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Journal of Electrochemistry Volume 17 Issue 3 Special Issue of Chemo/Biosensing Technology(Editor: Professor ZHANG Zongrang) 2011-08-28 Development of dehydrogenase-based bioanode using poly(phenosafranin)-functionalized SWCNT nanocomposites and its application to ethanol biosensor S. Saleh Farhana Okajima Takeyoshi Mao Lanqun Takeo Ohsaka Recommended Citation S. Saleh Farhana, Okajima Takeyoshi, Mao Lanqun, and Takeo Ohsaka (2011) "Development of dehydrogenase-based bioanode using poly(phenosafranin)-functionalized SWCNT nanocomposites and its application to ethanol biosensor," Journal of Electrochemistry: Vol. 17: Iss. 3, Article 2. Available at: https://jelectrochem.xmu.edu.cn/journal/vol17/iss3/2 This Special Issue Article is brought to you for free and open access by Journal of Electrochemistry. It has been accepted for inclusion in Journal of Electrochemistry by an authorized editor of Journal of Electrochemistry. ! 17 " !3 # Vol. 17 !"# 2011 $ 8 % ELECTROCHEMISTRY No. 3 Aug. 2011 Artical ID:10063471(2011)03026308 Development of DehydrogenaseBased Bioanode Using Poly ( Phenosafranin) Functionalized SWCNT Nanocomposites and its Application to Ethanol Biosensor Farhana S. Saleh1 , Takeyoshi Okajima1 , MAO Lanqun2 , Takeo Ohsaka1 (1. Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 2268502, Japan; 2. Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, the Chinese Academy of Sciences( CAS) , Beijing 100190, China) Abstract: A New type of dehydrogenasebased amperometric ethanol biosensor was constructed u sing alcohol dehydrogenase ( ADH) which was immobilized on the edgeplane pyrolytic graphite ( EP PG) electrode modified with poly( phenosafranin) functionalized singlewalled carbon nanotube ( PPS SWCNT) . The PPSSWCNT modified EPPG electrode was prepared by electropolymerization of phe nosafranin on the EPPG electrode which was previously coated with SWCNT. The performance of the ADH / PPSSWCNT / EPPG electrode was evaluated using cyclic voltammetry and amperometry in the presence of ethanol. The fabricated ethanol biosensor provided a reasonable sensitivity of 2. 0 μA· cm - 2 ·mmol - 1 ·L and a low detection limit (36 μmol·L - 1 ) for the electrocatalytic oxidation of eth anol with a linear concentration dependence upto ~ 1. 0 mmol·L - 1 at a detection potential of 0. 2 V. Key words: phenosafranin; electropolymerization; NADH; SWCNT; electrocatalysis CLC Number: O646; TP212. 3 Document Code: A 1 Introduction The quantitative measurement of alcohol is very important for clinical and forensic purposes in the an alyses of human breath [1] . Ethanol is an important compound in medicine, biotechnology and the food industry, etc. ,and it is often monitored for toxicologi cal and psychological effects [2] . In certain industrial fields, such as fermentation and distillation, the etha ven alcohol poisoning [3] . Some analytical methods have been developed during the years for the determi nation of ethanol. An amperometric enzymebased biosensor is one of the best choices for biochemical a nalysis due to their good selectivity, sensitivity, rapid response, miniature size, and reproducible results [4] . The good selectivity is attributed to the specific catal ysis by the enzyme. nol concentration can reach toxic levels, causing in In most cases, NAD + ( the oxidized form of β nicotinamide adenine dinucleotide ( NADH ) ) de junctiva, irritation of the skin and, at high levels, e reagent for their functioning. Electrode modification flammation of the nasal mucous membrane and con Received: 20100607 pendent enzymebased biosensors require NADH as a Corresponding author, Tel:(8145)9245404, Email:ohsaka@ echem. titech. ac. jp The present work was financially supported by GrantinAid for Scientific Research ( A) ( No. 19206079) from the Ministry of Edu cation, Culture, Sports, Science and Technology ( MEXT) , Japan, Tokyo Institute of Technology Global COE Program for Energy Science, and JapanChina Research Program on Enzymebased Biofuel Cells organized and sponsored by Japan Science and Tech nology ( JST) and Natural Science Foundation of China ( NSFC) . · 264· ! " 2011 $ # with carbon nanotubes ( CNTs ) has been shown to sensors [2528] . tion, thus allowing the preferable construction of de genasebased ethanol biosensor by immobilizing alco sors ( phenosafranin) ( PPS) functionalized SWCNT nano produce an electrocatalysis for NADH electrooxida hydrogenase enzyme based electrochemical biosen [57] . Concerning enzyme biosensors based on The present work aims at developing dehydro hol dehydrogenase ( ADH) on the surface of the poly CNTscomposite electrodes, they have been found to composite. Previously we have reported the electro quence of the unique properties of CNTs with the lytic graphite ( BPPG) modified with PPS film pre possess improved analytical performance as a conse catalytic oxidation of NADH by the basalplane pyro wellknown advantages of composite electrode designs pared by electropolymerization of phenosafranin ( PS) rent [8] . Many recent studies have aimed at the func potential of - 0. 458 V at pH 7. 0 [29] . We have also such as simple renewability and low background cur which is an Nsubstituted phenazine with a halfwave tionalization of CNTs with various molecules using co reported an excellent electrocatalysis of PPSfunction ches [1112] in order to obtain desired properties. The ( EPPG) electrode for the NADH oxidation [28] . Here, valent [910] or noncovalent bond formation approa alized SWCNT modified edgeplane pyrolytic graphite covalent functionalization is based on the formation of by immobilizing ADH onto the PPSSWCNT nanocom cules, while the noncovalent functionalization is PPSSWCNT / EPPG) biosensor was fabricated. The chemical bonds between CNTs and functional mole based on adsorption, wrapping, πstacking, or hy posite film, dehydrogenasebased ethanol ( ADH / obtained results showed that the ADH and PPSSWC drophobic / ππ interactions, etc. Among the many NT nanocompositebased bioanode possesses an effi chemical functionalization can provide an efficient, 2 different strategies to functionalize CNTs, electro clean, and more versatile alternative [13] . Fabrication of CNTs / conducting polymer composites has become of great interest since the CNTs can improve the elec trical and mechanical properties of polymers. It has been demonstrated that the CNTs / conducting polymer composites possess the original properties of the indi cient electrocatalytic activity for ethanol oxidation. 2. 1 Experimental Chemicals and Reagents Singlewalled carbon nanotube ( SWCNT) with a diameter = 1. 2 ~ 1. 5 nm and a length = 2 ~ 5 μm were purchased from Aldrich ( Tokyo, Japan) . Phenosafranin ( PS) was purchased from Acros Or vidual components with a synergistic function [1416] . ganics ( Geel, Belgium) . Oxidized form of βnicotin amide adenine dinuleotide ( NAD + , Oriental Yeast NTs) / poly (3octylthiophene) composites have been cohol dehydrogenase ( ADH) from Baker′s yeast, de For example, singlewalled carbon nanotubes ( SWC shown to have good photovoltaic properties [17] , and Co. Ltd. , Tokyo, Japan) was used as received. Al hydrated ethanol and 25% glutaraldehyde ( GA) so CNTs / polyaniline composites have been found to be a lution in water were purchased from Wako ( Japan) . charge capacity, lowcharge voltage and highdis Sigma ( USA) . TrisHCl buffer solution ( TBS; 0. 05 good material in batteries with a highchargedis charge voltage [18] . So far, several conducting poly mers such as polypyrrole, polyaniline, polyphenothia zine, polythiophene and their derivatives have been used to fabricate CNTs / polymer composites [1923] . However, there are limited studies on the CNTs / poly ( phenazine ) dye composites [24] , although poly ( phenazine) dyes are widely used as redox mediators in the construction of electrochemical sensors and bio Bovine serum albumin ( BSA ) was purchased from mol·L - 1 , pH 8. 2) was used as the supporting elec trolyte for electrochemical experiments. The solutions throughout this work were always prepared using dei onized water from a MilliQ water system ( Millipore, Japan ) . Edgeplane pyrolytic graphite ( EPPG ) ( Bioanalytical Systems Inc. ( BAS) ; 3 mm in diame ter) disk was used as the working electrode. All of the other chemicals were of analytical grade and were !3 # Farhana S. Saleh &:'()*+,-./0123456789:;<=>?@A used without further purification. 2. 2 Apparatus and Electrochemical Measurements Cyclic voltammetric and amperometric measure ments were carried out with an ALS CHI 750Cz elec trochemical analyzer ( Eco Chemie, Ultecht, The Netherlands) using a conventional twocompartment threeelectrode system with the prepared bioanodes as working electrodes, a spiral Pt wire as counter elec trode and a Ag | AgCl | KCl( sat. ) as reference elec · 265· trices. For attaching the enzyme layer, typically 4. 5 μL of ADHBSAGA mixture ( 2 μL of 20 mg / mL ADH solution in 0. 05 mol · L - 1 TBS, pH 8. 2 + 1 -1 μL 1% ( by mass) BSA solution in 0. 05 mol · L TBS, pH 8. 2 + 1. 5 μL 1% ( by mass) GA ( in wa ter) was put on the PPSSWCNT / EPPG electrode and airdried at room temperature for at least 1 h. The e lectrode assembly fabricated ( named as ADH / PPS SWCNT / EPPG electrode) is schematically shown in Scheme 1. trode. The amperometric measurements of the ethanol sensor fabricated were performed in 0. 1 mol · L - 1 PBS, stirred at ca. 300 r / min using a magnetic stir rer. A personal computer was used for data storage and processing. Electrolyte solutions were deaerated by bubbling Ar gas ( 99. 99% ) for at least 30 min prior to electrochemical measurements. All the meas urements were carried out at room temperature (25 ± 1 ℃ ). 2. 3 Electrode Preparation and Enzyme Immobilization The EPPG electrodes were first carefully pol ished with emery paper and then transferred to 0. 1 mol·L - 1 H2 SO4 solution and the potential scan was repeated between - 0. 2 and 1. 4 V at 100 mV·s - 1 Scheme 1 3 3. 1 Scheme of the ADHbased composite electrode for the electrocatalytic oxidation of ethanol Results and Discussion Electrocatalytic Characterization of the ADH / PPSSWCNT / EPPG Elec trode for Ethanol Oxidation The response of the ADH / PPSSWCNT / EPPG until a stable cyclic voltammetric response was at biosensor was electrochemically tested by cyclic volta DMF to form a 1 mg / mL SWCNT suspension solution 10 mmol · L - 1 NAD + in the absence (----) and ified EPPG ( SWCNT / EPPG) electrode was prepared No electrocatalytic response was observed at the tained. 1 mg of the SWCNT was dispersed in 1 mL with the aid of ultrasonic agitation. The SWCNT mod by casting 30 μL of the SWCNT suspension on the EPPG electrode surface and airdrying the casted sus pension solution. A film of PPS was prepared by im mmetry in 0. 05 mol·L - 1 TBS ( pH 8. 2) containing presence ( ———) of 20 mmol·L - 1 ethanol ( Fig. 1) . ADH / PPSSWCNT / EPPG electrode in 0. 05 mol · L - 1 TBS ( pH 8. 2) containing 10 mmol·L - 1 NAD + at a scan rate of a 5 mV·s - 1 in the potential range of mersing the SWCNT / EPPG electrode in 0. 2 mol · interest. Upon addition of 20 mmol·L - 1 of ethanol, 10 min and then by repeating the potential scan be a large anodic peak with a decrease in the reduction L - 1 H2 SO4 solution containing 0. 5 mmol·L - 1 PS for tween - 0. 5 and 1. 3 V at 50 mV · s . The thus -1 fabricated PPSSWCNT modified EPPG electrode is the cyclic voltammetric response was characterized by current. The onset potential of ca. - 0. 2 V vs. Ag | AgCl | KCl( sat. ) shows a reasonable relevance to that hereinafter abbreviated as PPSSWCNT / EPPG elec obtained for NADH oxidation at the PPSSWCNT immobilized by a crosslinking reaction using glutaral NADH oxidation is shown in the inset of Fig. 1. Upon trode. Finally, alcohol dehydrogenase ( ADH ) was dehyde ( GA) in bovine serum albumin ( BSA) ma nanocomposite modified electrode. The result of the addition of NADH, a dramatic enhancement in · 266· ! " 2011 $ # the anodic peak current is observed which comes from the mediated oxidation of NADH to NAD + . These re sults confirmed the efficient electrocatalytic property of the biocomposite film electrode which is associated with the oxidation of ethanol via ADH catalysis. ADH catalyzes, in the presence of NAD + as a coenzyme, the oxidation of ethanol to acetaldehyde and simulta neously, NAD + is reduced to NADH. Thus, the a nodic peak ( solid line) in Fig. 1 corresponds to the electrooxidation of NADH. Fig. 2 Effects of experimental parameters on the electrocat alytic response of the ADH / PPSSWCNT / EPPG e lectrode for the ethanol oxidation in 0. 05 mol·L - 1 TBS ( pH 8. 2) in the presence of 20 mmol·L - 1 ethanol a. SWCNT loading, b. ADH loading, c. NAD + concentration, d. pH in above cases, the anodic peak current ( I p a ) values were estimated at 5 mV ·s - 1 Fig. 1 CVs obtained at the ADH / PPSSWCNT / EPPG e lectrode in 0. 05 mol·L - 1 TBS ( pH 8. 2) contai ning 10 mmol · L - 1 NAD + in the presence ( ———) and absence ( - - -) of 20 mmol · L - 1 ethanol inset shows the voltammetric responses of 5 mmol ·L - 1 NADH at the bare EPPG (- - -) and PPS SWCNT / EPPG ( ———) electrodes in 0. 05 mol· L - 1 TBS ( pH 8. 2) scan rate: 5 mV·s 3. 2 -1 Optimization of Electrocatalysis for Ethanol Oxidation The performance of the ADH / PPSSWCNT / EP PG electrode towards the ethanol oxidation was found to be depended on the loading amounts of SWCNT and ADH, the NAD + concentration and pH ( Fig. 2) . As shown in Fig. 2a, the electrocatalytic current increases with the increasing loading amount of SWC NT and reaches a maximum value at 20 μL of SWC NT . When the loading amount of SWCNT is higher than 2 0 μL , the response remains same which means that this amount is effectively enough to observe a suf ficient response for the electrocatalysis of ethanol. The magnitude of the current response of the am perometric ethanol biosensor depends mainly on the enzyme kinetics which determines the rate at which NADH is generated enzymatically within the ADH / PPSSWCNT biocomposite and the electrochemical re action kinetics which controls the rate at which NADH can be converted to NAD + to generate the measured electric signal within the ADH / PPSSWCNT biocom posite. Fig. 2b shows the effect of the amount of en zyme dropped on the electrode surface on the voltam metric response of the enzyme electrode. With in creasing the loading of the ADHBSAGA mixture, the catalytic peak current increases and the current reaches an almost constant value at the loading of more than 10 μL, indicating the presence of an ade quate amount of enzyme in the PPSSWCNT modified electrode. Thus 10 μL of the ADHBSAGA mixture was used for preparation of the ethanol biosensor. !3 # Farhana S. Saleh &:'()*+,-./0123456789:;<=>?@A · 267· The NAD + concentration in the ethanol solution is another important parameter for ethanol detection. The effect of the NAD + cofactor concentration on the response of the biosensor for 20 mmol·L - 1 ethanol was investigated using the aboveoptimized enzyme loading. The NAD + concentrations in the solution ranged between 3 and 20 mmol · L - 1 , and the ob tained results are displayed in Fig. 2c. The signal in creases with the increase in the concentration of NAD + up to 10 mmol · L - 1 and remains practically constant at the higher concentration. As the enzyme activity is dependent upon the pH value of a buffer solution, the effect of the solution pH of 0. 05 mol·L - 1 TBS containing 10 mmol·L - 1 ue in the range of 6. 0 to 8. 2, and reaches a maxi Current ~ time response of the ADH / PPSSWCNT / EPPG electrode for the successive addition of 250 -1 -1 μmol·L ethanol at 0. 2 V in 0. 05 mol · L -1 + TBS ( pH 8. 2) containing 10 mmol·L NAD C ethanol / μmol·L - 1 : a. 250 , b. 500 , c. 750 , d. 1000, e. 1250, f. 1500, g. 1750,h. 2000, i. 2250 the solution was stirred with a magnetic stirrer at 300 r / min an increase in pH value above 8. 2. Therefore, a so · L - 1 ethanol to the solution . In this case , the elec  NAD on the current response of 20 mmol·L + -1 etha nol at the ADH / PPSSWCNT biocomposite film modi fied EPPG electrode was examined. A plot of the cur rent response against pH is shown in Fig. 2d. The current response increases with increasing the pH val mum value at pH 8. 2. It decreases dramatically with Fig. 3 lution of pH 8. 2 and 10 mmol · L - 1 NAD + in 0. 05 trode potential was kept at 0. 2 V vs. Ag | AgCl | KCl tivity of the ethanol biosensor. current increased steeply to a stable value within 5 s, mol·L 3. 3 -1 TBS were selected for examining the sensi Amperometric Determination of Eth anol Based on the good electrocatalysis of the ADH / PPSSWCNT / EPPG electrode for the ethanol oxidat  ion,its amperometric response as ethanol sensor was examined in a stirred 0. 05 mol·L - 1 TBS ( pH 8. 2) ( at 300 r / min ) . After stabilization of the baseline current, ethanol was injected into the buffer solution. ADH catalyzes the oxidation of ethanol and simultane ously the cofactor NAD + gets reduced to NADH ac cording to the following enzymatic reaction. ADH Ethanol + NAD + →Acetaldehyde + NADH + H + (1) ( sat. ) . Upon addition of an aliquot of ethanol, the demonstrating that the electrocatalytic response is very fast. The plot of the current vs. ethanol concentration gave a good calibration graph as shown in Fig. 4. The linear response range is upto ~ 1. 0 mmol · L - 1 which is found to be wider than the literature value (10 ~ 425 μmol · L - 1 ) [30] . From the slope of the linear portion, the sensitivity was calculated to be 2. 0 -2 -1 μA· cm · mmol · L and the limit of detection ( LOD) was estimated as 36 μmol·L - 1 . The LOD is lower than that obtained by immobilizing ADH on Au nanoparticles (49 μmol·L - 1 ) [31] and is much lower than 0. 1 mmol·L - 1 and 90 μmol·L - 1 reported for the sensors based on injection of the recognition ele ment [32] and the immobilization of ADH on SWCNT According to this reaction, the signal resulting via polyelectrolyte of poly ( dimethyldiallylammonium the concentration of ethanol. Fig. 3 shows the current The operational stability of the ADH / PPSSWC from the NADH oxidation increases with increasing time response to the successive addition of 250 μmol chloride) [33] , respectively. NT / EPPG sensor was also examined by a continuous · 268· ! " 2011 $ # i. e. , the electrode fouling. In addition, a large sur face area of the SWCNT allows a large amount of ADH to be immobilized within the carbon nanotubes assembly, resulting in a CNTsbased enzyme reser voir. 4 Conclusions A new kind of ethanol sensor based on the de hydrogenase enzyme immobilized onto PPSfunction alized SWCNT nanocomposite modified EPPG elec trode was successfully constructed. The fabricated ethanol biosensor provided a reasonable sensitivity of Fig. 4 The plot of the steadystate current against ethanol concentration using the data obtained from Fig. 3 measurement of 1. 0 mmol · L - 1 ethanol at the ap plied potential of 0. 2 V over a period of ca. 45 min. 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