. [6] G.P. Nikoleli, M.Q. Israr, N. Tzamtzis, D.P. Nikolelis, M. Willander, N. Psaroudakis, Structural characterization of graphene nanosheets for miniaturization of potentiometric urea lipid film based biosensors, Electroanalysis 24 (2012) 1285–1295. [7] A. Ali, M. AlSalhi, M. Atif, A.A. Ansari, M.Q. Israr, J. Sadaf, E. Ahmed, O. Nur, M. Willander, Potentiometric urea biosensor utilizing nanobiocomposite of chitosan-iron oxide magnetic nanoparticles, in: Journal of Physics: Conference Series, IOP Publishing, 2013, pp. 012024.
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