Carboxylated graphene-alcohol oxidase thin films modified graphite electrode as an electrochemical sensor for electro-catalytic detection of ethanol

Journal Pre-proofs Carboxylated Graphene-Alcohol Oxidase Thin Films Modified Graphite Electrode as an Electrochemical Sensor for Electro-catalytic Detection of Ethanol S. Prasanna kumar, L. Parashuram, D.P. Suhas, Prakash krishnaiah PII: DOI: Reference:

S2589-2991(19)30116-8 https://doi.org/10.1016/j.mset.2019.10.009 MSET 129

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Materials Science for Energy Technologies

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14 July 2019 1 October 2019 2 October 2019

Please cite this article as: S. Prasanna kumar, L. Parashuram, D.P. Suhas, P. krishnaiah, Carboxylated GrapheneAlcohol Oxidase Thin Films Modified Graphite Electrode as an Electrochemical Sensor for Electro-catalytic Detection of Ethanol, Materials Science for Energy Technologies (2019), doi: https://doi.org/10.1016/j.mset. 2019.10.009

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Carboxylated Graphene-Alcohol Oxidase Thin Films Modified Graphite Electrode as an Electrochemical Sensor for Electro-catalytic Detection of Ethanol S. Prasanna kumara*, L. Parashuramb*, D. P. Suhasc, Prakash krishnaiahb aIndian

Academy Degree College Autonomous, Hennur Cross, Hennur Main Road, Kalyan

nagar, Bangalore-560043, India. bDepartment

of Chemistry, New Horizon College of Engineeering, Bangalore-560103, India.

cDepartment

of Chemistry, St.Joesph’s College, Langford gardens, Bangalore-560027, India.

__________________________ * Corresponding author Telephone: +91-80-6629 7777 Fax no: +91-80-2844 0770 E-mail address: [email protected] (Prasanna Kumar S.)

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Abstract A new enzyme-electrode is developed for the determination of ethanol by immobilizing carboxylated graphene (CG) and alcohol oxidase (AOX) on poly diallyldimethylammonium chloride (PDDA) modified graphite electrode (Gr). The biosensor showed high sensitivity and rapid detection of ethanol in the concentration range 250 μM to 1500 μM. Addition of ethanol reduced the reduction current of oxygen, which is due to the consumption of molecular oxygen for the oxidation of ethanol. The effect of temperature and pH on the performance of the biosensor has been studied. The excellent performance of the biosensor is attributed to large surface to volume ratio and high conductivity of graphene. This promotes direct electron transfer between redox enzymes and electrode surface. Low operating potential and specificity of the enzyme will restrict the interference of ascorbic acid, acetaminophen, and glucose. The biosensor was characterized by field emission scanning electron microscopy (FESEM) and cyclic voltammetry (CV) using Fe2+/Fe3+ as an electrochemical probe.

Keywords: Alcohol Oxidase (AOX), Ethanol sensor, Carboxylated Graphene (CG), Poly diallyldimethylammonium chloride (PDDA)

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1. INTRODUCTION Nanoscale materials in combination with the enzymes present unique properties as electrocatalysts, catalysts and sensors for various needs of society and industry. Most of the properties rely on high specificity of these enzymes and also the quantum confinement effects in case of nanoscale materials. Also, these enzymes anchored to the carbonaceous materials exhibit high mechanical and electrochemical stability. In particular the noble metals supported on carbon support were found to be excellent materials for the non-enzymatic detection of ethanol. However, low oxidation kinetics of ethanol on platinum supported carbon catalyst was still a challenge; hence it pushes the limit of exploring new materials and modifications for the development of ethanol sensors. Many researchers proposed palladium supported on carbon as model electrocatalyst for the electrochemical oxidation of ethanol. Several experimental strategies were used to improve the oxidation kinetics of ethanol, this includes using combinational catalysts. Recently, Rosana A. Gonçalves et al. reported palladium supported on antimonic oxide as a promising electrocatalyst for the amperometric detection of ethanol [1]. Vijay K. Tomer et al. reported silver loaded cubic mesoporous graphitic carbon nitride for room temperature detection of ethanol, the excellent response towards ethanol was attributed to high surface area of carbon nitride and planar morphology [2], few more metal modified electrodes and metal composite electrodes are proposed for the determination of ethanol without using enzymes [3–6].. However, enzyme based sensors show high precision and selectivity towards a particular substrate and prove superior over non-enzymatic electrodes, also enzymes are essential components of living system catalyzing most of the chemical transformations during cell metabolism [7]., Nataliya Stasyuk et al. a combinational

platinum-ruthenium

modified

with

alcohol

oxidase

as

an

robust

electrochemical probe for the quantification of primary alcohols in real food samples [8]. Detection of ethanol is of paramount importance in the field of beverages and food, health

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and fuel quality parameters, [9–13] as per the Occupational safetly and health administration (OSHA), 1000 ppm is the permissible limit of ethanol. Ethanol sensors are broadly divided into two i) enzyme ii) non-enzymatic nanomaterials based sensors. Many researchers reported non-enzymatic sensors for the detection of ethanol and other biomolecules [14–23]. Due to practical issues like temperature sensitivity, pH sensitivity and chemical stability in case of enzyme based sensors, search for hybrid electrodes fabricated using enzyme in combination with nanomaterials have attracted considerable interest owing to their higher thermal stability, chemical resistance and improved electrochemical properties . Since, highly active ethanol detection probes find direct application as an anodic catalyst in ethanol fuel cells and also for electrochemical quantification of ethanol. Various electrodes modified with AOX are reported [24–30]. Recently, CNT/poly (brilliant green) and CNT/poly (3,4-ethylenedioxythiophene) based electrochemical enzyme biosensors were used for glucose sensing [31], also a non-enzymatic electrochemical sensor based on copper (I) stabilized zirconia for glucose sensing was developed by our group [32]. Functionalized graphene modified graphite electrode and ionic liquid-functionalized graphene electrodes were used for the electrochemical determination of NADH and ethanol [33,34]. Thus, there are constant efforts by the electrochemists for fabricating ethanol sensors with improved electrocatalytic properties, we developed carboxylated graphene-alcohol oxidase ultra thin films modified graphite electrode via layer-by-layer technique (LBL). This technique is mainly based on adsorption of oppositely charged materials in alternate layers onto solid surface [35]. It has been proved that layer-by-layer method is a simple and elegant method for the preparation of ultrathin films with defined composition and uniform thickness. Finely controlled multilayer films were constructed by LBL method using enzymes and polyelectrolyte and excellent retention of its activity in multilayer assembly was reported [36].

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Direct electron transfer (DET) from an enzyme is a significant research for developing reagentless amperometric biosensor [37]. Various studies have been performed on DET of glucose oxidase [38] and cholesterol oxidase [39–41]. In the current research we have shown the DET of AOX using CG through LBL technique. In this work positively charged AOX and negatively charged graphene are immobilized on graphite electrode using LBL technique. DET of AOX was observed after graphite electrode was modified with CG and enzyme. The modified electrode showed electrocatalytic activity towards reduction of oxygen, the reduction current of oxygen decreases linearly with increase of ethanol concentration, this shows its possible application in mediator-free (reagentless) ethanol biosensor. 2. EXPERIMENTAL DETAILS 2.1. Reagents and solutions Alcohol oxidase from Hansenula polymorpha (E.C. 1.1.3.13) and PDDA (Mw: 200,000350,000), were purchased from sigma aldrich. Ethanol is received from changshu yangyuan chemical, China. Phosphate buffer solution was prepared using stock solutions of 0.1 M K2HPO4 and 0.1 M KH2PO4, acetate buffer was prepared using 0.2 M acetic acid and sodium acetate then pH was adjusted using 0.1 M HCl and 0.1 M NaOH. All other chemicals used were of analytical grade are used without further purification and all solutions were prepared using milli-Q water. Graphene sheets were prepared by solvothermal reduction of colloidal suspension of graphite oxide and carboxylated according to the procedure described in our earlier work [42]. 2.2. Construction of ethanol biosensor Alcohol oxidase (E.C. 1.1.3.13 sigma A0438) solution was prepared in 0.1 M acetate buffer of pH 5.0. An electrode was fabricated by inserting 6 mm diameter cylindrical graphite into a Teflon tube with same internal diameter and electrical contact was made with a copper wire passing through the center of Teflon tube. The electrode was polished with emery papers

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of different of grades i.e. 1000, 800, 6/0, 4/0 and finally with 2/0 until a mirror shining surface was obtained. It was then ultrasonicated for several minutes to remove adsorbed carbon, rinsed with pure distilled water and dried in nitrogen atmosphere. The electrode was immersed in PDDA (1% w/v) with 0.5 M NaCl solution for an hour to provide sufficient time for the deposition of PDDA on graphite surface. The surface is then rinsed with distilled water to remove excess, loosely bound polymer material and further dried to get adherent layer. The positively charged graphite electrode is then immersed in aqueous solution containing CG (2mg/ml) followed by AOX prepared in 0.1 M acetate buffer solution (pH 5.0) the process is repeated five times to obtain five bilayers as shown in Fig. 1 and the obtained electrode is labeled as Gr/(PDDA-[CG- AOX]5). After each modification step, the sensor was immersed in pure water and dried. Hereafter, the biosensor is designated as Gr/(PDDA-[CG- AOX]5). Multilayer films of CG and AOX were formed on PDDA modified graphite electrode surface due to electrostatic interaction between negatively charged graphene and positively charge AOX. 2.3. Electrochemical measurements Cyclic voltammetry and chronoamperometry experiments were performed using Versa stat 3 (Princeton applied research, USA). All experiments were done in a three electrode electrochemical cell with Gr/(PDDA-[CG- AOX]5 as working electrode, a saturated calomel as reference electrode and a platinum electrode as auxillary electrode. 3. RESULTS AND DISCUSSION 3.1. Field emission scanning electron microscopy and XRD of Gr/(PDDA-[CG- AOX]5. The modified electrode is characterized by FESEM. Fig. 2a shows the SEM micrographs of the surface of bare graphite (A) and modified electrode (B). The bare graphite electrode exhibits high heterogeneity with many cavities and stacked flakes. The surface of the

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electrode shows decrease in cavities and stacked flakes after modification with graphene and alcohol oxidase. Also, the CG and PDDA modification showed carboxylated graphene structure with random agglomeration, crampled sheets closely associated with each other. A random distribution of the PDDA polymer over the CG and graphitic layer was visualized in the SEM image. XRD spectrum of Gr/(PDDA-CG) electrode is shown in Fig. 2b. A broad peak observed in the range of 18.7 ˚ (002), the broad peak indicates amorphous nature and due to the presence of oxygen containing carboxyl functional group in carboxylated graphene. Also, this decreased 2θ value of 18.7 compared to pure graphene and carboxylated graphene indicates increase in d-spacing in case of PDDA-CG modified graphite electrode. 3.2. Ethanol bio-sensing 3.2.1. Cyclic voltammetric characterization of Gr/(PDDA-[CG- AOX]5 Cyclic voltammetric technique will provide the details about the role of electrode material, its surface modification by organic materials, solvent and analyte in a electrochemical system. Fig. 3 shows the cyclic voltammetric behavior of bare graphite and modified electrode. The electrochemical activity of Gr/(PDDA-[CG- AOX]5 was examined using Fe(CN)6

4-/3-

as an electrochemical probe. The cyclic voltammogram of bare graphite

electrode in 1 mM Fe(CN)6

4-/3-

shows a pair of quasi reversible redox peaks with peak to

peak separation (ΔEp) of 142 mV (curve a). The CV of graphite electrode modified with PDDA shows a negative shift in the potential of the redox peak of Fe(CN)6 4-/3-, this is due to the fact that the positive surface of PDDA could attract Fe(CN)6

4-/3-

redox couple, which

results in the reduction of over potential and increase in peak current. The CVs of Gr/PDDACG electrode shows a ΔEp of 98 mV also shows a pronounced improvement in the peak current, these changes are attributed to the electrocatalytic activity of CG. Immobilization of AOX enzyme on Gr/PDDA-CG increased the ΔEp value to 104 mV along with decrease in

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peak current; this is due to the hindrance offered by the enzyme for electron transfer towards electrode surface. 3.2.2. Effect of pH on the enzyme activity In the enzymatic reactions, pH plays an important role for the transfer of protons from one chemical species to another. Hence the solution pH has a considerable effect on the performance of the prepared ethanol biosensor. Owing to the fact that AOX sufferes denaturation in more acidic medium (
AOX (FADH2)

(3)

The redox potential E0 was obtained by taking the average of E pa and E pc and found to be –0.480 V, this value is close to the redox potential of -0.505 V (vs. SCE) for 8

FAD/FADH2 redox couple at pH 7.0 [43]. The data indicates that, AOX retained electrochemical activity even after immobilizing on CG surface, thus DET of AOX in CG film was successfully achieved. Uniform increase in peak current indicates that the amount of AOX adsorbed in each layer on the electrode surface by layer-by-layer method is almost same and it is shown in Fig. 3b. Thus Gr/(PDDA-[CG-AOX]5) electrode was used for further studies and for ethanol detection. The surface coverage (Γ) was calculated for one layer of AOX according to equation Γ= Q/nFA [44], and was found to be 6×10-9 mol cm-2, where Q is charge, n is the no of electrons, F is faraday constant and A is the geometric area of working electrode. Surface coverage increases with increase in number of bilayers, reached maximum value of 1.0×10-8 mol cm-2 for Gr/(PDDA-[CG-AOX]5) electrode and attained saturation at fifth bilayer which as shown in the inset of Fig. 5(b). This indicates that CG can offer large surface area for the enzyme to get accommodated on its surface. Also, the present Gr/PDDA-[CG-AOX]5 sensor showed good retention of its activity even after four cycles of studies, the marginal lose in the activity will show high retention of electrocatalytic activity of the present sensor Fig. 5(c) 3.2.4. Effect of scan rate at Gr/(PDDA-[CG- AOX]5) electrode The influence of scan rate on the voltammetric response of Gr/PDDA-[CG-AOX]5 in 0.1 M phosphate buffer solution at pH 7.0 was studied in the range of 0.025 -0.150 mV/s and this provides the information about kinetics of the electrode reactions. Fig. 6 shows that the increase in scan rate increases the anodic and cathodic peak currents, but the anodic peak potentials are slightly shifted to positive potential and the cathodic peak potentials are shifted to the negative potential. Integration of area under reduction peaks gave nearly constant charge (Q) values independent of scan rate, these characteristics suggest that the redox reaction of AOX in CG film is a quasi-reversible surface controlled electrochemical process. The linear regression equations are.

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ipa(A) = -1.8846×10-5 + 1.81634×10-4 ν (Vsˉ1), (R=0.9984) ipc(A)= -7.986 ×10-5 –2.877 ×10-6 ν (Vsˉ1), (R = 0.9956) 3.2.5. Gr/(PDDA-[CG- AOX]5) electrode for ethanol determination Compared with N2 saturated buffer (curve a), a pair of well defined AOX redox peak were also observed in air saturated buffer (curve c) in absence of ethanol with an increased reduction peak current and decreased oxidation peak current. After addition of ethanol (3 mM) the reduction peak current decreased significantly (curve b), the reason is that AOX utilizes dissolved oxygen for the oxidation of ethanol as evident from Fig. 7. In the presence of oxygen the reduced enzyme AOX (FADH2) is quickly oxidized to AOX (FAD) and the electro-catalytic regeneration of AOX (FADH2) enzyme as per the scheme 3 causes the loss of reversibility and also enhances cathodic peak current. Due to the presence of ethanol in contact with the substrate AOX may result an enzyme–catalyzed reaction which will decrease the concentration of the oxidized form of AOX on electrode surface and leads to the decrease of reduction peak current. These results provide evidence that the AOX encapsulated on the electrode retains its biocatalytic activity towards ethanol oxidation. 3.2.6. Chronoamperometric biosensing of ethanol As shown in Fig. 8(a), addition of ethanol aliquots to static air saturated phosphate buffer solution (pH 7.0), the chronoamperometric curves showed decreasing response, the current quickly reached the steady value and the time taken to reach 95% of the steady value was less than 10 s. The chronoamperometric response decreased linearly with ethanol concentration ranging from 250 μM to 1500 μM. The Gr/(PDDA-[CG-AOX]5) electrode displayed a correlation coefficient of 0.9900 and a slope of 31.9 nA/mM. Due to the high selectivity and low detection potential (-0.4 V) of Gr/(PDDA-[CG-AOX]5) towards oxidase substrate, the common interferants like ascorbic acid, acetaminophen and glucose did not

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interfere as shown in Fig. 8(b). A table comparing the performance of the proposed biosensor with that reported in the literature was shown in Table 1. The data clearly indicates that, the current biosensor is a good competitor among the sensors listed with respect to linear range and detection limit. The apparent Michelis-Menten (Kmapp) constant corresponding to the value of b from the equation y = ax/(b+x) was found to be 151 µM. Quantification of ethanol real alcoholic beverages was carried out and the data is presented in the Table 2. 3.2.7. Effect of temperature on the enzyme activity To perform the temperature effect experiment, the electrochemical cell containing buffer solution was placed in a thermostat at a constant pH 7.0., and then the effect of temperature on the current response of biosensor in presence of ethanol has been recorded. The electrochemical response increased with increase in temperature from 30 ºC to 50 ºC, which may be due to the faster enzyme reaction and analyte diffusion. Further increase in temperature decreased the response, which may be due to denaturation of enzymes due to the heat and is known to be a continuous process. The rate of denaturation increases with increase in temperature beyond the optimum temperature. From the experiment it is found that the modified electrode showed maximum current at 48 ºC as shown in Fig. 9. From the literature also it is evident that AOX shows optimum performance at 50 ºC and a pH of 7.5 [45]. Based on the experimental investigation carried out for the detection of ethanol, a plausible mechanism for electrochemical detection of ethanol at Gr/(PDDA-[CG-AOX]5) electrode has been given in Figure 10. 4. CONCLUSION Unique multicomponent ultrathin films of AOX and CG were constructed with on PDDA modified graphite electrode, the oppositely charged assembly of layers resulted in a stable and robust electrode. The biosensor fabrication process was very simple, less costly and eco friendly. From the SEM images deposition of these layers on the graphite layer has

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been shown. The effect of deposited layers and immobilization of AOX on electrochemical performance has been clearly demonstrated, it is noted biosensor with five bi-layer construction showed maximum electrochemical activity for the detection of ethanol. The biosensor shows rapid and highly sensitive amperometric response to ethanol at a lower reduction potential under non-stirring conditions with a good linear range of 250 μM to 1500 μM. Also, the biosensor was showed highly stable and selective electrochemical response for ethanol in real alcoholic beverages with percentage recoveries ranging from 96 % - 101.4 %, this indicates practical application of the present biosensor for direct detection of ethanol. In conclusion in this work we have successfully fabricated and demonstrated an interferents free, mediator less (reagent less) ethanol biosensor. 5. ACKNOWLEDGEMENTS We thank Sri. A.V.S. Murthy, Honorary Secretary, Rashtreeya Sikshana Samiti Trust, Bangalore, for encouragement and providing the facility for the successful completion of this work. References [1]

R.A. Gonçalves, M.R. Baldan, E.G. Ciapina, O.M. Berengue, Nanostructured Pd/Sb2O3: A new and promising fuel cell electrocatalyst and non-enzymatic amperometric sensor for ethanol, Appl. Surf. Sci. 491 (2019) 9–15. doi:10.1016/j.apsusc.2019.06.116.

[2]

V.K. Tomer, R. Malik, K. Kailasam, Near-Room-Temperature Ethanol Detection Using Ag-Loaded Mesoporous Carbon Nitrides, ACS Omega. 2 (2017) 3658–3668. doi:10.1021/acsomega.7b00479.

[3]

N. Lu, C. Shao, X. Li, T. Shen, M. Zhang, F. Miao, X. Zhang, P. Zhang, K. Wang, Y. Zhang, Y. Liu, CuO/Cu2O nanofibers as electrode materials for non-enzymatic glucose sensors with improved sensitivity, RSC Adv. 4 (2014) 31056–31061.

12

doi:10.1039/c4ra03258f. [4]

C.T. Carvalho-filho, E.L. Franz, J. Arboleda, A. Echavarrı, A. Bueno-l, K.I.B. Eguiluz, G.R. Salazar-banda, Platinum tin/carbon catalysts for ethanol oxidation : Influence of Sn content on the electroactivity and structural characteristics, Int J Hydrog. Energy. 40 (2015) 12674–12686. doi:10.1016/j.ijhydene.2015.07.135.

[5]

J. Thepkaew, S. Therdthianwong, A. Kucernak, A. Therdthianwong, Electrocatalytic activity of mesoporous binary/ternary PtSn-based catalysts for ethanol oxidation, J. Electroanal. Chem. 685 (2012) 41–46. doi:10.1016/j.jelechem.2012.09.006.

[6]

M.M. Pereira, S. Neves, M. Bego, P. Bobes, D. Hern, A non-enzymatic ethanol sensor based on a nanostructured catalytic disposable electrode, Anal. Methods,. 9 (2017) 5108–5114. doi:10.1039/C7AY01078H.

[7]

A.M. Azevedo, D.M.F. Prazeres, J.M.S. Cabral, P. Fonseca, Ethanol biosensors based on alcohol oxidase, Biosens. Bioelectron. 21 (2005) 235–247. doi:10.1016/j.bios.2004.09.030.

[8]

N. Stasyuk, G. Gayda, A. Zakalskiy, O. Zakalska, R. Serkiz, M. Gonchar, Amperometric Biosensors Based on Oxidases and PtRu Nanoparticles as Artificial Peroxidase, Food Chem. 285 (2019) 213–220. doi:10.1016/j.foodchem.2019.01.117.

[9]

N.L.T. Nguyen, S.H. Baek, Z.A. Akbar, S.Y. Jang, S. Ha, J.P. Park, T.J. Park, Rapid Determination of Ethyl Alcohol in Alcoholic Beverages Using a Fluorescent Nanofiber Film, BioChip J. 12 (2018) 240–248. doi:10.1007/s13206-017-2305-6.

[10]

T. Arakawa, T. Suzuki, M. Tsujii, K. Iitani, P.J. Chien, M. Ye, K. Toma, Y. Iwasaki, K. Mitsubayashi, Real-time monitoring of skin ethanol gas by a high-sensitivity gas phase biosensor (bio-sniffer) for the non-invasive evaluation of volatile blood compounds, Biosens. Bioelectron. 129 (2019) 245–253. doi:10.1016/j.bios.2018.09.070.

13

[11]

D. Farina, M. Zinellu, M. Fanari, M.C. Porcu, S. Scognamillo, G.M.G. Puggioni, G. Rocchitta, P.A. Serra, L. Pretti, Development of a biosensor telemetry system for monitoring fermentation in craft breweries, Food Chem. 218 (2017) 479–486. doi:10.1016/j.foodchem.2016.09.092.

[12]

A. Rydosz, E. Maciak, K. Wincza, S. Gruszczynski, Microwave-based sensors with phthalocyanine films for acetone, ethanol and methanol detection, Sens. Actuator. B. Chem. 237 (2016) 876–886. doi:10.1016/j.snb.2016.06.168.

[13]

S. Cinti, M. Basso, D. Moscone, F. Arduini, A paper-based nanomodified electrochemical biosensor for ethanol detection in beers, Anal. Chim. Acta. 960 (2017) 123–130. doi:10.1016/j.aca.2017.01.010.

[14]

S. Park, S. Kim, G. Sun, C. Lee, Synthesis, structure and ethanol gas sensing properties of In2O3 nanorods decorated with Bi2O3 nanoparticles Synthesis , structure and ethanol gas sensing properties of In2O3 nanorods decorated with Bi2O3 nanoparticles, ACS Appl. Mater. Interfaces. 7 (2015) 8138–8146.

[15]

V.B. Kamble, A.M. Umarji, Gas sensing response analysis of p-type porous chromium oxide thin films, J. Mat. Chem. C. 1 (2013) 8167–8176. doi:10.1039/c3tc31830c.

[16]

X. Li, Y.D. Jiang, G.Z. Xie, X.S. Du, H.L. Tai, J.F. Yan, S.Q. Fu, Ethanol gas sensors based on copper phthalocyanine thin-film transistors, 2010 Int. Conf. Apperceiving Comput. Intell. Anal. ICACIA 2010 - Proceeding. (2010) 470–473. doi:10.1109/ICACIA.2010.5709943.

[17]

A. Benali, S. Azizi, M. Bejar, E. Dhahri, M.F.P. Graça, Structural, electrical and ethanol sensing properties of double-doping LaFeO3 perovskite oxides, Ceram. Int. 40 (2014) 14367–14373. doi:10.1016/j.ceramint.2014.06.029.

[18]

V. Kamble, A. Umarji, Analyzing the kinetic response of tin oxide-carbon and tin oxide-CNT composites gas sensors for alcohols detection, AIP Adv. 5 (2015).

14

doi:10.1063/1.4916755. [19]

N.P. Shetti, S.D. Bukkitgar, K.R. Reddy, C.V. Reddy, T.M. Aminabhavi, ZnO-based nanostructured electrodes for electrochemical sensors and biosensors in biomedical applications, Biosens. Bioelectron. 141 (2019) 111417. doi:10.1016/j.bios.2019.111417.

[20]

N.P. Shetti, S.D. Bukkitgar, K.R. Reddy, C.V. Reddy, T.M. Aminabhavi, Nanostructured titanium oxide hybrids-based electrochemical biosensors for healthcare applications, Colloids Surfaces B Biointerfaces. 178 (2019) 385–394. doi:10.1016/j.colsurfb.2019.03.013.

[21]

N.P. Shetti, D.S. Nayak, S.J. Malode, R.R. Kakarla, S.S. Shukla, T.M. Aminabhavi, Sensors based on ruthenium-doped TiO 2 nanoparticles loaded into multi-walled carbon nanotubes for the detection of flufenamic acid and mefenamic acid, Anal. Chim. Acta. 1051 (2019) 58–72. doi:10.1016/j.aca.2018.11.041.

[22]

S. Kumar, S.D. Bukkitgar, S. Singh, Pratibha, V. Singh, K.R. Reddy, N.P. Shetti, C. Venkata Reddy, V. Sadhu, S. Naveen, Electrochemical Sensors and Biosensors Based on Graphene Functionalized with Metal Oxide Nanostructures for Healthcare Applications, ChemistrySelect. 4 (2019) 5322–5337. doi:10.1002/slct.201803871.

[23]

S.D. Bukkitgar, N.P. Shetti, R.M. Kulkarni, K.R. Reddy, S.S. Shukla, V.S. Saji, T.M. Aminabhavi, Electro-catalytic behavior of Mg-doped ZnO nano-flakes for oxidation of anti-inflammatory drug, J. Electrochem. Soc. 166 (2019) B3072–B3078. doi:10.1149/2.0131909jes.

[24]

H.B. Yildiz, L. Toppare, Biosensing approach for alcohol determination using immobilized alcohol oxidase, Biosens. Bioelectron. 21 (2006) 2306–2310. doi:10.1016/j.bios.2005.11.006.

[25]

M.M. Barsan, C.M.A. Brett, An alcohol oxidase biosensor using PNR redox mediator

15

at carbon film electrodes, Talanta. 74 (2008) 1505–1510. doi:10.1016/j.talanta.2007.09.027. [26]

L. Wu, J. Lei, X. Zhang, H. Ju, Biosensors and Bioelectronics Biofunctional nanocomposite of carbon nanofiber with water-soluble porphyrin for highly sensitive ethanol biosensing, Biosens. Bioelectron. 24 (2008) 644–649. doi:10.1016/j.bios.2008.06.009.

[27]

C. Gouveia-caridade, R. Pauliukaite, C.M.A. Brett, Development of electrochemical oxidase biosensors based on carbon nanotube-modified carbon film electrodes for glucose and ethanol, Electrochim. Acta. 53 (2008) 6732–6739. doi:10.1016/j.electacta.2008.01.040.

[28]

M. Akin, M. Yuksel, C. Geyik, D. Odaci, A. Bluma, T. Ho, S. Beutel, T. Scheper, Alcohol Biosensing by Polyamidoamine ( PAMAM )/ Cysteamine / Alcohol OxidaseModified Gold Electrode, Biotechnol. Prog. 26 (2010) 896–906. doi:10.1002/btpr.372.

[29]

L. Wu, M. McIntosh, X. Zhang, H. Ju, Amperometric sensor for ethanol based on onestep electropolymerization of thionine-carbon nanofiber nanocomposite containing alcohol oxidase, Talanta. 74 (2007) 387–392. doi:10.1016/j.talanta.2007.09.023.

[30]

M. Hnaien, F. Lagarde, A rapid and sensitive alcohol oxidase / catalase conductometric biosensor for alcohol determination, Talanta. 81 (2010) 222–227. doi:10.1016/j.talanta.2009.11.061.

[31]

M.M. Barsan, V. Pifferi, L. Falciola, C.M.A. Brett, New CNT/poly (brilliant green ) and CNT/poly (3, 4- ethylenedioxythiophene) based electrochemical enzyme biosensors, Anal. Chim. Acta. 927 (2016) 35–45. doi:10.1016/j.aca.2016.04.049.

[32]

L. Parashuram, S. Sreenivasa, S. Akshatha, V. Udayakumar, S. Sandeep kumar, A non-enzymatic electrochemical sensor based on ZrO2: Cu(I) nanosphere modified carbon paste electrode for electro-catalytic oxidative detection of glucose in raw Citrus

16

aurantium var. sinensis, Food Chem. 300 (2019) 125178. doi:10.1016/j.foodchem.2019.125178. [33]

S.P. Kumar, R. Manjunatha, C. Nethravathi, S. Suresh, Electrocatalytic Oxidation of NADH on Functionalized Graphene Modified Graphite Electrode, Electroanal. 23 (2011) 842–849. doi:10.1002/elan.201000552.

[34]

C. Shan, H. Yang, D. Han, Q. Zhang, A. Ivaska, L. Niu, Biosensors and Bioelectronics Electrochemical determination of NADH and ethanol based on ionic liquidfunctionalized graphene, Biosens. Bioelectron. 25 (2010) 1504–1508. doi:10.1016/j.bios.2009.11.009.

[35]

G. Decher, J. Hong, Anonymous, Build-up of ultrathin multilayer films by a selfassembly process, J. Makromol Chem Macromol Symp. 46 (1991) 321. doi:10.1016/0040-6090(92)90417-A.

[36]

H. Zheng, H. Okada, S. Nojima, S. Suye, T. Hori, H. Zheng, H. Okada, S. Nojima, S. Suye, T. Hori, Layer-by-layer assembly of enzymes and polymerized mediator on electrode surface by electrostatic adsorption Layer-by-layer assembly of enzymes and polymerized mediator on electrode surface by electrostatic adsorption, Sci. Technol. Adv. Mater. 5 (2004) 371–376. doi:10.1016/j.stam.2004.01.007.

[37]

J. Haccoun, B. Piro, L.D. Tran, L.A. Dang, M.C. Pham, Reagentless amperometric detection of L-lactate on an enzyme-modified conducting copolymer poly(5-hydroxy1,4-naphthoquinone-co-5-hydroxy-3- thioacetic acid-1,4-naphthoquinone), Biosens. Bioelectron. 19 (2004) 1325–1329. doi:10.1016/j.bios.2003.11.006.

[38]

G.P. Keeley, M.E.G. Lyons, The Effects of Thin Layer Diffusion at Glassy Carbon Electrodes Modified with Porous Films of Single-Walled Carbon Nanotubes, Int. J. Electrochem. Sci. 4 (2009) 794–809.

[39]

H.A. Hill, Bio-electrochemistry, Pure Appl. Chem.,. 59 (1987) 743–748.

17

[40]

A.A. Abdelwahab, M. Won, Y. Shim, Direct Electrochemistry of Cholesterol Oxidase Immobilized on a Conducting Polymer: Application for a Cholesterol Biosensor, Electroanal. 22 (2010) 21–25. doi:10.1002/elan.200900363.

[41]

R. Manjunatha, D. Hanumantharayudu, G. Shivappa, J. Savio, S.F.D. Souza, T. Venkatarangaiah, Direct electrochemistry of cholesterol oxidase on MWCNTs, J. Electroanal. Chem. 651 (2011) 24–29. doi:10.1016/j.jelechem.2010.11.009.

[42]

S. Prasannakumar, R. Manjunatha, C. Nethravathi, G.S. Suresh, M. Rajamathi, T.V. Venkatesha, Graphene-carbon nanotubes modified graphite electrode for the determination of nicotinamide adenine dinucleotide and fabrication of alcohol biosensor, J. Solid State Electrochem. 16 (2012) 3189–3199. doi:10.1007/s10008-0121754-z.

[43]

W. Zhang, Y. Huang, H. Dai, X. Wang, C. Fan, G. Li, Tuning the redox and enzymatic activity of glucose oxidase in layered organic W lms and its application in glucose biosensors, Anal. Biochem. 329 (2004) 85–90. doi:10.1016/j.ab.2004.01.032.

[44]

A.H. Suroviec, Determining Surface Coverage of Self-Assembled Monolayers on Gold Electrodes Determining Surface Coverage of Self-Assembled Monolayers on Gold Electrodes, Chem. Educ. 17 (2014) 83–85.

[45]

S. Suye, Purification and Properties of Alcohol Oxidase from Candida methanosorbosa M-2003, Curr. Microbiol. 34 (2003) 374–377.

[46]

G. Wen, Y. Zhang, S. Shuang, C. Dong, M.M.F. Choi, Application of a biosensor for monitoring of ethanol, Biosens. Bioelectron. 23 (2007) 121–129. doi:10.1016/j.bios.2007.03.024.

[47]

M. Das, P. Goswami, Direct electrochemistry of alcohol oxidase using multiwalled carbon nanotube as electroactive matrix for biosensor application, Bioelectrochemistry. 89 (2013) 19–25. doi:10.1016/j.bioelechem.2012.08.007.

18

[48]

S. Soylemez, E. Kanik, D. Uzun, O. Hacioglu, Development of an efficient immobilization matrix based on a conducting polymer and functionalized multiwall carbon nanotubes: synthesis and its application to ethanol biosensors, J. Mater. Chem. B. 2 (2014) 511–521. doi:10.1039/c3tb21356k.

[49]

L. V. Shkotova, A.P. Soldatkin, M. V. Gonchar, W. Schuhmann, S. V. Dzyadevych, Amperometric biosensor for ethanol detection based on alcohol oxidase immobilised within electrochemically deposited Resydrol film, Mater. Sci. Eng. C. 26 (2006) 411– 414. doi:10.1016/j.msec.2005.10.031.

[50]

X. Jiang, L. Zhu, D. Yang, X. Mao, Y. Wu, Amperometric Ethanol Biosensor Based on Integration of Alcohol Dehydrogenase with Meldola s Blue/Ordered Mesoporous Carbon Electrode, Electroanal. 21 (2009) 1617–1623. doi:10.1002/elan.200804586.

19

Table 1: Comparison of ethanol biosensors reported in the literature with the present work

Chitosan/AOx-eggshell membrane

Method of modification Casting

MWCNTNafion/AOx/Au electrode

Casting

f-MWCNT/ poly(BIPN)/AOx

Electro deposition and casting

Resydrol immobilized on alcohol oxidase

Electrochemci al method

Alcohol Dehydrogenase with Meldola s Blue/Ordered Mesoporous Carbon Electrode

Cross-linking method

Gr/(PDDA-[CG- AOx]5) electrode

Layer by Layer technique

Medium

Phosphate buffer solution

Modified electrode

(NP: Not presented)

20

Linear range

Ref.

60–800 µM

Detection limit µM 30

8.0–42 µM

5

[47]

855 -1197 µM

0.17

[48]

NP

3500

[49]

19-6000 µM

0.64

[50]

250 µM – 1500 µM

50

This work

[46]

Table 2: Detection of alcohol in real sample using Gr/(PDDA-[CG- AOX]5) sensor. Real sample

Percentage of ethanol Detected using Gr/(PDDA-[CG- Percentage (mentioned) (% v/v) AOX]5) sensor (% v/v) recovery (%)

White wine

10

9.6±0.1

96

Red wine

14

14.2±0.2

101.4

Whisky

40

40.3±0.1

100.75

vodka

40

39.9±0.1

99.75

21

5 bi-layers

Modified

AOX CG PDDA

electrode

Bare graphite surface

Graphite surface

Polished to mirror finish

Electrode fabrication process

Washing

PDDA (1% w/v) in 0.5 M NaCl

Washing

Carboxylated graphene (2mg/ml)

AOX in 0.1 M acetate buffer

Till 5 bi-layers

Fig. 1: Procedure followed for the fabrication of biosensor

22

Fig. 2a. FESEM image of A. Bare graphite electrode B. CG and AOX modified Gr/PDDA biosensor 400

Intensity (a. u.)

C (002) 300

C (100) 200

100

0

20

40

2

60

80

Fig. 2b. XRD spectrum of Gr/(PDDA-CG)

23

0.6 0.5

d

0.4

c

I / mA

0.3 0.2

b

0.1

a

0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.1

0.0

0.1

0.2

0.3

0.4

E / V vs. SCE Fig. 3. Cyclic voltammograms of bare Gr (a), Gr/PDDA (b), Gr/PDDA-CG (c) and Gr/PDDA-[CG-AOX] (d) electrodes in 1 mM Fe(CN)6

4-/3-

phosphate buffer solution

containing 0.1 M KCl (pH 7.0) at a scan rate of 0.1 V s-1.

0.28

I / mA

0.26 0.24 0.22 0.20 0.18 4

5

6

pH

7

8

9

Fig. 4. Effect of pH on the enzyme electrode in presence of 0.1 M phosphate buffer solution containing 0.1 m KCl.

24

0.8 A c b a

I / mA

0.4

-0.4

-0.8 -0.8

-0.6

-0.4

-0.2

0.0

E / V vs. SCE

0.4 0.2

B

0.00036

I / A

0.6

0.00027 0.00018 0.00009 0.00000

1 2 3 4 5 6 Number of bilayers

I / mA

0.0 -0.2 -0.4

a

-0.6 -0.8 -1.0

f -0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

E / V vs. SCE Fig. 5. A. Cyclic voltammograms of bare Gr (a), Gr/PDDA-CG (b) Gr/PDDA-[CG-AOX] (c) electrodes in 0.1 M Phosphate buffer containing 0.1 M KCl (pH 7.0). B. Cyclic voltammograms of [CG-AOX]5 multilayer films assembled on Gr/PDDA electrode in phosphate buffer (pH 7.0). The number of multilayer of [CG-AOX] increasing from (a to f) at a scan rate of 0.1 V s-1.

25

0.20 0.15

Current (mA)

0.10 0.05 0.00 -0.05 -0.10 -0.15 -0.20 1

2

3

4

Cycle number

Fig. 5c: Stability of Gr/PDDA-[CG-AOX]5 biosensor

0.6 20

I / mA

I

0.4

0.2

Pa

0

-20

I

Pb

-40 0.02

0.04

0.06

0.10

0.08

0.12

0.14

-1

I / mA

  mVs

0.0

a

-0.2

-0.4

-0.6

f -0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

E / V vs. SCE Fig. 6. Cyclic voltammograms of

Gr/(PDDA-[CG-AOX]5) electrode in phosphate buffer

containing 0.1 M KCl (pH 7.0) at different scan rates; 0.025, 0.05, 0.075, 0.1, 0.125 and 0.15 V s-1 (a-f). Inset shows the plot of peak current vs. scan rate.

26

0.4 0.2

I / mA

0.0 -0.2

a c b

-0.4 -0.6 -0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

E / V vs. SCE Fig. 7. Cyclic voltammetric measurements at Gr/PDDA-[CG-AOX]5 biosensor in phosphate buffer saturated with nitrogen saturation (a) and air saturation (b) and air saturation containing 3 mM ethanol (c) in 0.1 M phosphate buffer (pH 7.0), at a scan rate of 0.1 V s-1.

27

A

I / mA

0.5 0.4

0 250 500 750 1000 1250 1500

0.00015 0.00014 0.00013

I/A

0.6

0.00012 0.00011 0.00010 0.00009

0

400

800

Ethanol (M)

1200

1600

0.3 0.2 0.1 0

5

10

15

20

25

30

Time / S 0.16

B

0.14

I / mA

0.5 mM Ethanol 1.0 mM Ethanol

0.12

1.0 mM Glucose 20 M AA

0.10

20 M ACT

0.08 0

30

60

90

120

Time / S Fig. 8. Chronoamperograms of Gr/PDDA-[CG-AOX]5 biosensor in air saturated 0.1 M phosphate buffer solution with 0.1 M KCl (pH 7.0) containing 0, 250, 500, 750, 1000, 1250 and 1500 μM ethanol (a-g). Inset shows the calibration curve. 6b. Effect of interfering species at Gr/PDDA-[CG-AOX]5 sensor, applied potential - 0.4 V in a stirred 0.1 M phosphate buffer solution saturated with air containing 0.1 M KCl.

28

0.45

I / mA

0.40

0.35

0.30

0.25

0.20 30

35

40

45

50

o

Temperature ( C) Fig. 9. Effect of temperature on current response of biosensor in 0.1 M phosphate buffer solution containing 0.1 m KCl.

AOX (FAD) + 2e- + 2H+

AOX (FADH2)

Direct electrochemistry

*Ethanol sensing

5 bilayers

Alcohol oxidase Carboxylated graphene PDDA Graphite

CH3CH2OH + O2

AOX

CH3CHO + H2O2

Fig. 10. Plausible mechanism for electrochemical detection of ethanol at Gr/(PDDA-[CGAOX]5) biosensor

Carboxylated graphene-alcohol oxidase thin films modified graphite electrode as an electrochemical sensor for electro-catalytic detection of ethanol S. Prasanna kumara*, L. Parashuramb*, D. P. Suhasc 29

Graphical abstract

Electrochemical workstation

Graphite surface

0.6

A

0.00015 0.00014

0.5 I/A

0.00013 0.00012 0.00011

0.4

0.00010

I / mA

Graphite surface

PDDA PDDA

0.00009

0

0.3

400

800

Ethanol (M)

1200

1600

0.2 0.1 0

5 bi-layers

CG Carboxylated graphene

Alcohol Oxidase AOX CH3CH2OH + O2

AOX

5

10

15

20

25

30

Time / S

CH3CHO + H2O2

Highlights  Fabrication of ethanol sensor by simple Layer-by-Layer adsorption of carboxylated graphene and alcohol oxidase on graphite.

 Direct electrochemistry of alcohol oxidase in multi layers.  Excellent electrocatalytic activity of ethanol biosensor, high sensitivity and stability.

Declaration of interests

30

All the authors of the manuscript titled “Carboxylated Graphene-Alcohol Oxidase Thin Films Modified Graphite Electrode as an Electrochemical Sensor for Electro-catalytic Detection of Ethanol” declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported. Dr. S. Prasanna Kumar

31