Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer

G Model

ARTICLE IN PRESS

SNB-23313; No. of Pages 15

Sensors and Actuators B xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer Reza Rahmanian, Sayed Ahmad Mozaffari ∗ , Hossein Salar Amoli, Mohammad Abedi Thin Layer and Nanotechnology Laboratory, Department of Chemical Technology, 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 28 April 2017 Received in revised form 25 September 2017 Accepted 2 October 2017 Available online xxx Keywords: Hierarchical nanostructure Nanoporous ZnO thin film Disposable urea biosensor Reactive DC magnetron sputtering Impedimetric sensing

a b s t r a c t 3D hierarchical Nano-ZnO/TiO2 on conductive fluorinated-tin oxide (FTO) layer was fabricated by reactive direct current (DC) magnetron sputtering of ZnO, at the optimized instrumental deposition conditions, on a pre-covered TiO2 surface with Polyvinyl Alcohol (PVA) as an omissible polymer in a pattern of parallel strips (Nano-ZnO/PVA/TiO2 /FTO) following by PVA omission via annealing process, which resulted in an efficient porous media for urease (Urs) enzyme immobilization (Urs/Nano-ZnO/TiO2 /FTO) designed for urea biosensing. The criteria for TiO2 selection as substrate was based on: (i) its ability to promote electron transfer between ZnO to FTO substrate, (ii) affording high electronic density to the biosensor surface as an electrostatic repulsion layer for the anionic interferents at the biological media, and (iii) enhancement of urea biosensing by the formation of heterojunctions with ZnO. Characterization of the surface morphology of 3D hierarchical Nano-ZnO/TiO2 film by field emission-scanning electron microscopy (FE-SEM) exhibits cavities of nanoporous ZnO film as an effective biosensing area for Urs enzyme immobilization. Step by step monitoring of Urs/Nano-ZnO/TiO2 /FTO biosensor fabrication was performed using electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Fabricated Urs/Nano-ZnO/TiO2 /FTO biosensor was used for urea determination using impedimetric assessment. The impedimetric results show high sensitivity for urea detection within 5–205 mg dl−1 and limit of detection as 2 mg dl−1 . A fast response of fabricated biosensor can usually allow a real-time analysis. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Urea (carbonyl diamide) with the chemical formula (NH2 )2 CO is a vital biomolecule which exists in nature and its approximation is of utmost relevance not only for clinical purposes but also from environmental viewpoints. In the human body, urea is present in specific quantities in various pathological fluids such as blood, serum, and urine. Increase in urea level in blood (normal range is 15–40 mg dl−1 ) [1] and urine causes a number of health concerns including urinary tract obstruction, dehydration, renal failure, etc. while the reduced level of urea may be responsible for the hepatic failure, cachexia, and nephritic syndrome [2]. Therefore, the growth of cost effective procedure for efficient recognition of urea levels is of utmost importance. Numerous direct

∗ Corresponding author. E-mail address: [email protected] (S.A. Mozaffari).

spectroscopic methods are accessible for urea quantification, but they usually involve pretreatment or conditioning [3–5]. Nowadays, urea biosensors are favored because of their easy, fast, cost effective, and well-organized performance. During recent years potentiometric [6–9], conductometric [10–12], coulometric [13], thermal, and amperometric [14] techniques have been described for urea determination. Thermal biosensors with complex fabrication and narrow detection range for conductometric biosensors make them less popular. Various types of electrochemical biosensors [15–20] have been used for the detection of urea concentration based on urease (Urs) enzyme which utilizes the catalyzed hydrolysis of urea [21–25], followed by potentiometric assessments. Potentiometric biosensors based on Urs have been the most preferred choice due to the simple mode of detection based on either detection of NH4 + or pH change. The high K+ interference in sensing media is the major disadvantage of these types of biosensors [26–28]. In addition, amperometric biosensor [13,29,30] based on Urs is a talented technique because of its efficiency, simplicity, and

https://doi.org/10.1016/j.snb.2017.10.009 0925-4005/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS

2

R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

ease of calibration. In our research, the electrochemical impedance spectroscopy (EIS) has been employed as a powerful candidate for biosensing assessment due to its reliability via electrochemical signal transduction. By taking advantages of the EIS technique such as the capability to see electrode surface modifications just by looking impedance curves, and opportunity to design the electrical circuit according to obtained Nyquist plots, the impedimetric assessment exhibited excellent analytical performance towards the quantification analysis over the other electrical measurement techniques [31–36]. The most significant aspect of a biosensor design with enhanced sensing properties are the strategy of enzyme immobilization, the construction and morphology of transducer media [37–41], and surface interactions at the transducer in order to reduce interferents. To achieve this goal, nanostructured semiconducting metal oxides have concerned significant interest in the bioanalytical area as they possess non-toxicity, ease of fabrication, high surface area, good optical transmission in the visible region, chemical stability, and unique ability to promote electron transfer between electrode and active site of desired enzyme [42]. In the previous studies of our research group in thin film and nanotechnology lab at IROST, we reported a series of urea biosensors based on different metal oxides with moderate urea detection range [43–45]. As a novel approach, in this study, heterostructured transducer media with novel and distinct characteristic was employed. The combination of TiO2 nanoparticles and nanoporous ZnO film provides a family of hierarchical nanostructures which is expected to hold an excessive potential for future utilization in biosensing. When these dissimilar nanomaterials with different isoelectric point (IEP) are integrated together, they enable the integration of multiple important functions and allow exhibition of more complex and fascinating novel properties as the foundation for the new transducing substrate. These functions and properties can affect the enzyme immobilization strategy and biosensor interface interactions. In light of these facts, we have focused on designing and engineering a new class of hierarchical metal oxidebased nanostructures for improving biosensor efficiency, detection range, and selectivity. Herein, in comparison with our previous report based on single semiconductor transducer [43–45], threedimensional (3D) hierarchical Nano-ZnO/TiO2 was engineered and applied for efficient loading of enzyme, efficient transforming of surface chemical species into electrical signals by surface depletion layers, and repulsion of anionic interferents from the exposed TiO2 surface [42]. In this work, 3D hierarchical Nano-ZnO/TiO2 on the conductive fluorinated-tin oxide (FTO) layer was fabricated via a facile approach in five steps: (i) deposition of TiO2 paste on FTO surface using the doctor-blade technique (TiO2 /FTO), (ii) covering TiO2 surface by Polyvinyl Alcohol (PVA) as an omissible polymer in a pattern of parallel strips (PVA/TiO2 /FTO), (iii) deposition of ZnO thin film onto PVA/TiO2 /FTO electrode via reactive DC magnetron sputtering (Nano-ZnO/PVA/TiO2 /FTO), (iv) PVA omission from Nano-ZnO/PVA/TiO2 /FTO by annealing, which afford 3D hierarchical nanoporous ZnO thin film along with exposed TiO2 substrate to electrolyte (Nano-ZnO/TiO2 /FTO), and (v) preparation of a Urs/Nano-ZnO/TiO2 /FTO biosensor by Urs immobilization on 3D hierarchical Nano-ZnO/TiO2 film, which was forced by the difference in their isoelectric points (IEP). As a unique performance, urea measurements were carried out by an impedimetric method utilizing Urs/Nano-ZnO/TiO2 /FTO electrode served as biosensing electrode. The proposed Urs/NanoZnO/TiO2 /FTO biosensor showed a fast response time of fewer than 4 s and retained good enzymatic activity for more than 20 days when kept at 4 ◦ C temperature when not in use. The 3D hierarchical Nano-ZnO/TiO2 film demonstrated higher sensitivity, a lower detection limit, a more rapid response and a better selectivity,

in comparison to pure ZnO [42]. The unique heterostructure of TiO2 /ZnO is responsible for the enhanced performance. 2. Experimental 2.1. Chemicals and reagents PVA (Alfa Aesar, 87%, high molecular weight, MW 88000–97000), 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. Deionized (DI) water (resistivity = 18M, Milli-Q system, Millipore Inc.) was used for rinsing and preparing all aqueous solutions. A fluorine-doped SnO2 conductive glass (FTO, resistivity = 15  cm−2 ) was purchased from Dye Sol Company. The FTO plate was cleaned in a detergent solution using an ultrasonic bath for 15 min, rinsed with DI water and ethanol, and then dried. The TiO2 paste was deposited on the FTO using the doctor-blade technique to obtain the suitable layer of TiO2 . 2.2. Apparatus Surface analysis was done using a low vacuum Tescan Mira II Field emission scanning electron microscope (FE-SEM, Czech Republic) after coating the samples with a thin layer of gold by magnetron sputtering. Energy-dispersive X-ray spectrometry (EDX) is an important non-destructive analytical tool mostly applied for the chemical composition analysis. The spectrophotometer (Perkin Elmer Lambda 25 UV–vis, United States) was applied for evaluation of absorbance intensity. For all X-ray diffraction (XRD) patterns reported in this study, XRD was performed under atmospheric conditions with a Philips X-Pert. The deposition of ZnO thin films was carried out in a reactive DC magnetron sputtering system (Nanostructured Coatings Co., Tehran, Iran) using 99.999% pure Zn target under different conditions. Voltammetric and impedimetric experiments were performed with a potentiostat/galvanostat (PGSTAT. 302N, Autolab, EcoChemie, The Netherlands). All electrochemical experiments were carried out in a conventional three electrode system at ambient temperature. The Urs/Nano-ZnO/TiO2 /FTO electrode served as the 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 peakto-peak AC amplitude was applied, a range of frequencies from 100 kHz to 1000 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 [46].

3. Results and discussion 3.1. Step by step designing of 3D hierarchical nanostructure based biosensor As represented in Scheme 1, the surface engineering of biosensor based on 3D hierarchical Nano-ZnO/TiO2 on FTO was performed

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

3

via a new strategy in five steps. Each step will be presented and discussed in details hereinafter. 3.1.1. Step (i): Fabrication of TiO2 /FTO by doctor-blade technique In order to fabricate TiO2 /FTO electrode, the TiO2 paste was deposited on the FTO conductive glass using the doctor-blade technique to obtain the suitable thickness of the TiO2 film, as the substrate. Scotch tape was used as a spacer to control the TiO2 film thickness and to provide non-coated areas for electrical contact. After being coated, the TiO2 film was air-dried for about 5 min to reduce the surface irregularities. The TiO2 film coated on FTO glass was sintered at 500 ◦ C for 1 h to increase the compactness of the internal voids of film organization. 3.1.2. Step (ii): Preparation of PVA/TiO2 /FTO In this step, the TiO2 surface was covered by PVA as an omissible polymer in a pattern of parallel strips (PVA/TiO2 /FTO), in order to protect some parts of the TiO2 surface from covering by ZnO in the next step. After the sputtering of the whole surface by ZnO, these strips will be omitted by annealing in step iv, which afford 3D hierarchical nanoporous ZnO thin film containing exposed TiO2 substrate to the electrolyte.

Scheme 1. Step by step designing of hierarchical nanostructure based Urs/NanoZnO/TiO2 /FTO biosensor.

3.1.3. Step (iii): Nano-ZnO/PVA/TiO2 /FTO fabrication by reactive DC magnetron sputtering The basic principles of DC magnetron sputtering have been described elsewhere [47–49]. In this study, reactive DC magnetron sputtering was carried out in reactive Ar/O2 gas atmosphere by supplying DC power for the covering of Nano-ZnO on PVA/TiO2 /FTO electrode. In the evacuated sputtering chamber, the Zn-cathode plate was bombarded by Ar+ ions generated in the glow discharge plasma situated in front of the Zn-target. Oxide films are often fabricated using reactive sputtering [50]. In reactive sputtering, the deposited ZnO thin film is formed by chemical reaction between the sputtered Zn atoms from the target surface and O2 reactive gas which is introduced into the vacuum chamber. The composition of the thin film can be controlled by varying the relative pressures of the inert (Ar) and reactive (O2 ) gases. In order to obtain very uniform nanoporous ZnO thin film, five sets of sputtering experiments were designed and carried out at various instrumental parameters such as: sputtering voltage (250–400 volts), deposition time (2–30 min), distance between target and substrate (5–9 cm), mixed gases pressure (total gas pressure from 3 × 10−3 to 4.5 × 10−2 Torr), and Ar:O2 gas flow ratio (5:5 to 9:1 sccm/min). In every set of experiments, only one of the above-mentioned parameters was varied while keeping other parameters constant. The sputtering conditions are chosen to be optimum for NanoZnO thin film formation with the lowest electrical resistivity to achieve more efficient transducer. DC sputtering voltage strongly affects the electrical resistivity of ZnO thin film (Fig. 1A). Lower DC sputtering voltage 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 the higher electrical resistivity of the thin film. As much as the DC sputtering voltage increases up to 350 V, the amounts of non-reacted Zn sputtered atoms and the deficiencies of oxygen in the deposited film increase and lead to achieve lower electrical resistivity. However, the excessive supply of DC sputtering voltage over 350 V may cause thin film degradation by the bombardment of highly energized particles, resulting in an inhomogeneous thin film. The deposition time is one of the effective parameters in reactive DC magnetron sputtering for obtaining the uniform Nano-ZnO thin film with lower electrical resistivity. By increasing the time of deposition, the lower electrical resistivity of the thin film will be observed (Fig. 1B). Increasing film thickness, by increasing the time

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS

4

R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

Fig. 1. Effect of (A) DC magnetron sputtering voltage, (B) deposition time, (C) target to substrate distance, (D) total gas pressure, and (E) Ar:O2 gases ratio on NanoZnO/PVA/TiO2 /FTO characteristics.

of deposition, reduces defects in the thin film and causes low thin film resistivity [51]. For this study, to save energy, the deposition time of about 25 min was selected as the optimum deposition time (Fig. 1B).

In Fig. 1C the dependence of Nano-ZnO thin film electrical resistivity on distance between target and substrate from 5 to 9 cm was shown. Increasing of the distance between target and substrate from 5 to 9 cm resulted in increasing the electrical resistivity of the film based on the lower deposited ZnO nanoparticles while lower

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

5

Fig. 2. FE-SEM images of the uniform deposited TiO2 film (A), and its size distribution histogram diagram (B); the sputtered ZnO on the TiO2 substrate before PVA elimination (C), and after PVA elimination (D); Nano-ZnO size distribution histogram diagram (E), and the EDX spectrum of the Nano-ZnO/TiO2 heterostructure (F).

distance caused substrate damage by highly energized Ar+ . For this study the distance between target and substrate of about 6 cm was selected as the optimum distance.

Total gas pressure has a vital effect on plasma formation, and directly influences the deposition rate and accordingly the porosity and physical homogeneity of ZnO thin film (Fig. 1D). Total gas pres-

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS

6

R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

Fig. 3. The XRD spectra for sputtered Nano-ZnO thin films (A) before, and (B) after annealing process.

sure and deposition rate are very important to control concurrently to achieve better homogenous thin film. The Ar:O2 gas flow ratio is a very effective parameter in reactive DC magnetron sputtering for obtaining the nanoporous ZnO thin film with lower electrical resistivity. By decreasing the amount of oxygen in gas flow ratio, the lower electrical resistivity of the thin film was observed (Fig. 1E). 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. The optimum conditions for obtaining uniform nanoporous ZnO thin film were considered in continue as 350 V, 25 min, 6 cm, 6 × 10−3 Torr, and 9:1 for sputtering voltage, deposition time, distance between target and substrate, mixed gases pressure (total pressure), and Ar:O2 gas flow ratio, respectively.

3.1.4. Step (iv): Fabrication of 3D hierarchical Nano-ZnO/TiO2 on FTO In this stage, in order to obtain a 3D hierarchical Nano-ZnO/TiO2 , the PVA content in the Nano-ZnO/PVA/TiO2 /FTO electrode was ◦ eliminated by annealing it at 400 C for 2 h. The PVA elimination will afford a nanostructure substrate (Nano-ZnO/TiO2 /FTO) containing

both Nano-ZnO and TiO2 as an efficient nanoporous transducer media for enzyme immobilization. 3.1.5. Step (v): Urs immobilization on 3D hierarchical Nano-ZnO/TiO2 film An important part of biosensor fabrication is to immobilize enzyme on the transducer without changing its structural conformation and its activity. The immobilization strategy can govern the performance and reliability of the obtained urea biosensor. Nanomaterials offer unique advantages in immobilizing enzymes due to enhanceing surface reactivity, preserving enzyme activity due to the microenvironment. In this work, Urs was immobilized by soaking the NanoZnO/TiO2 /FTO in 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 attachment of Urs with 3D hierarchical Nano-ZnO/TiO2 was expected to be forced by the difference in their isoelectric points (IEP), which provides Urs/Nano-ZnO/TiO2 /FTO biosensor [52,53]. The fabricated Urs/Nano-ZnO/TiO2 /FTO was then washed and kept in PBS until use. Step by step monitoring of Urs/Nano-ZnO/TiO2 /FTO biosensor fabrication was performed using electrochemical methods such as current vs. potential (I–V curve), cyclic voltammetry (CV) and EIS techniques. After completing these steps, the fabricated biosensors

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

7

were applied as the working electrode for urea determination using impedimetric assessment. 3.2. Structural characterization of fabricated Urs/Nano-ZnO/TiO2 /FTO biosensor As described schematically, monitoring of 3D hierarchical NanoZnO/TiO2 fabrication was performed using FE-SEM, XRD, and UVvis. The surface characterization results of the uniform deposited TiO2 film using FE-SEM technique and the size distribution histogram of TiO2 nanoparticles was shown in Fig. 2A, B. The FE-SEM images of sputtered ZnO on the partially covered TiO2 with PVA strips before and after PVA elimination were shown in Fig. 2C, D. The comparison of Fig. 2C with D clearly shows the availability of both ZnO and TiO2 surface area after PVA elimination. In addition, ◦ annealing of ZnO thin film at 400 C causes better aligned textured films which are required for a homogeneous matrix for enzyme immobilization in biosensor fabrication. The revealed feature confirms the uniform Nano-ZnO pores are open and extend through the surface into the bulk which is believed to play an important role in enzyme immobilization. In addition, the surface shows the nanosize distribution of ZnO particles provide high surface area nanostructured matrix which can lead to a greater amount of an immobilized enzyme on the surface (Fig. 2E). These results indicate that the heterojunctions are well formed between the TiO2 nanoparticles and the Nano-ZnO. The EDX spectrum of the NanoZnO/TiO2 heterostructure on FTO, shown in Fig. 2F indicates that the obtained heterostructure is composed of Ti, Zn, Sn, Si, F and O elements, which are related to TiO2 , ZnO, fluorinated SnO2 on the silicone substrate. For investigation of the crystal structure of Nano-ZnO before and ◦ after annealing process at 400 C, the sputtered Nano-ZnO thin film was prepared separately without the presence of TiO2 and FTO and ◦ ◦ studied using XRD patterns. The angle 2␪ ranged from 10 to 109 , ◦ with a step of 0.02 . The scan step time was 0.5 s and measurement temperature was 25 ◦ C. The XRD spectra for sputtered Nano-ZnO thin films before and after annealing process were shown in Fig. 3A ◦ and B, respectively. Broad peak around 30 in Fig. 3A in comparison with International Center for Diffraction Data (ICDD) refers to amorphous nanostructured ZnO while the sharp peak in Fig. 3B corresponds to crystalline nanostructured ZnO which is prepared after annealing at 400 ◦ C in the furnace and cooling down to room temperature. The results revealed that the effect of annealing process will cause better aligned textured films for achieving a homogeneous matrix for enzyme immobilization. Fig. 4 shows typical optical absorption spectra recorded for TiO2 , Nano-ZnO, and Nano-ZnO/TiO2 thin films. The sharp absorption peaks at wavelengths 312 nm, and 320 nm was demonstrated for TiO2 , and Nano-ZnO thin films, respectively. The experimental results revealed that the Nano-ZnO/TiO2 hetrostructure exhibited a strong absorption for UV–vis light when compared to pure ZnO and TiO2 .

Fig. 4. Typical optical absorption spectra recorded for TiO2 , Nano-ZnO, and NanoZnO/TiO2 thin films.

were prepared in PBS and used as an electrolyte. The 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 sensitivity curves indicating the variation due to urea, as presented in Fig. 5. The slope of I–V curve (I/V) was used as a measure of enzyme activity of the biosensor [43–45]. A monotonous increase after immobilization in the current with applied voltage is clearly evident. As can be seen in Fig. 5, it is the clear evidence that the change in response is due 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 the same as that after Urs immobilization [17,54,55]. This increase is attributed to the catalytic reaction of Urs· · ·urea as below; Urs

NH2 CONH2 + 2H2 O →2NH4 + + CO3 2−

(1)

This reaction results two NH4 + and CO3 2− from uncharged urea which raises the conductivity of the Urs/Nano-ZnO/TiO2 /FTO by providing the 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 Urs/Nano-ZnO/TiO2 /FTO is due to the high surface area of nanoporous ZnO thin film as a family of the efficient nanostructure [55]. As result, the sensitivity of this biosensor is higher due to higher surface area of hierarchical nanostructured. 3.4. Impedimetric and voltammetric characterization of Urs/Nano-ZnO/TiO2 /FTO biosensor in the absence/presence of urea

3.3. Determination of enzyme activity after immobilization The electrical property of Urs/Nano-ZnO/TiO2 /FTO biosensor, after Urs immobilization, was studied to determine the enzyme activity. For this study, a cell was made, which consists of gold wire ( 0.3 mm, 5 cm length) as an electrode and Urs/NanoZnO/TiO2 /FTO as another electrode. A 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 was 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, 5.0, 55, 105, 155 and 205 mg dl−1

The EIS and CV techniques were used to trace the events during the formation of Urs/Nano-ZnO/TiO2 /FTO biosensor [31–36,56]. Fig. 6A and B shows the impedance spectra as Nyquist plots (-Z” vs. Z’) and cyclic voltammograms, respectively, upon the layer by layer assembly of Urs/Nano-ZnO/TiO2 /FTO and their characterization in urea-free PBS containing 5.0 mM [Fe(CN)6 ]3−/4− . The monitoring of layer by layer assembly of biosensor in the EIS spectrum and cyclic voltammogram is reflected by the appearance of a semicircle part [57] corresponding to charge transfer resistance (Rct ) [58,59] and magnitude of anodic peak current (ipa ) of the marker ([Fe(CN)6 ]3−/4− ) in electrolyte solution, respectively.

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS

8

R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

Fig. 5. Typical I–V curves (vs. SCE) showing the effect of Urs immobilization on Nano-ZnO/TiO2 /FTO films with increasing urea concentration, (A) before and (B) after Urs immobilization.

Four layers of (a) FTO, (b) TiO2 /FTO, (c) Nano-ZnO/TiO2 /FTO, and (d) Urs/Nano-ZnO/TiO2 /FTO result in a change of the electrontransfer resistance and magnitude of anodic peak current from (a) Rct = 0.96 k, ipa = 2.09 mA to (b) Rct = 13 k, ipa = 0.07 mA, (c) Rct = 6 k, ipa = 0.09 mA, and (d) Rct = 20 k, ipa = 0.04 mA, respectively. The increases in Rct and decreases in ipa values of the TiO2 film (Fig. 6A, B; curve b) with respect to FTO electrode (Fig. 6A, B; curve a) is related to the lower electrical conductivity of the TiO2 uniform film. FTO exhibited a low electrical resistivity due to the high carrier concentration (Nd ) caused by the oxygen vacancies and the substitutional fluorine dopant, while the reverse change in Rct and ipa values of ZnO film (Fig. 6A, B; curve c) with respect to TiO2 electrode (Fig. 6A, B; curve b) is related to the higher electrical conductivity of ZnO nanoporous film. ZnO sputtered thin film fabricated by DC magnetron sputtering contains ZnO:Zn (metal oxide:metal) phases, which finally leads to a lower electrical resistivity respect to the TiO2 semiconductor. However, the Rct value further increases and ipa value decreases (Fig. 6A, B; curve c) after the immobilization of

Fig. 6. (A) The Nyquist plots (-Z” vs. Z’) obtained in the range of frequencies from 100 kHz to 1000 mHz, at E = 0.1 V, for the impedance measurements in urea-free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3−/4− on FTO (a), TiO2 /FTO (b), NanoZnO/TiO2 /FTO (c), and Urs/Nano-ZnO/TiO2 /FTO (d). Dash-symbol lines indicate experimental data and solid lines approximated results; (B) Cyclic voltammograms (vs. SCE) obtained in urea-free PBS (pH 7.4) containing 5 mM [Fe(CN)6 ]3−/4− on FTO (a), TiO2 /FTO (b), Nano-ZnO/TiO2 /FTO (c), and Urs/Nano-ZnO/TiO2 /FTO (d) at the scan rate of 0.1 V s−1 . The inset in (A) shows the magnified Nyquist plot of curve a accompanied with electrochemical cell geometry, while the inset in (B) shows the magnified cyclic voltammograms of curves b, c, and d.

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

9

Table 1 The metal oxide/interferents charge for hierarchical nanostructures to major interferents in pH 7.4. Metal oxide

TiO2 ZnO

Interferents Ascorbic acid

Uric acid

−/− +/−

−/− +/−

At first, it is important to give details that TiO2 film shows the characteristic charging/discharging currents assigned to electron injection into its sub-band gap states at -1.1 V which is not refer to urea hydrolysis. The Rct and ipa values at Nano-ZnO film (Rct = 49.7 k, ipa = 0.008 mA, curve c) are comparable with the values obtained at FTO and TiO2 surface (Fig. 7A, B; curves b and c), which imply a partial hydrolysis of urea at ZnO surface. Further decreasing in Rct and increasing in ipa values (Rct = 8.1 k, ipa = 0.027 mA, curve d) were observed after immobilization of Urs onto Nano-ZnO/TiO2 /FTO matrix due to fast hydrolysis of urea by enzyme [17,54,55]. Fig. 7 B displays the difference in the typical voltammetric behaviour, at a scan rate of 0.1 V s−1 . Since this measurement was carried out just to understand the oxidation-reduction behaviour in order to support the I–V characteristic, hence the scan rates were not varied. In case of PBS, the electrochemical response did not show any current peak related to oxidation-reduction process (data in not shown). Addition of urea resulted in a typical electrochemical biosensor response with roughly symmetrical peak. The formal potential ((Eo = Epa + Epc )/2) of −0.75 V was observed. It is very clear that process is a redox-dominated process. The current variation is analogous to that of the sensitivity curves derived from I–V characteristic (Fig. 5). The cyclic voltammetric studies clearly indicate that the process is a reversible process and confirms the Urs· · ·urea interaction as a function of applied potential i.e. an enzymatic catalysis The quick response shows that porous structure of ZnO matrix yields a very low mass transport barrier and results in a rapid diffusion from solution to the enzyme and retains the enzyme bioactivity. This also designates that ZnO matrix (isoelectric point (IEP) ∼9.0) not only provides a friendly microenvironment for negatively charged Urs (IEP ∼5.9) to keep its activity but also promotes fast electron transfer. 3.5. Hierarchical Nano-ZnO/TiO2 and expected sensing mechanism Fig. 7. (A) The Nyquist plots (-Z” vs. Z’) obtained in the range of frequencies from 100 kHz to 1000 mHz, at E = -0.8 V, for the impedance measurements in PBS (pH 7.4) containing 5 mg dL−1 urea on FTO (a), TiO2 /FTO (b), Nano-ZnO/TiO2 /FTO (c), and Urs/Nano-ZnO/TiO2 /FTO (d). Dash-symbol lines indicate experimental data and solid lines approximated results; (B) Cyclic voltammograms (vs. SCE) obtained at 0.1 V s−1 scan rate in PBS (pH 7.4) containing 5 mg dL−1 urea on FTO (a), TiO2 /FTO (b), Nano-ZnO/TiO2 /FTO (c), and Urs/Nano-ZnO/TiO2 /FTO (d). The inset in (B) shows first (solid line), second (dash line), and third (dot line) cycles of cyclic voltammogram of Urs/Nano-ZnO/TiO2 /FTO (d) in PBS (pH 7.4) containing 5 mg dL−1 urea, which completely overlapped together.

Urs onto Nano-ZnO/TiO2 /FTO electrode due to insulating characteristics of Urs (Fig. 6A, B; curve d) [17,54,55]. The Rct and ipa values (Fig. 7A, B) at the interface of the Urs/NanoZnO/TiO2 /FTO biosensor in the presence of urea were obtained by using EIS and CV techniques, respectively. Modified Randles model was exploited for the EIS data approximation. For any fit performed a ␹2 factor of about 10−3 was obtained. In addition, various equivalent circuits for the EIS data approximation presented in the Supplementary data (section. 2) and properly discussed.

The primary attempt to immobilize Urs onto the electrode was through spontaneous adsorption, which occurs by electrostatic adsorption of negatively charged Urs (IEP ∼ 5.9) and positively charged ZnO (IEP ∼ 9.0) at biological pH 7.4. The optimum buffer pH for hydrolysis of urea by Urs was considered as 7.4, at which Urs enzyme retains its activity and natural structure that is required to improve detection limit and sensitivity for urea detection [60]. In addition, TiO2 (IEP ∼ 5.8) as negatively charged substrate reduces the anionic interferents. Table 1 shows the charge of substrates in pH 7.4, which indicate that TiO2 can repel anionic interferents from the biosensor surface during the urea measurements, while ZnO can be employed as a porous media for Urs immobilization [17,61–64]. Henry and Peter (1979) were amongst the first few to report the microscopic model for the operation of SnO2 -based carbon monoxide (CO) sensor. The physical basis of the model they presented is the oxidation of CO on sensor surface by chemisorbed oxygen and subsequent emission of an electron from the chemisorbed species into the conduction band of the sensor. Based on this model, many reports explain the change in the electrical conductance of a highly

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS

10

R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

porous semiconducting thick or thin film sensor in the presence of toxic gases due to the reactions occurring on the surface [65]. The results show semiconductor surfaces can be used as a base for the design of new sensor architectures with high electron transfer facility. The electron transfer at biosensor surface during urea determination can be explained by the following mechanism [65]. Compared to the sensing properties of pure sputtered NanoZnO [43–45], the Nano-ZnO/TiO2 hierarchal nanostructure displays better response values, caused by heterojunctions created at the interface between TiO2 and ZnO, as shown in the SEM images [42]. Initially, in the open air, the oxygen adsorbates (Oads − ) form on the surface of TiO2 and ZnO, resulting in an electron-depleted surface layer due to the electron transfer from TiO2 and ZnO to oxygen, and thus a reduced conductivity (Scheme 2A) [55]: O2 + 2e− →2Oads −

(2)

Upon exposing Urs/Nano-ZnO/TiO2 /FTO to urea, an enzymatic reaction takes place between Urs and urea which can be represented as [53]: Urs

NH2 CONH2 + 2H2 O →2NH4 + + CO3 2− When the Nano-ZnO/TiO2 heterostructures are exposed to NH3 (R: as reducing gas), the reaction takes place between the adsorbed oxygen ions and the NH3 molecules (Scheme 2B): R + Oads − →RO + e−

(3)

The free electrons will flow into the conduction band of the Nano-ZnO/TiO2 heterostructures of the biosensor; cause a decrease in the width and height of the barrier potential at the interfaces. The change in the heterojunction barrier in different NH3 atmospheres may point to the enhanced urea sensing mechanism. Characteristics of the Nano-ZnO/TiO2 in urea biosensing can be attributed to an extra depletion layer at the interface between the TiO2 and the ZnO. At the areas near to the Nano-ZnO/TiO2 interface, the electrons are released more simply from the surface reaction back into the conduction band, which really enhances the conductivity of the Nano-ZnO/TiO2 nanostructure in NH3 gas. Therefore, these regions close to the Nano-ZnO/TiO2 interface make the sensors more sensitive in urea detection compared with the biosensor based on pure ZnO. Besides, compared with pure ZnO, the hierarchal NanoZnO/TiO2 induces more active sites for the adsorption of oxygen molecules [42–45]. For extra confirmation to validate our proposed biosensing mechanism, the impedimetric Nano-ZnO/TiO2 /FTO response as a function of NH3 concentration was determined in a urea-free NH3 solution (detailed information on the proposed mechanism can be found in the Supplementary data). The magnitude of Rct decreases on the addition of NH3 concentration from 0.001 to 0.01 M. When reducing molecules (R = ammonia) reacts with pre-adsorbed negatively charged oxygen adsorbates (Eq. (3)), the trapped electrons are given back to conduction band of semiconductors, and as a result the decrease of Rct is perceived. The outcomes attained by impedimetric studies of Rct variation as a function of NH3 concentration support the proposed mechanism. 3.6. Working curve for impedimetric measurements The electrochemical response of Urs/Nano-ZnO/TiO2 /FTO biosensor has been impedimetrically measured as a function of urea concentration. Modified Randles model was used for the approximation of EIS data for impedimetric urea detection (Fig. 8A). For any fit performed, a ␹2 factor of about 10−3 was obtained. As shown in Fig. 8B, the magnitude of Rct decreases on the addition of urea concentration from 5 to 205 mg dL−1 (curve a to curve u). The quick response indicates that porous structure of ZnO matrix

Scheme 2. The proposed mechanism of urea sensing on metal oxides semiconductor in the presence of oxygen adsorbates (A), and reducing gas (B).

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

11

Fig. 8. (A) Modified Randles equivalent circuit model used for impedance data approximation, (B) the Nyquist plots (-Z” vs. Z’) obtained in the range of frequencies from 100 kHz to 1000 mHz, at E = -0.8 V, on the Urs/Nano-ZnO/TiO2 /FTO biosensor in the presence of different concentration of urea: (a) to (u) from 5.0 to 205.0 mg dL−1 . Dashsymbol lines indicate experimental data; (C) variation of charge transfer resistance (Rct ) as a function of urea concentration represents calibration curve, (D) the graph shows the effect of interferents on the response of Urs/Nano-ZnO/TiO2 /FTO, and (E) the Nyquist plots (-Z” vs. Z’) obtained in the range of frequencies from 100 kHz to 1000 mHz, at E = -0.8 V, on the Urs/Nano-ZnO/TiO2 /FTO biosensor in the presence of interferents with different concentrations + 205.0 mg dL−1 urea, Dash-symbol lines indicate experimental data.

yields a very low mass transport barrier and results in a rapid diffusion from solution to the enzyme while keeping its bioactivity. At physiological pH (7.4), ZnO thin film (isoelectric point (IEP) ∼ 9) possesses a positive charge surface which provides a friendly micro-environment for negatively charged Urs (IEP ∼ 5.9) to retain its activity and also promote fast electron transfer. 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 biosensor can be re-used. The reversible impedimetric investigation involves system has to be stable during the measurement and should not alter because of the perturbation (10 mV peak-to-peak AC amplitude) applied. In this case, impedance was measured at the so-called open circuit potential (OCP) −0.75 V which has equilibrated at the working electrode. This means a situation which has equilibrated at the electrode depending on the solution composition with stability during the measurements. The other reason of considering −0.75 V DC poten-

tial is related to Rct , which is the kinetic component of the resistance determined by EIS. Therefore to attain a kinetic controlled interface process, the DC potential should be designated to match the kinetic region of voltammogram in Fig. 7B. In addition, the biosensor was deliberated by monitoring the CV peak currents after continuous scanning for 500 cycles, 96.5% of the initial current response was retained, indicating that biosensor was stable in buffer solution. CV peak currents after continuous 3 scanning were shown in Fig. 7B inset. The inset showed no reversibility constrain, no oxygen limitation or other mechanisms causing signal saturation due to high surface area of nanoporous thin film as a family of the richest nanostructure and no kinetic restraint. The calibration curve for urea at the Urs/Nano-ZnO/TiO2 /FTO biosensor was obtained by monitoring its Rct responses (|Rct | = |Rct urea -Rct buffer(blank) |). Fig. 8C displays a working curve shown in a linear scale [Rct (k) = 1.163 + 0.02 [urea]; R2 = 0.998] in a concentration range of 5–205 mg dL−1 of urea. The Urs/NanoZnO/TiO2 /FTO biosensor exhibits detection limit of 2 mg dL−1 for

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS

12

R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

Table 2 Determination of urea level in different solution. Sample number

urea (mg dL−1 ) Spiked urea added (mg dL−1 ) Measured by Urs/Nano-ZnO/TiO2 /FTO biosensor (mg dL−1 ) a %Recovery a

%Recovery =

hp −hreal hadded

1

2

3

4

5

20 0 19.9 –

15 3 18.3 110%

17 4 20.9 97%

28 7 35.1 101%

36 5 40.5 90%

× 100; where hp is predicted concentration (sum of real concentration and added amount), hreal is real sample concentration without any added

amount, and hadded is concentration amount which is added to real concentration. Table 3 Determination of urea level in blood serum. Method

Sample number

−1

Determined by Spectrophotometry (mg dL ) Measured by Urs/Nano-ZnO/TiO2 /FTO biosensor (mg dL−1 ) R.S.D.a (n = 5) (%) a

1

2

3

4

5

24.0 23.8 1.3

27.7 26.5 1.1

31.0 31.2 2.2

47.2 47.6 1.4

64.0 63.2 2.1

R.S.D.: Relative Standard Deviation.

Table 4 Analytical characteristics of Urs/Nano-ZnO/TiO2 /FTO biosensor compared with other reported values of urea biosensor in the literature. Response Characteristics Matrix

D.R.

D.L.

R.T.

ZnO nanoparticles Polyaniline Nafion/Au/ceramic composite film Gelatin Polyaniline-poly (n-butylmethacrylate) Cellulose membrane Dead cellulose Whatman paper Nano-porous silicon Gold-coated plastic substrates Polyaniline Graphite and platinum composite electrode Carboxylic poly(vinylchloride) Polyethylenimine Nanoporous Alumina Tetramethylorthosilicate (TMOS) Laser ablated ZnO thin film Cs-Co3 O4 nanocomposite NiO nanoparticles Zinc oxide-chitosan Mn2+ doping in TiO2 thin films Silicalite BSA embedded surface modified polypyrrole film TiO2 film ZnO nanorods Sputtered ZnO thin film Electrodeposited ZnO-PVA hybrid film Electrodeposited nanoporous ZnO 3D hierarchical nanoporous Nano-ZnO/TiO2

10–80 mg dL-1 3–30 mg dL-1 5–140 mg dL-1 20–120 mg dL-1 – 10–400 mg dL-1 12–660 mg dL-1 10–99 mg dL-1 10–100 m mol L-1 0.1–100 m mol L-1 10-6 –10-1 mol L-1 10–250 ␮ mol L-1 10-5 –10-1 mol L-1 10-2.5 –10-1.5 mol L-1 0.5 ␮M–3.0 m mol L-1 0.01–30 m mol L-1 5–200 mg dL-1 10-4 -8 × 10-2 mol L-1 0.83–16.65 m mol L-1 5–100 mg dL-1 0–6.5 mg mL-1 5–850 ␮ mol L-1 6.6 × 10-6 –7.5 × 10-4 mol L-1 8 ␮ mol L-1 –3 m mol L-1 0.001–24.0 m mol L-1 5–140 mg dL-1 5–125 mg dL-1 8–110 mg dL-1 5–205 mg dL-1

13.5 mg dL-1 0.3 mg dL-1 5 mg dL-1 20 mg dL-1 6 mg dL-1 10 mg dL-1 12 mg dL-1 10 mg dL-1 – 0.1 m mol L-1 – 3 ␮ mol L-1 0.28 m mol L-1 10-2.5 mol L-1 0.2 ␮ mol L-1 52 ␮g mL-1 – – – 3 mg dL-1 – – – 5 ␮ mol L-1 10 ␮ mol L-1 2.42 mg dL-1 3 mg dL-1 5 mg dL-1 2 mg dL-1

– – 2 min 10 min 1–5 min – 5 min 2 min <1 min 4s – 2 min – 15–30 s 30 s – – 12 s 5s 10 s – 3 min 70-90 s 25 s – 4s 3s 10 s 4s

Detection method

Ref.

Cyclic Voltammetry Amperometric Potentiometric Conductometric Optical Colorimetric Manometric Potentiometric Amperometric Potentiometric Potentiometric Amperometric Potentiometric Potentiometric Conductometric Potentiometric Amperometric Potentiometric Cyclic Voltammetry Cyclic Voltammetry Chronoamperometry Conductometric Potentiometric Potentiometric Cyclic Voltammetry Impedimetric Impedimetric Impedimetric Impedimetric

[66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [43] [44] [45] This work

Detection Range [D.R.], Detection Limit [D.L.], Response Time [R.T.].

urea and sensitivity of 0.02 k per mg dL−1 with a linear range within 5–205 mg dL−1 . These results confirm that ZnO thin film matrix provides the attractive microenvironment for the enzyme immobilization to retain its desired natural activities. For studying the selectivity of biosensor towards the determination of urea, EIS studies have been carried out by adding normal concentration of the possible interferents present in human serum such as uric acid (0.1 mM), ascorbic acid (0.05 mM), and lactic acid (0.05 mM), separately along with concentration of urea (205 mg dL−1 ). The EIS analysis for all the interferents shows a maximum interference of 1% with the presence of ascorbic acid (Fig. 8D, E). The results illustrate the importance of prepared biosensor (Urs/Nano-ZnO/TiO2 /FTO) in selective detection of urea.

Matrix spiking is a technique that is used to evaluate the performance of an analytical procedure when testing a specific sample (matrix) type. A matrix spike is generated by adding a known amount (a spike) of the analyte to a sample, testing the spiked sample, and determining if we have recovered the amount that we added. In this study, urea samples were considered to test the accuracy of the proposed biosensor, known concentrations of analytes were spiked to some samples as reported in Table 2 and the recovery of analytes was calculated. The results show the proposed Urs/Nano-ZnO/TiO2 /FTO biosensor can be considered as an accurate biosensor for urea sensing. Several assays were made on blood serum to test the precision of the Urs/Nano-ZnO/TiO2 /FTO biosensor. The urea concentration

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

was determined by the calibration curve (Table 3). 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 to be useful for application in real samples with good precision and accuracy. Table 4 compares response characteristics of the Urs/NanoZnO/TiO2 /FTO biosensor with the other reported urea biosensors [43–45,66–90]. The biosensor of the present work clearly shows good linear range, detection limit and response time with respect to results published in previous studies. The proposed Urs/NanoZnO/TiO2 /FTO biosensor has a fast response time of less than 4 s and retained good enzymatic activity for more than 3 weeks when kept at 4 ◦ C temperature and not in use. In addition, the stability of the Urs/Nano-ZnO/TiO2 /FTO 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%, 3% and 8% of their original activity after storing time of 2 days, 10 days and 4 weeks, respectively. 4. Conclusions 3D hierarchical nanoporous Nano-ZnO/TiO2 was designed and made using the sputtering and doctor-blade techniques, which was exploited as an efficient transducer for fabrication of urea biosensor. The presence of TiO2 as a part of hierarchical heterostructure shows a vital effect on biosensor performance via promoting electron transfer between ZnO to FTO substrate, as well as affording high electronic density to the biosensor surface as an electrostatic repulsion layer to reduce anionic interferents at the biological media. Porous ZnO nanomaterials provide a larger surface area available for enzyme immobilization and decrease the diffusion distance for the substrate to access the immobilized enzyme, which can promote faster detection kinetics, once the electrochemically detectable species (ammonia) is produced closer to the transducer, reducing diffusion resistance, and thus increasing sensitivity and decreasing response times. As a result, the biosensor exhibits a comparable sensing and a faster response time in the presence of urea than most other biosensors, which is most expected due to the formation of the heterojunction between TiO2 and ZnO. 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. (Tehran, Iran) for this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2017.10.009. References [1] M. Dervisevic, E. Dervisevic, M. Senel, Design of amperometric urea biosensor based on self-assembled monolayer of cystamine/PAMAM-grafted MWCNT/Urease, Sens. Actuators B 254 (2018) 93–101. [2] O. Mapazi, P.K. Matabola, R.M. Moutloali, C.J. Ngila, A urea-modified polydiacetylene-based high temperature reversible thermochromic sensor: characterisation and evaluation of properties as a function of temperature, Sens. Actuators B 252 (2017) 671–679. [3] D. Liu, K. Ge, K. Chen, L. Nie, S. Yao, Clinical analysis of urea in human blood by coupling a surface acoustic wave sensor with urease extracted from pumpkin seeds, Anal. Chim. Acta 307 (1995) 61–69. [4] M. Mascini, Enzyme-based optical-fibre biosensors, Sens. Actuators B 29 (1995) 121–125. [5] X. Xie, A.A. Suleiman, G.G. Guilbault, Determination of urea in serum by a fiber-optic fluorescence biosensor, Talanta 38 (1991) 1197–1200.

13

[6] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Polypyrrole-based potentiometric biosensor for urea Part 1. Incorporation of urease, Anal. Chim. Acta 281 (1993) 611–620. [7] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Polypyrrole-based potentiometric biosensor for urea: part 2. Analytical optimization, Anal. Chim. Acta 281 (1993) 621–627. [8] M.F. Esteve, S. Alegret, The modern student laboratory: a practical approach to chemical sensors through potentiometric transducers: determination of urea in serum by means of a biosensor, J. Chem. Educ. 71 (1994) 67–70. [9] D. Liu, M.E. Meyerhoff, H.D. Goldberg, R.B. Brown, Potentiometric ion and bioselective electrodes based on asymmetric polyurethane membranes, Anal. Chim. Acta 274 (1993) 37–46. [10] K. Chen, D. Liu, L. Nie, S. Yao, Determination of urea in urine using a conductivity cell with surface acoustic wave resonator-based measurement circuit, Talanta 41 (1994) 2195–2200. [11] A.S. Jdanova, S. Poyard, A.P. Soldatkin, N. Jaffrezic-Renault, C. Martelet, Conductometric urea sensor: use of additional membranes for the improvement of its analytical characteristics, Anal. Chim. Acta 321 (1996) 35–40. [12] H. Sangodkar, S. Sukeerthi, R.S. Srinivasa, R. Lal, A.Q. Contractor, A biosensor array based on polyaniline, Anal. Chem. 68 (1996) 779–783. [13] R.E. Adams, P.W. Carr, Coulometric flow analyzer for use with immobilized enzyme reactors, Anal. Chem. 50 (1978) 944–950. [14] E. Dervisevic, M. Dervisevic, J.N. Nyangwebah, M. Senel, Development of novel amperometric urea biosensor based on Fc-PAMAM and MWCNT bio-nanocomposite film, Sens. Actuators B 246 (2017) 920–926. [15] M.S. Wilson, Electrochemical immunosensors for the simultaneous detection of two tumor markers, Anal. Chem. 77 (2005) 1496–1502. [16] P. D’Orazio, Biosensors in clinical chemistry, Clin. Chim. Acta 334 (2003) 41–69. [17] S.G. Ansari, Z.A. Ansari, G.S. Kim, Y.S. Kim, G. Khang, H.S. Shin, Urea sensor based on tin oxide thin films prepared by modified plasma enhanced CVD, Sens. Actuators B 132 (2008) 265–271. [18] P.R. Solanki, A. Kaushik, A.A. Ansari, G. Sumana, B.D. Malhotra, Zinc oxide-chitosan nanobiocomposite for urea sensor, Appl. Phys. Lett. 93 (2008) 163903–163907. [19] A.P. Soldatkin, J. Montoriol, W. Sant, C. Martelet, N. Jaffrezic-Renault, A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs, Biosens. Bioelectron. 19 (2003) 131–135. [20] S.K. Jha, A. Topkar, S.F. D’Souza, Development of potentiometric urea biosensor based on urease immobilized in PVA-PAA composite matrix for estimation of blood urea nitrogen (BUN), J. Biochem. Bioph. Methods 70 (2008) 1145–1150. [21] R. Singhal, A. Gambhir, M.K. Pandey, S. Annapoorni, B.D. Malhotra, Immobilization of urease on poly(n-vinyl carbazole)/stearic acid Langmuir–Blodgett films for application to urea biosensor, Biosens. Bioelectron. 17 (2002) 697–703. [22] A. Sehitogullari, A.H. Uslan, Preparation of a potentiometric immobilized urease electrode and urea determination in serum, Talanta 57 (2002) 1039–1044. [23] I. Vostiar, J. Tkac, E. Sturdik, P. Gemeiner, Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe, Bioelectrochemistry 56 (2002) 113–115. [24] S. Lee, W. Lee, Determination of heavy metal ions using conductometric biosensor based on sol-gel immobilized urease, Bull. Korean Chem. Soc. 23 (2002) 1169–1172. [25] P.C. Pandey, G. Singh, Tetraphenylborate doped polyaniline based novel pH sensor and solid-state urea biosensor, Talanta 55 (2001) 773–782. [26] M.H. Keyes, R. Barabino, Development of immobilized urease for the Owens-Illinois bun analyzer, Enzyme Eng. 1 (1978) 51–56. [27] P. Bertocchi, D. Compagnone, G. Palleschi, Amperometric ammonium ion and urea determination with enzyme-based probes, Biosens. Bioelectron. 11 (1996) 1–10. [28] P.V. Sundaram, W.E. Hornby, Preparation and properties of urease chemically attached to nylon tube, FEBS Lett. 10 (1970) 325–327. [29] D. Kirstein, L. Kirstein, F. Scheller, Enzyme electrode for urea with amperometric indication: part I-basic principle, Biosensors 1 (1985) 117–130. [30] M.L. Seo, J.S. Kim, S.S. Lee, Z.U. Bae, H.L. Lee, Amperometric enzyme electrode for the determination of NH4 + , J. Korean Chem. Soc. 37 (1993) 937–942. [31] R.K. Shervedani, S.A. Mozaffari, Preparation and electrochemical characterization of a new nanosensor based on self-assembled monolayer of cysteamine functionalized with phosphate groups, Surf. Coat. Technol. 198 (2005) 123–128. [32] R.K. Shervedani, S.A. Mozaffari, Impedimetric sensing of uranyl ion based on phosphate functionalized cysteamine self-assembled monolayers, Anal. Chim. Acta 562 (2006) 223–228. [33] R.K. Shervedani, M. Bagherzadeh, S.A. Mozaffari, Determination of dopamine in the presence of high concentration of ascorbic acid by using gold cysteamine self-assembled monolayers as a nanosensor, Sens. Actuators B 115 (2006) 614–621. [34] R.K. Shervedani, S.A. Mozaffari, Copper (II) nanosensor based on a gold cysteamine self-assembled monolayer functionalized with salicylaldehyde, Anal. Chem. 78 (2006) 4957–4963. [35] S.A. Mozaffari, T. Chang, S.-M. Park, Diffusional electrochemistry of cytochrome c on mixed captopril/3-mercapto-1-propanol self-assembled

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS

14

R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47] [48] [49] [50] [51]

[52] [53]

[54]

[55]

[56]

[57] [58]

[59]

[60] [61]

[62]

[63]

monolayer modified gold electrodes, J. Phys. Chem. C 113 (2009) 12434–12442. S.A. Mozaffari, T. Chang, S.M. Park, Self-assembled monolayer as a pre-concentrating receptor for selective serotonin sensing, Biosens. Bioelectron. 26 (2010) 74–79. R. Ahmad, M.S. Ahn, Y.B. Hahn, ZnO nanorods array based field-effect transistor biosensor for phosphate detection, J. Colloid Interface Sci. 498 (2017) 292–297. R. Viter, A. Tereshchenko, V. Smyntyna, N. Starodub, R. Yakimova, V. Khranovskyy, A. Ramanavicius, Toward development of optical biosensors based on photoluminescence of TiO2 nanoparticles for the detection of Salmonella, Sens. Actuators B 252 (2017) 95–102. A. Tereshchenko, V. Fedorenko, V. Smyntyna, I. Konup, A. Konup, M. Eriksson, R. Yakimova, A. Ramanavicius, S. Balme, M. Bechelany, ZnO films formed by atomic layer deposition as an optical biosensor platform for the detection of grapevine virus A-type proteins, Biosens. Bioelectron. 92 (2017) 763–769. R. Viter, I. Iatsunskyi, V. Fedorenko, S. Tumenas, Z. Balevicius, A. Ramanavicius, S. Balme, M. Kempiski, G. Nowaczyk, S. Jurga, M. Bechelany, Enhancement of electronic and optical properties of ZnO/Al2 O3 nanolaminate coated electrospun nanofibers, J. Phys. Chem. C 120 (2016) 5124–5132. Q. Humayun, M. Kashif, U. Hashim, Area-selective ZnO thin film deposition on variable microgap electrodes and their impact on UV sensing, J. Nano Mat. 2013 (2013) 1–5. Z. Lou, J. Deng, L. Wang, A class of hierarchical nanostructures: ZnO surface-functionalized TiO2 with enhanced sensing properties, RSC Adv. 3 (2013) 3131–3136. S.A. Mozaffari, R. Rahmanian, M. Abedi, H.S. Amoli, Urea impedimetric biosensor based on reactive RF magnetron sputtered zinc oxide nanoporous transducer, Electrochim. Acta 146 (2014) 538–547. R. Rahmanian, S.A. Mozaffari, Electrochemical fabrication of ZnO-polyvinyl alcohol nanostructured hybrid film for application to urea biosensor, Sens. Actuators B 207 (2015) 772–781. S.A. Mozaffari, R. Rahmanian, M. Abedi, Disposable urea biosensor based on nanoporous ZnO filmfabricated from omissible polymeric substrate, Mater. Sci. Eng. C 146 (2015) 387–396. J.R. Macdonald, J. Schoonman, A.P. Lehnen, The applicability and power of complex nonlinear least squares for the analysis of impedance and admittance data, J. Electroanal. Chem. 131 (1982) 77–95. P.J. Kelly, R.D. Arnell, Magnetron sputtering: a review of recent developments and applications, Vacuum 56 (2000) 159–172. J. Musil, P. Baroch, J. Vlˇcek, K.H. Nam, J.G. Han, Reactive magnetron sputtering of thin films: present status and trends, Thin Solid Films 475 (2005) 208–218. I. Safi, Recent aspects concerning DC reactive magnetron sputtering of thin films: a review, Surf. Coat. Technol. 127 (2000) 203–218. L. Yao, F. Lu, Low vacuum deposition of aluminum nitride thin films by sputtering, Int. J. Appl. Ceram. Technol. 10 (2013) 51–59. K.W. Seo, H.S. Shin, J.H. Lee, K.B. Chung, H.K. Kim, The effects of thickness on the electrical optical, structural and morphological properties of Al and Ga co-doped ZnO films grown by linear facing target sputtering, Vacuum 101 (2014) 250–256. A. Sassolas, L. Blum, Immobilization strategies to develop enzymatic biosensor, Biotechnol. Adv. 30 (2011) 489–511. S.M.U. Ali, Z.H. Ibupoto, S. Salman, O. Nur, M. Willander, B. Danielsson, Selective determination of urea using urease immobilized on ZnO nanowires, Sens. Actuators B 160 (2011) 637–643. Z.A. Ansari, H. Mazhar Ul, H.K. Seo, A. Umar, A. Al-Hajry, S.A. Al-Sayari, H.S. Shin, S.G. Ansari, Urea sensing properties of Cu-doped titanate nanostructures, Adv. Sci. Lett. 4 (2011) 3451–3457. S.G. Ansari, R. Wahab, Z.A. Ansari, Y.S. Kim, G. Khang, A. Al-Hajry, H.S. Shin, Effect of nanostructure on the urea sensing properties of sol-gel synthesized ZnO, Sens. Actuators B 137 (137) (2009) 566–573. R.P. Janek, W.R. Fawcett, A. Ulman, Impedance spectroscopy of self-assembled monolayers on Au(111): Evidence for complex double-layer structure in aqueous NaClO4 at the potential of zero charge, J. Phys. Chem. 101 (1997) 8550–8558. A. Lasia, A. Rami, Kinetics of hydrogen evolution on nickel electrodes, J. Electroanal. Chem. 294 (1990) 123–141. S. Ameur, H. Maupas, C. Martelet, N. Jaffrezic-Renault, H. Ben Ouada, S. Cosnier, P. Labbe, Impedimetric measurements on polarized functionalized platinum electrodes: application to direct immunosensing, Sci. Eng. C 5 (1997) 111–119. E.T. Mcadams, A. Lackermeier, J.A. McLaughlin, D. Macken, J. Jossinet, The linear and non-linear electrical properties of the electrode-electrolyte interface, Biosens. Bioelectron. 10 (1995) 67–74. J.B. Sumner, D.B. Hand, The isoelectric point of crystalline urease, J. Am. Chem. Soc. 51 (1929) 1255–1260. M.P. Massafera, S.I.C.d. Torresi, Urea amperometric biosensors based on a multifunctional bipolymeric layer: comparing enzyme immobilization methods, Sens. Actuators B 137 (2009) 476–482. S.G. Ansari, S.W. Gosavi, S.A. Gangal, R.N. Karekar, R.C. Aiyer, Characterization of SnO2 -based H2 gas sensors fabricated by different deposition techniques, J. Mater. Sci. Mater. Electron. 8 (1997) 23–27. S.G. Ansari, Z.A. Ansari, R. Wahab, Y.S. Kim, G. Khang, H.S. Shin, Glucose sensor based on nano-baskets of tin oxide templated in porous alumina by plasma enhanced CVD, Biosens. Bioelectron. 23 (2008) 1838–1842.

[64] N.J. Dayan, S.R. Sainkar, R.N. Karekar, R.C. Aiyer, Formulation and characterization of ZnO:Sb thick-film gas sensors, Thin Solid Films 325 (1998) 254–258. [65] X. Fang, L. Wu, Handbook of Innovative Nanomaterials from Syntheses to Applications, Pan Stanford Publishing Pte Ltd, Singapore, 2012. [66] A. Ali, A.A. Ansari, A. Kaushik, P.R. Solanki, A. Barik, M.K. Pandey, B.D. Malhotra, Nanostructured zinc oxide film for urea sensor, Mater. Lett. 63 (2009) 2473–2475. [67] Y.C. Luo, J.S. Do, Urea biosensor based on PANi(Urease)-Nafion/Au composite electrode, Biosens. Bioelectron. 20 (2004) 15–23. [68] P.K. Srivastava, A.M. Kayastha, Srinivasan, Characterization of gelatin-immobilized pigeonpea urease and preparation of a new urea biosensor, Biotechnol. Appl. Biochem. 34 (2001) 55–62. [69] M.M. Castillo-Ortega, D.E. Rodriguez, J.C. Encinas, M. Plascencia, F.A. Méndez-Velarde, R. Olayo, Conductometric uric acid and urea biosensor prepared from electroconductive polyaniline–poly(n-butyl methacrylate) composites, Sens. Actuators B 85 (2002) 19–25. [70] M. Krysteva, M.A. Hallak, Optical enzyme sensor for urea determination via immobilized pH indicator and urease onto transparent membranes, Sci. World J. 3 (2003) 585–592. [71] K.R. Reddy, P.K. Srivastava, P.M. Dey, A.M. Kayastha, Immobilization of pigeonpea (Cajanus cajan) urease on Dead-cellulose paper strips for urea estimation, Biotechnol. Appl. Biochem. 39 (2004) 323–327. [72] D.M. Jenkins, M.J. Delwiche, Manometric biosensor for on-line measurement of milk urea, Biosens. Bioelectron. 17 (2002) 557–563. [73] N. Verma, M. Singh, A disposable microbial based biosensor for quality control in milk, Biosens. Bioelectron. 18 (2003) 1219–1224. [74] J.H. Jin, S.H. Paek, C.W. Lee, N.K. Min, S.I. Hong, Fabrication of amperometric urea sensor based on nano-porous silicon technology, J. Korean Phys. Soc. 42 (2003) 735–738. [75] S.M.U. Ali, Z.H. Ibupoto, S. Salman, O. Nur, M. Willander, B. Danielsson, Selective determination of urea using urease immobilized on ZnO nanowires, Sens. Actuators B 160 (2011) 637–643. [76] B. Lakard, D. Magnin, O. Deschaume, G. Vanlancker, K. Glinel, S. Demoustier-Champagne, B. Nysten, A.M. Jonas, P. Bertrand, S. Yunus, Urea potentiometric enzymatic biosensor based on charged biopolymers and electrodeposited polyaniline, Biosens. Bioelectron. 26 (2011) 4139–4145. [77] A. Pizzariello, M. Stredansky, S. Stredanska, S. Miertus, Urea biosensor based on amperometric pH-sensing with hematein as a pH-sensitive redox mediator, Talanta 54 (2001) 763–772. [78] Z. Wu, L. Guan, G. Shen, R. Yu, Renewable urea sensor based on a self-assembled polyelectrolyte layer, Analyst 127 (2002) 391–395. [79] B. Lakard, G. Herlem, S. Lakard, A. Antoneou, B. Fahys, Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films, Biosens. Bioelectron. 19 (2004) 1641–1647. [80] Y. Zhengpeng, S. Shihui, D. Hongjuan, Z. Chunjing, Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes, Biosens. Bioelectron. 22 (2007) 3283–3287. [81] R. Sahney, S. Anand, B. Puri, A. Srivastava, A comparative study of immobilization techniques for urease on glass-pH-electrode and its application in urea detection in blood serum, Anal. Chim. Acta 578 (2006) 156–161. [82] N. Batra, M. Tomar, P. Jain, V. Gupta, Laser ablated ZnO thin film for amperometric detection of urea, J. Appl. Phys. 114 (2013) 124702–124708. [83] A. Ali, A.A. Ansari, A. Kaushik, P.R. Solanki, A. Barik, M.K. Pandey, B.D. Malhotra, Nanostructured zinc oxide film for urea sensor, Mater. Lett. 63 (2009) 2473–2475. [84] A. Ali, M. Israr-Qadir, Z. Wazir, M. Tufail, Z. Ibupoto, S. Jamil-Rana, M. Atif, S. Khan, M. Willander, Cobalt oxide magnetic nanoparticles-chitosan nanocomposite based electrochemical urea biosensor, Indian J. Phys. 89 (2015) 331–336. [85] M. Tyagi, M. Tomar, V. Gupta, NiO nanoparticle-based urea biosensor, Biosens. Bioelectron. 41 (2013) 110–115. [86] D. Marshal, Using nano-arrayed structures in sol-gel derived Mn2+ doped TiO2 for high sensitivity urea biosensor, J. Biosens. Bioelectron. 1 (2010) 1000101–1000104. [87] I. Kucherenko, O. Soldatkin, B.O. Kasap, S. Öztürk, B. Akata, A. Soldatkin, S. Dzyadevych, Elaboration of urease adsorption on silicalite for biosensor creation, Electroanalysis 24 (2012) 1380–1385. [88] T. Ahuja, I.A. Mir, D. Kumar, Potentiometric urea biosensor based on BSA embedded surface modified polypyrrole film, Sens. Actuators B 134 (2008) 140–145. [89] X. Chen, Z. Yang, S. Si, Potentiometric urea biosensor based on immobilization of urease onto molecularly imprinted TiO2 film, J. Electroanal. Chem. 635 (2009) 1–6. [90] R. Ahmad, N. Tripathy, Y.B. Hahn, Highly stable urea sensor based on ZnO nanorods directly grown on Ag/glass electrodes, Sens. Actuators B 194 (2014) 290–295.

Biographies Reza Rahmanian received his PhD of chemistry from department of chemical technologies of Iranian research organization for science and technology (IROST). His main areas of interests include thin film based sensors and biosensors, application of

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009

G Model SNB-23313; No. of Pages 15

ARTICLE IN PRESS R. Rahmanian et al. / Sensors and Actuators B xxx (2017) xxx–xxx

electrochemical impedance spectroscopy (EIS) in bioelectroanalysis, Physical Vapor Deposition (PVD) such as sputtering, spin coating, spray pyrolysis. Sayed Ahmad Mozaffari received his PhD of chemistry under supervision of Professor Reza Karimi Shervedani from university of Isfahan in 2006. Then he undertook post-doctoral studies at Pohang University of science and technology (POSTECH) in South Korea under supervision of professor Su-moon Park (2008–2009). His main areas of interests include electroanalytical chemistry, thin layers and nanotechnology, sensors and biosensors, electrochemistry of solar cells and fuel cells, electrochemical impedance spectroscopy (EIS) and Fourier transform EIS.

15

Hossein Salar Amoli received his PhD of chemistry from university of Salford in 1990. His main areas of interests include electroanalytical chemistry, thin layers and nanotechnology, sensors and biosensors, electrochemistry of solar cells, and separation. Mohammad Abedi received his PhD of chemistry from university of Isfahan in 2006. His main areas of interests include green chemistry, thin layers and nanotechnology, and solar cells.

Please cite this article in press as: R. Rahmanian, et al., Development of sensitive impedimetric urea biosensor using DC sputtered Nano-ZnO on TiO2 thin film as a novel hierarchical nanostructure transducer, Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.009