Accepted Manuscript Title: Design of amperometric urea biosensor based on Self-Assembled Monolayer of Cystamine/PAMAM-grafted MWCNT/Urease Authors: Muamer Dervisevic, Esma Dervisevic, Mehmet S¸enel PII: DOI: Reference:
S0925-4005(17)31180-2 http://dx.doi.org/doi:10.1016/j.snb.2017.06.161 SNB 22632
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
14-2-2017 11-6-2017 23-6-2017
Please cite this article as: Muamer Dervisevic, Esma Dervisevic, Mehmet S¸enel, Design of amperometric urea biosensor based on Self-Assembled Monolayer of Cystamine/PAMAM-grafted MWCNT/Urease, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.161 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Design of amperometric urea biosensor based on Self-Assembled Monolayer of Cystamine/PAMAM-grafted MWCNT/Urease Muamer Dervisevica, Esma Dervisevica, Mehmet Şenelb* a
Mehmed-pase-Sokolovica No 21, Bihac 77000, Bosnia and Herzegovina
EMC Technology Inc, Asik Veysel Mah. Talatpasa Caddedi, Esenyurt, Istanbul 34510, Turkey
*Corresponding author; E-mail: [email protected]
Highlights > Novel amperometric urea biosensor based on immobilization of Urease onto self-assembled
MWCNT-PAMAM dendrimers > The biosensor seems to be fast to respond and sensitive. > The bionanocomposite film used in this study is an effective platform to produce reliable biosensors.
Abstract In this work, we report novel amperometric urea biosensor based on immobilization of Urease enzyme onto self-assembled polyamidoamine grafted multiwalled carbon nanotube (MWCNT-PAMAM) dendrimers (from G1, first generation up to G5) on Au electrode. Biosensing electrode’s performance parameters such as optimum pH, temperature, and applied potential together with reproducibility were investigated. The electrode based on fifth
generation of MWCNT-PAMAM and urease enzyme showed excellent performance in urea detection at an applied potential of 0.45 V with short response time of 3s, wide linear range of 1-20 mM, detection limit of 0.4 mM, and sensitivity of 6.6 nA/mM. No significant response was detected in the presence of possible interferon molecules. Non-diluted human blood plasma has been used for real sample application where electrode showed excellent performance in urea detection.
Keywords: Urea, Urease, PAMAM dendrimers, MWCNT, amperometric biosensor.
1. Introduction Urea is non-toxic, nitrogenous organic end product of protein metabolism enabling 8090% of nitrogen elimination from human body [1, 2]. Normal blood serum/plasma urea level is in the range of 3.3 - 6.7 mM  while above 30 mM concentration signify the requirement of dialysis in addition to other possible factors such as saline, water depletion, and gastrointestinal tract destruction [2, 4, 5]. On the other hand, decreased blood urea level could
be pointing to the impaired renal function, inadequate protein diet, or alcohol abuse [6, 7]. The urea evaluation has been performed via different techniques such as chromatographic , chemiluminometric [11, 12], colorimetric [13-15], spectrophotometric [16,17], fluorimetric , and electrochemical [19-21]. The electrochemical techniques, specifically amperometric ones, are distinguished in many aspects from the above listed methods in terms of low fabrication cost, short operation time with no necessity of highly skilled personnel, detecting the analyte with high selectivity and sensitivity . Biosensors are analytical devices integrating target analyte recognition element, physical or chemical signal transduction element, and signal display unit. The demand of sensitive, selective, inexpensive, and rapid detection of biologically significant analytes has been attracted attention on biosensors development. Among other types of electrochemical biosensors, the key components in the design of amperometric biosensors are the recognition and signal transduction elements where various materials have been employed to enhance the sensing capacity in terms of faster electron transfer through the sensing layer to the transducer including nanoparticles, carbon based nanomaterials, conductive polymers, mediators etc . Due to the outstanding mechanical, chemical, and physical properties of nano-scaled materials, they have been widely used for the amperometric biosensors fabrication . Carbon
electrochemical properties such as high surface area to volume ratio, electrical conductivity, and thermal stability together with biocompatibility explains the reason of being widely studied since their discovery in 1991 [29-31]. Successful employment of CNT within various electrochemical biosensors have been reported where biological recognition element such as proteins (enzyme), cofactors, or nucleic acids were either physically adsorbed or covalently bound to the CNTs [32-37]. Magar et al.,  reported highly sensitive choline oxidase enzyme inhibition biosensor for led ions which is based on MWCNT study, Ibanez et al., 
reported work on electrochemical lactate biosensor based upon chitosan/carbon nanotubes for determination of lactate in embryonic cell cultures, and Kangkamano et al., reports study on highly sensitive flow based non-enzymatic glucose sensor based on chitosan cryogel with embedded gold nanoparticles decorated MWCNT . The covalent modification of carbon nanotubes with polymers or small molecules enables further surface to volume ratio increase and helps to overcome the dispersibility problem by enhancing solubility. Poly(amidoamine) (PAMAM) dendrimers are well designed polymers with internal gaps and amine group terminated controllable highly branched structures being successfully covalently linked on the CNTs and applied for drug delivery, bio-imaging, biosensing, and CO2 absorption purposes [32, 41-46]. The proposed work illustrates the application of urease enzyme attached to PAMAM grafted MWCNTs (MWCNT-PAMAM G(x)) for the amperometric determination of urea in human blood plasma sample. The electrochemical characterization of bio-sensing electrodes constructed separately with five dendrimer generations has shown that the maximum response having electrode is fifth generation dendrimer modified electrode (MWCNT-PAMAM G-5). Analytical performance of G5 electrode has been evaluated in terms of optimum operating pH, temperature, and applied potential, reusability, and storage characteristics. The results show that MWCNT-PAMAM G-5 electrode has a linear range of 1-20 mM, 0.4 mM detection limit, 6.6 nA/mM sensitivity, and a short response time of 3s together with negligible interference response in the presence of uric acid, ascorbic acid, glucose, cholesterol, or lactic acid. 2. Experimental Part 2.1. Materials Urease (EC 188.8.131.52. from jack beans), urea, glucose (G), cholesterol (C), ascorbic acid (AA), uric acid (UA), lactic acid (LA), cysteamine, ethylenediamine, methylacrylate, and
gluteraldehyde 25% were purchased from Sigma Aldrich. Methanol was purchased from Merck. Amine functional Multi-walled CTNs (MWCNT) was obtained from Cheap Tubes (USA). Different generations of dendrimer-modified MWCNTs were prepared similar to previous report . 2.2. Instrumentation Electrochemical impedance spectroscopy (EIS) measurements were performed on CHI Model 6005 electrochemical analyzer workstation where other electrochemical measurements were recorded on IVIUM CompactStat portable electrochemical interface and impedance analyzer (Ivium Technologies, B.V., Netherlands) in an electrochemical cell containing Au plate electrode as working, Platinum wire as counter, and Ag/AgCl as reference electrode at ambient conditions. The determination of urea from selected samples was performed after the electrode reached the steady state status by adding certain amount of urea while being in a permanently stirred buffer filled cell. 2.3. Preparation of enzyme electrode Before any modification, Au plate electrode was kept in piranha solution (sulfuric acid:hydrogen peroxide, 3:1) and then sonicated in distilled water (dH2O) for 1 min to remove all chemically and physically attached molecules. In order to obtain amine functionalized electrodes, cleaned electrodes were immersed in 0.2M cysteamine solution for 2 h (Fig. 1). The MWCNT/PAMAM dendrimer (10mg/ml dissolved in methanol, from G1 to G5) modification of electrode was achieved by immersing electrodes in 2% gluteraldehyde (GA) for 45 min before and after dendrimer modification. As the last step, electrodes were kept in 20mg/ml urease solution prepared in 10 mM PBS with a pH of 7.4 for 3 h aiming the covalent attachment of enzyme to the amine groups of the dendrimers through carboxyl ends of enzyme whereas whole steps are schematically summarized in Fig. 1B. All immersion steps were conducted on an orbital shaker set at 200 rpm while between each modification step, the
electrodes were carefully rinsed with dH2O and stored at 4°C in 10 mM PBS, pH 7.4 when not being in use.
(Figure 1.) 2.4. Real sample application Real sample measurements were performed using human blood which was obtained from 36-year-old healthy male laboratory volunteer. Urea measurements were performed in blood plasma alone and 6.0 mM and 12.0 mM urea added plasma samples for the biosensing electrode performance analysis. 3. Results and Discussion 3.1. Electrochemical Characterizations
(Figure 2.) Cyclic voltammetry was used in order to investigate the electron transfer rate between the ferrocyanide solution and the electrode surface modified separately with each dendrimer generation as illustrated in Fig. 2A. The results show that the current transfer efficiency through the modified layer reduced as the generation of dendrimer increased. Oxidation peak for dendrimer G1 was obtained at 0.8µA after which it is gradually decreasing as dendrimer generations were increasing and eventually declines to 0.6µA for G5 where at reduction point current change was 0.15µA. Similar results were obtained from EIS measurements being given in Fig. 2B. The charge transfer resistance (Rct) is well known as a useful parameter reflecting the facility of electrode reaction which can be measured by EIS from the semicircle diameter in the Nyquist plots . EIS method is very useful in monitoring the interfacial reaction mechanism on electrode surface and it is often used in study if chemical transformations and processes associated to conductivity changes in the equivalent circuit
. The semicircular part in EIS curves at higher frequencies correspond to the electrontransfer-limited process and its diameter is equal to the electron transfer resistance . Fitting procedure, after EIS measurements and in order to obtain Rct values, was preformed using designed equivalent circuit model (insert of Fig. 2B). Adjusting most suitable equivalent circuit in EIS measurements is important point which is well explained by Ramanavicius et al.,  in study on electrochemical impedance spectroscopy based evaluation of glucose oxidase modified electrode. The circuit is composed of Rct which represents charge transfer resistance through electrode surface, RS represents resistance of electrolytes and all connections, C represents capacitance of the bioactive layer, and Zw stands for Warburg impedance which shows redox probe diffusion through surface of biosensing electrode . Rct values shown in insert of Fig. 2B are obtained using equivalent circuit model and CHI 6005 instrument software. The results show that the electron transfer resistance increases in the order of the increasing the generation of PAMAM dendrimers on MWCNTs, which indicates that PAMAM dendrimers on the MWCNT decrease the conductivity of the gold electrode. Although the conductivity is decreased by the growing amine branches from the bio-electrode surface available for the covalent modification of biological recognition elements such as enzyme, DNA, antibody, etc., the amperometric signal in the presence of the target analyte is enhanced because of the increased recognition element number . (Figure 3.) Figure 3 illustrates electrochemical characterization of Au/MWCNT-PAMAM G1 to G5 modified Au electrode with cyclic voltammogram by applying different scan rates from 50 mVs-1 to 500mVs-1 within the range of -0.1V to 0.6V in 0.5mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1M KCl which served as redox probe. Observed current transfer rate between the ferrocyanide solution and electrode surface decreases as dendrimer
generation increases which lowers electron transfer efficiency. Considering the peak currents, each of the dendrimer has good electron transfer rate of redox couples. However, as scan rate was increasing, the anodic peak potential slightly shifts towards the positive potential and cathodic peak shifts in the reverse direction. The inset of figures 3A-E shows linear correlation between peak currents and square root of scan rate which increases with scan rate increase. This behavior was observed in all dendrimer generations which shows a characteristic of a diffusion controlled redox process. 3.2. Determination of experimental variables (Figure 4.) Optimum potential for Au/MWCNT-PAMAM (G5) bio-sensing electrode has been investigated in the range of 0.3 to 0.5 V. As it can be seen in Figure 4A, amperometric response of fabricated electrode was gradually increasing as applied potential was increasing from 0.3 to 0.45 where it reaches its maximum response. Further increase in applied potential resulted in decrease in current response where at 0.5 V electrode losses 15% of its response obtained at 0.45V. Applied potential of 0.45V for amperometric urea biosensor was reported in several works which used different modification of electrode surface [54, 55]. Optimum applied potential for Au/MWCNT-PAMAM (G5) based electrode was selected to be 0.45V, as well it is important to note that low applied potential is useful in avoiding interference of molecules which can easily oxidize at higher potentials. Effect of pH on amperometric response of enzyme electrode has been investigated over the range of pH 5.0 to 9.0. Figure 4B shows results obtained from Au/MWCNTPAMAM (G5) electrode where current responses increase gradually as pH increases reaching plateau at pH 8.0 at its maximum response. After increasing pH from 8.0 to 9.0, there was a high loss in current response, approximately 50% of response obtained at pH 8.0. As can be
concluded form Figure 4B, optimum working pH for Au/MWCNT-PAMAM (G5) based electrode is 8.0. Similar results in investigation of optimum pH for urea biosensor can be seen in other works reported by Hao et al., where optimum pH fore urea biosensor based on metal catalysts immobilized by a polyion complex was 8.0 , as well in our previous studies on urea amperometric biosensors optimum pH of 8.0 can be observed . Figure 4C represents optimum temperature study for proposed electrode in the range of 25oC to 60oC with a step size of 5oC. As can be seen from figure 4C, amperometric response drastically increases up to 50oC after which it decreased probably caused by the thermal deactivation of the enzyme at high temperatures and decrement in the molecular oxygen in the solution. As a result, optimum temperature for the G5 modified electrode was determined to be 50oC. 3.3. Amperometric response of enzyme electrode (Figure 5.) Amperometric response of proposed bio-sensing electrodes were examined by successive addition of 1mM urea in continuously stirred 10mM PBS with a pH of 8.0 at applied potential of 0.45V. Experiments were performed using Au/MWCNT-PAMAM G1 to G5/ Urease electrodes having the results represented by current vs. time plots as seen in figure 5A. Comparison among the five different electrodes shows that the electrode modified with fifth generation dendrimer has the highest current response to the consecutive addition of the analyte urea reaching the 95% of steady-state current within approximately 3 s. Current response obtained by electrodes towards 1mM urease was in the following sequence: G1-G5; 0.0034 µM, 0.0042 µM, 0.0046 µM, 0.0052 µM, and 0.0060 µM. Figure 5B being the current vs. concentration plot illustrates calibration plots of amperometric responses displayed in Fig. 5A. Obviously, best performance in urea detection is obtained by Au/MWCNT-PAMAM
(G5)/ Urease having a linear range of 1 to 20 mM, response time of 4s, detection limit of 0.4mM, and sensitivity of 6.60 nA/mM. The regression equation was calculated as ∆I(µA)=0.0002-0.0065[Urea (mM)] with a regression coefficient of (R) 0.9986. Table 1 summarizes analytical performances of electrodes used in this study bringing to the conclusion that electrode based on G5 demonstrated best performance characteristics in terms of linear range, sensitivity, and detection limit due to the increased enzyme bonding capability of the highest PAMAM dendrimer generation. (Table 1.)
Table 2 enables the comparison of the performance of Au/MWCNT-PAMAM (G5)/Urease based electrode with previously reported urea detecting bio-electrodes. According to the Table 2, proposed bio-sensing electrode possesses superior analytical characteristics in comparison with most of the reported studies thanks to the short response time and wide linear range. It is notable that in contrast to most of the reported studies, the bio-electrode’s linear range is wide enough for the blood samples containing abnormal urea concentration to be processed without any further treatments.
3.4. Operational and storage stability (Figure 6.) Operational stability of Au/MWCNT-PAMAM (G5)/Urease based bio-sensing electrode was performed by recording amperometric response of 15 consequent measurements carried out in 10mM PBS with pH 8.0 at applied potential of 0.45V. Figure 6A illustrates results obtained from amperometric measurements from which it can be seen that
proposed electrode kept 90% of its initial response in first 7 measurements after which it gradually decreased down to 75% at the end of 15th measurement. Au/MWCNT-PAMAM (G5)/Urease based electrode demonstrates good results in operational stability since the current response loss after 7 measurements was only 10%. Figure 6B shows results obtained from long term stability measurements which were performed in15 day time period while the electrode was kept in 10mM PBS, pH 8.0 at 4oC when not being in use. According to figure 6B, 8 days of storage caused approximately 10% decreased response of the initial current response where at the end of 15 days lasting storage experiments, the Au/MWCNT-PAMAM (G5)/Urease electrode was able to retain 83% of its initial response. PAMAM dendrimers played important role in giving stability to enzyme immobilization as well good storage results can be supported in previous reported study on amperometric urea biosensor . 3.5. Interference, analytical recovery and real sample measurements (Figure 7.) Interference study is highly significant in the characterization of biosensors in terms of high requirement to avoid false positive response caused by the biomolecules other than target ones present in the sample environment. Although UA (up to 457.9 µM ) and AA sort of molecules are in much smaller concentration when compared to blood urea level, they are first candidates studied as interferons based on the fact that they undergo self-oxidation at high applied potential. Figure 7 illustrates the results from interference effect investigation of 1 mM of UA, AA, LA, G, and C on amperometric response of Au/MWCNT-PAMAM (G5)/Urease bio-sensing electrode when compared to the response to 1 mM urea. Results show that with 0.293% for UA and 0.701% for AA of 1 mM urea caused response and no detectable response at all for LA, G, and C, the proposed bio-electrode is highly selective
towards the target analyte displaying the adoptability to be used for blood samples. These results can be attributed to low working potential of 0.45V and selectivity of Urease enzyme. (Table 3.)
Analytical recovery for Au/MWCNT-PAMAM (G5)/Urease bio-sensing electrode was determined by being tested for three urea concentrations; 2.0 mM, 5.0 mM, and 10 mM where the results including found urea concentration, standard deviation, and recovery are shown in Table 3. Recovery was in the range of 108.8 to 111.5% and RSD form 2.79 to 3.87% indicating that electrodes analytical recovery is acceptable. The results conduce to the possession of excellent analytical performance of sensing electrode with high reliability in electrochemically detecting urea. (Table 4.) After the approval of proposed bio-sensing electrode’s high performance through analytical recovery and interference studies, human blood plasma samples were analyzed in terms of urea level. Au/MWCNT-PAMAM (G5)/Urease based electrode was used to electrochemically detect the urea concentration in three plasma samples; absolute, 6 mM urea added, and 12 mM urea added. Table 4 illustrates results obtained from real sample measurements where sample 1 represents absolute plasma urea level being 3.44 mM falling in the range of normal blood urea level of 3.3 to 6.7 mM reported by Food and Drug Administration (FDA) in Investigations operations manual 2015 . The recovery of 94.0 to 107.16 % with RSD of 2.58 to 3.77% for n=3 calculated for urea spiked samples measurements showed the bio-sensing electrode’s high reliability and accuracy when being applied to blood urea level assessment. 4. Conclusions
We demonstrated an amperometric urea biosensing electrode fabricated by covalent immobilizing of urease on self-assembled PAMAM grafted MWCNTs on Au electrode. Polyamidoamine (PAMAM) dendrimers up to the fifth generation (G-5) were grown onto the surface of functionalized multiwall carbon nanotubes (MWCNTs-NH2) by a divergent method resulting in the PAMAM-g-MWCNTs hybrid materials. The G-5 of PAMAM-gMWCNTs shows a significant improvement in biosensor performance when compared to the other PAMAM generations that is due to the increased enzyme immobilization efficiency of the higher generations. The fabricated urea biosensor implies good analytical characteristics such as short response time, low detection limit, high sensitivity, stability and reproducibility. On the basis of these results, this biosensor successfully could be applied for the quantification of the urea in blood samples.
 F. Kurzer, P.M. Senderson, Urea in the history of organic chemistry: Isolation from natural sources, J. Chem. Educ. 33 (1956) 452-459.
 A.J. Taylor, P. Vadgama, Analytical reviews in clinical biochemistry: The estimation of urea, Ann. Clin. Biochem. 29 (1992) 245-264.  Food and Drug Administration (FDA), Investigations operations manual. Appendix C. Bool serum chemistry. 2015, pp. 443  G. Dhawan, G. Sumana, B.D. Malhotra, Recent developments in urea biosensors Biochem. Eng. J. 44 (2009) 42–52  N. Amin, R.T. Mahmood, M.J. Asad, M. Zafar, A.M. Raja, Evaluating urea and creatinine levels in chronic renal failure pre and post dialysis: A prospective study, J. Cardiovasc. Diseas. 2 (2014) 182-185.  J.F.T. Glasgow, E.M. Hicks, J.G. Jenkins, S.R. Keilty, G.W. Black, T.F. Fannin, Reye's syndrome, BrJ. Hosp. Med. 34 (1985) 42-45.  G. Lum, S. Leal-Khouri, Significance of low serum urea nitrogen concentrations, Clin. Chem. 35 (1989) 639-640  S. Kodama, T. Suzuki, Highly sensitive method for urea detection in wine, J. Food Sci. 60 (1995) 1097-1099  T. Miyauchi, Y. Miyachi, M. Takahashi, N. Ishikawa, H. Mori, Determination of urea in serum based on the combination of an enzymatic reaction with immobilized urease and ion chromatographic analysis, Anal. Sci. 26 (2010) 847-851.  Y.Q. Wang, S.S. Wang, J. Zhu, L. Wang, B.H. Jiang, W.J. Zhao, Determination of urea content in urea cream by centrifugal partition chromatography, J. Food and Drug Anal. 24 (2016) 399-405.
 M. Tabata, T.J. Murachi, A Chemiluminometric method for the determination of urea in serum using a three-enzyme bioreactor, Biolumin. Chemilumin. 2 (1988) 63-67.  X. Hu, N. Takenaka, M. Kitano, H. Bandow, Y. Maeda, M. Hattori, Determination of trace amounts of urea by using flow injection with chemiluminescence detection, Analy. 119 (1994) 1829-1833.  L. Goeyens, N. Kindermans, M. AbuYusuf, M. Elskens, A room temperature procedure for the manual determination of urea in seawater, Estuar. Coast. Shelf Scien. 47 (1988) 415418.  D.M. Sullivan, J.L. Havlin, Flow injection analysis of urea nitrogen in soil extracts, Soil Sci. Soc. Am. J. 55 (1991) 109-113.  H.H. Deng, G.L. Hong, F.L. Lin, A.L. Liu, X.H. Xia, W. Chen, Colorimetric detection of urea, urease, and urease inhibitor based on the peroxidase-like activity of gold nanoparticles, Anal. Chimica Acta. 915 (2016) 74–80.  T. K. With, T.D. Petersen, B. Petersen, A simple spectrophotometric method for the determination of urea in blood and urine, J. Clin. Pathol. 14 (1961) 202–204.  M.A. Fuertes, J.M. Pérez, C. Alonso, Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialysed protein solutions, J. Biochem. Biophys. Meth. 59 (2004) 209–216.  F. Roch-Ramel, An enzymic and fluorophotometric method for estimating urea concentrations in nanoliter specimens, Anal. Biochem. 21 (1976) 372-381.
 W.Y. Lee, S.R. Kim, T.H. Kim, K.S. Lee, M.C. Shin, J.K. Park, Sol–gel-derived thickfilm conductometric biosensor for urea determination in serum, Anal. Chim. Acta. 404 (2000) 195-203.  A. Tiwari, S. Aryal, S. Pilla, S. Gong, An amperometric urea biosensor based on covalently immobilized urease on an electrode made of hyperbranched polyester functionalized gold nanoparticles. Talanta. 78 (2009) 1401–1407.  N.S. Nguyen, G. Das, H.H. Yoon, Nickel/cobalt oxide-decorated 3D graphene nanocomposite electrode for enhanced electrochemical detection of urea, Biosens. Bioelectron. 77 (2016) 372-377.  R. Freeman, Y. Li, R. Tel-Vered, E. Sharon, J. Elbaz, I. Willner, Self-assembly of supramolecular aptamer structures for optical or electrochemical sensing. Analyst. 134 (2009) 653–656.  S. Park, S. Kim, H. Kheel, S.K. Hyun, C. Jin, C. Lee, Enhanced H2S gas sensing performance of networked CuO-ZnO composite nanoparticle sensor, Mater. Res. Bullet. 82 (2016) 130-135.  B. Liu, L. Wang, B. Tong, Y. Zhang, W. Sheng, M. Pan, S. Wang, Development and comparison of immunochromatographic strips with three nanomaterial labels: Colloidal gold, nanogold-polyaniline-nanogold microspheres (GPGs) and colloidal carbon for visual detection of salbutamol, Biosens. Bioelectron. 85 (2016) 337-342.  E. Bayram, E. Akyilmaz, Development of a new microbial biosensor based on conductive polymer/multiwalled carbon nanotube and its application to paracetamol determination, Sens. Actuat. B: Chem. 233 (2016) 409-418.
 M. Dervisevic, E. Çevik, M. Şenel, Development of glucose biosensor based on reconstitution of glucose oxidase onto polymeric redox mediator coated pencil graphite electrodes Enzy. Microb. Techno. 68 (2015) 69-76.  T. Hoshino, S. Sekiguchi, H. Muguruma, Amperometric biosensor based on multilayer containing
phenothiazine, and glucose dehydrogenase, Bioelectrochem. 84 (2012) 1-5.  X. Zhang, Q. Guo, D. Cui, Recent advances in nanotechnology applied to biosensors. Sensors 9 (2009) 1033-1053.  S. Iijima, Helical microtubules of graphitic carbon, Nature. 354 (1991) 56-58.  P.M. Ajayan, Nanotubes from Carbon, Chem. Rev. 99 (1999) 1787–1800.  M.C. Hersam, Progress towards monodisperse single-walled carbon nanotubes, Nat. Nanotechnol. 3 (2008) 387–394.  F. Li, J. Peng, Q. Zheng, X. Guo, H. Tang, S. Yao, Carbon Nanotube-Polyamidoamine dendrimer hybrid-modified electrodes for highly sensitive electrochemical detection of microRNA24, Anal. Chem. 87 (2015) 4806-4813  L. Meng, P. Wu, G. Chen, C. Cai, Y. Sun, Z. Yuan, Low potential detection of glutamate based on the electrocatalytic oxidation of NADH at thionine/single-walled carbon nanotubes composite modified electrode, Biosens. Bioelectron. 24 (2009) 1751-1756.  B. Liang, L. Li, X. Tang, Q. Lang, H. Wang, F. Li, J. Shi, W. Shen, I. Palchetti, M. Mascini, A. Liu, Microbial surface display of glucose dehydrogenase for amperometric glucose biosensor, Biosens. Bioelectron. 45 (2013) 19-24.
 F. Hu, S. Chen, C. Wang, R. Yuan, Y. Xiang, C. Wang, Multi-wall carbon nanotubepolyaniline biosensor based on lectin–carbohydrate affinity for ultrasensitive detection of Con A, Biosens. Bioelectron. 34 (2012) 202-207.  M. Dervisevic, E. Custiuc, E. Çevik, M. Senel, Construction of novel xanthine biosensor by using polymeric mediator/MWCNT nanocomposite layer for fish freshness detection, Food Chem. 181 (2015) 277–283.  M. Dervisevic, E. Çevik, Z. Durmus, M. Şenel, Electrochemical sensing platforms based on the different carbon derivative incorporated interface, Mater. Scien. Eng. C. 58 (2016) 790 –798.  H.S. Magar, M.E. Ghica, M.N. Abbas, C.M.A. Brett, Highly sensitive choline oxidase enzyme inhibition biosensor for lead ions based on multiwalled carbon nanotube modified glassy carbon electrode. Electroanaly. (2017) 10.1002/elan.201700111  N.H. Ibanez, L.G. Cruz, V. Montiel, C.W. Foster, C.E. Banks, J. Iniesta, Electrochemcial lactate biosensor based upon chitosan/carbon nanoubes modified screen-printed graphite electrodes for the determination of lactate in embryonic cell cultures. Biosens. Bioelectron. 77 (2016) 1168-1174  T. Kangkamano, A. Numnuam, W. Limbut, P. Kanatharana, P. Thavarungkul, Chitosan cryogel with embedded gold anoparticles decorated multiwalled carbon nanotubes modified electrode for highly sensitive flow based non-enzymatic glucose sensor. Sens. Actuat. B: Chem. 246 (2017) 854-863  T.N. Pourianazar, P. Mutlu, U. Gunduz, Bioapplications of poly(amidoamine) (PAMAM) dendrimers in nanomedicine, J. Nanopart. Res. 16 (2014) 2342-2380.
 B.F. Pan, D.X. Cui, P. Xu, T. Huang, Q. Li, R. He, F. Gao, Cellular uptake enhancement of polyamidoamine dendrimer modified single walled carbon nanotubes, J. Biomed. Pharmaceu. Eng. 1 (2007) 13-16  X. Shi, S.H. Wang, M. Shen, M.E. Antwerp, X. Chen, C. Li, E.J. Petersen, Q. Huang, W.J. Weber, J.R. Baker, Multifunctional dendrimer-modified multiwalled carbon nanotubes: synthesis, characterization, and In-Vitro cancer cell targeting and imaging, Biomacromol. 10 (2009) 1744–1750  J. Yang, L. Huang, Y. Zhou, F. Chen, M. Zhong, Multiwalled carbon nanotubes grafted with polyamidoamine (PAMAM) dendrimers and their influence on polystyrene supercritical carbon dioxide foaming, J. Supercrit. Fluids. 82 (2013) 13–21.  Y. Zeng, Y. Huang, J. Jiang, X. Zhang, C. Tang, G. Shen, R. Yu, Functionalization of multi-walled carbon nanotubes with poly(amidoamine) dendrimer for mediator-free glucose biosensor, Electrochem. Commun. 9 (2001) 185–190.  Q. Chen, S. Ai, X. Zhub, H. Yina, Q. Maa, Y. Qiu, A nitrite biosensor based on the immobilization of Cytochrome c on multi-walled carbon nanotubes–PAMAM–chitosan nanocomposite modified glass carbon electrode, Biosens. Bioelectron. 24 (2009) 2991–2996.  Y. Zhang, Y. Li, P. Zhang, De novo growth of poly(amidoamine) dendrimers on the surface of multi-walled carbon nanotubes, J. Mater. Sci. 49 (2014) 3469–3477.  N. Kaur, H. Thakur, N. Prabhakar, Conducting polymer and multi-walled carbon nanotubes nanocomposites based amperometric biosensor for detection of organophosphate, J. Electroanal. Chem. 775 (2016) 121–128.
 A. Ramanavicius, A. Finkelsteinas, H. Cesiulis, A. Ramanaviciene, Electrochemical impedance
Bioelectrochem. 79 (2010) 11-16  M. Şenel, Simple method for preparing glucose biosensor based on in-situ polypyrrole cross-linked chitosan/glucose oxidase/gold bionanocomposite film, Mater. Scien. Eng. C. 48 (2015) 287–293.  A. Ramanavicius, P. Genys, A. Ramanaviciene, Electrochemical impedance spectroscopy based evaluation of 1,10-Phenanthroline-5,6-dione and glucose oxidase modified graphite electrode, Electrochim. Acta. 146 (2014) 659-665  M. Dervisevic, M. Senel, T. Sagir, S. Isik, Boronic acid vs Folic acid: A comparison of the biorecognition performance by impedimetric cytosensor based on ferrocene cored dendimer. Biosens. Bioelectron. 91 (2017) 680-686.  C.S.R. Vusa, V. Manju, S. Berchmans, P. Arumugam, Electrochemical amination of graphene using nanosized PAMAM dendrimers for sensing applications, RSC Adv. 6 (2016) 33409-33418.  W. Hao, G. Das, H.H. Yoon, Fabrication of an amperometric urea biosensor using urease and metal catalysts immobilized by a polyion complex. J. Electroanal. Chem. 747 (2015) 143148  M. Dervisevic, E. Dervisevic, M. Senel, E. Cevik, H.B. Yildiz, P. Camurlu, Construction of ferrocene modified conducting polymer based amperometric urea biosensor. Enzy. Microb. Technol. 102 (2017) 53-59
 E. Dervisevic, M. Dervisevic, J.N. Nyangwebah, M. Senel, Development of novel amperoemtric urea biosensor based on Fc-PAMAM and MWCNT bio-nanocomposite film. Sens. Actuat. B: Chem. 246 (2017) 920-926  Y. Velichkova, Y. Ivanov, I. Marinov, R. Ramesh, N.R. Kamini, N. Dimcheva, E. Horozova, T. Godjevargova, Amperometric electrode for determination of urea using electrodeposited rhodium and immobilized urease, J. Mol. Catal. B: Enzym. 69 (2011) 168– 175.  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.  A. Kaushik, P. R. Solanki, A. A. Ansari, G. Sumana, S. Ahmad, B. D. Malhotra, Iron oxide-chitosan nanobiocomposite for urea sensor, Sens. Actuat. B Chem. 138 (2009) 572-580. 
polyaniline−perfluorosulfonated ionomer composite electrode, Anal. Chem. 70 (1998) 39463951.  P. Bertocchi, D. Compagnone, G. Palleschi, Amperometric ammonium ion and urea determination with enzyme-based probes, Biosens. Bioelectron. 11 (1996) 1-10.  M. Tak, V. Guptaa, M. Tomar, Zinc oxide–multiwalled carbon nanotubes hybrid nanocomposite based urea biosensor, J. Mater. Chem. B. 1 (2013) 6392–6401.  Y.C. Luo, J.S. Do, Urea biosensor based on PANi(urease)-Nafion®/Au composite electrode, Biosens. Bioelectron. 20 (2004) 15-23.
 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.  V.R.. Bisht, W. Takashima, K. Kaneto, An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly (N-3aminopropyl pyrrole-co-pyrrole) film, Biomater. 26 (2005) 3683-3690.  M. Dervisevic, E. Dervisevic, M. Senel, E. Cevik, H.B. Yildiz, P. Camurlu, construction of ferrocene modified conducting polymer based amperometric urea biosensor. Enz. Microb. Technol. 102 (2017) 53-59  M.A. Williamson, L.M. Snyder, J.B. Wallach JB, Wallach's Interpretation of Diagnostic Tests. 9th ed. Philadelphia, 2011.
Biographies Muamer Dervisevic finished his BSc and MSc studies in the Department of Genetics and Bioengineering, Faculty of Engineering, Fatih University/Turkey and currently he is a PhD candidate at Monash University, Faculty of Pharmacy and Pharmaceutical Sciences, Australia. His current researches are enzyme immobilization, biosensors, and electrochemical cancer cell and cancer markers detection. Esma Dervisevic finished her BSc studies in the Department of Genetics and Bioengineering, Faculty of Engineering, Fatih University/Turkey, currently she is a PhD candidate at Monash University, Faculty of Engineering, Australia. Her current researches are enzyme immobilization, electrochemical and photonic biosensors. Mehmet Senel is a R&D director at EMC Technology Company, Istanbul, Turkey. He is performing research in the Biotechnology Research Laboratory on biomaterials, enzyme immobilization, biosensors, and electrochemical cancer cell detection.
Figure Captions Figure 1. A) Experimental route of synthesis and modification of PAMAM-MWCNT. B) Schematic illustration of preparation of urea biosensing electrode. Figure 2. A) Comparison of Cyclic Voltammograms of Au/MWCNT-PAMAM G1 to G5 based bio-sensing electrodes in 0.5mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1M KCl. B) Nyquist plot of Electrochemical Impedance Spectroscopy measurements of Au/MWCNT-PAMAM G1 to G5 electrodes in 0.5mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1M KCl. Figure 3. Cyclic voltammetry with different scan rates of A) Au/MWCNT-PAMAM (G1), B) Au/MWCNT-PAMAM (G2), C) Au/MWCNT-PAMAM (G3), D) Au/MWCNT-PAMAM (G4), and E) Au/MWCNT-PAMAM (G5) base electrodes. Figure 4. A) Optimum applied potential of proposed bio-sensing electrode, B) Effect of pH on Au/MWCNT-PAMAM(G5) based electrode on amperometric response in 10mM PBS with applied potential of 0.45 V, C) Optimum temperature of Au/MWCNT-PAMAM(G5) based enzyme electrode in 10mM PBS, pH 8.0, with applied potential of 0.45 V. Figure 5. A) Amperometric current response of Au/MWCNT-PAMAM based bio-sensing electrodes constructed with each five generations in 10mM PBS pH 8.0 at applied potential of +0.45V, B) Amperometric calibration curve of Au/MWCNT-PAMAM G1 to G5 based electrodes. Figure 6. A) Operational stability and B) Storage stability of Au/MWCNT-PAMAM (G5)/Urease based electrode in 10mM PBS pH 8.0 under applied potential of 0.45V.
Figure 7. Interference study on effect of uric acid (UA), ascorbic acid (AA), lactic acid (LA), glucose (G), and cholesterol (C) on Au/MWCNT-PAMAM (G5)/Urease based electrode.
Table 1. Comparison of analytical performance of fabricated electrodes Electrode
MWCNT-PAMAM (G1) MWCNT-PAMAM (G2) MWCNT-PAMAM (G3) MWCNT-PAMAM (G4) MWCNT-PAMAM (G5)
1-18mM 1-18mM 1-17mM 1-15mM 1-20mM
DL 0.87mM 0.80mM 0.78mM 0.74mM 0.40mM
LR, Linear Range; DL, Detection Limit; RT, Response Time
Sensitivity 3.61nA/mM 3.948nA/mM 4.34nA/mM 5.94nA/mM 6.60nA/mM
RT(s) 3 4 3 3 3
R2 0.9996 0.9988 0.9957 0.9991 0.9986
Table 2. Comparison of analytical performance of Au/MWCNT-PAMAM (G5)/Urease based bio-sensing electrode with studies reported in literature. Electrode
Rhodinized polymer membrane
Graphite and platinum composite
Polyaniline-Nafion/Au/ceramic composite -
ZnO thin film
LR, Linear Range ; DL, Detection Limit; NR, Not Reported
Table 3. Analytical performance of proposed bio-sensing electrode (n=3) Urea added (mM)
Urea found (mM)
Table 4. Real sample application of proposed bio-sensing electrode in human blood (n=3) Blood Sample
Urea added (mM)
Urea found (mM)