Construction of the functionalized graphene- alcohol oxidase modified graphite electrode Characterization

Transcription

1 Biosensing approach for ethanol determination using alcohol oxidase and functionalized graphene modified graphite electrode by layer-by-layer technique i^m^-^siftv^t-'f^err^iivc-' Construction of the functionalized graphene- alcohol oxidase modified graphite electrode Characterization Direct electrochemistry of alcohol oxidase in multilayer films Applications in Ethanol detection 128

2 I Ethanol Sensor Abstract A new enzyme-electrode for the determination of ethanol is developed by immobilizing negatively charged graphene and positively charged alcohol oxidase (AOX) on poly diallyldimethylammonium chloride (PDDA) modified graphite electrode through electrostatic interaction between graphene and AOX. Effects of temperature and ph on the performance of modified electrode have been studied in detail. The excellent performance of the biosensor is attributed to large surface to volume ratio and high conductivity of graphene. This promotes direct electron transfer I! M between redox enzymes and electrode surface. Ascorbic acid, acetaminophen, and glucose did not interfere due to a very low operating potential and specificity of the enzyme. 129

3 8.1. Introduction In order to control the fermentation process and the product quauty in the beverages, food and other industries hke medicine, many analytical methods have been developed for the determination of ethanol and other aliphatic alcohols. The use of biosensors, have overcome the disadvantages of chemical methods like Colorimetry, refractometry, chromatographic and spectroscopic techniques such as mass spectroscopy, nuclear magnetic resonance and infra red spectroscopy [1, 2]. Enzymes are one of the essential components of living system, catalyzing almost all chemical transformations that occur during cell metabolism [3]. The specificity and nature of their catalytic activity make them excellent tool for chemical analysis. Major advantage is that enzyme catalyzed reaction can be followed by simple, widely available spectroscopic or electrochemical methods. Two enzymes have been extensively used in the determination of alcohols namely alcohol dehydrogenase (ADH) and alcohol oxidase (AOX). Alcohol dehydrogenase based biosensors [4-7] catalyze the reversible oxidation of primary aliphatic alcohol and aromatic alcohols other than methanol according to equation RCHjOH + NAD^ ^^^ ^ R-CHO + NADH + H^ 8.1 Alcohol Oxidase is an oligomeric enzyme consisting of eight identical sub units arranged in quasi-cubic arrangement, each containing a strongly bound co-factor, flavin adenine dinucleotide (FAD) molecule. AOX is responsible for the oxidation of low molecular weight alcohols to the corresponding aldehyde, using molecular oxygen (O2) as electron acceptor according to the equation. AOX RCH2OH + O2 R-CHO + H2O2 8.2 The classical ways to follow an oxidase-caltalysed reaction have been by measuring either the decrease in O2 tension or the increase in H2O2 concentration [8-14]. 130

4 8.2. The construction of the functionalized graphene - alcohol oxidase modified electrode is discussed in the chapter three, Section Characterization Atomic force microscopy AFM images were recorded in contact mode using silicon cantilevers with an elastic constant of 0.2 N/m over the scan areas of 50 xmx49.5 j.m to identify the immobilization by LBL and the surface roughness was calculated as root mean square (RMS) roughness. Figure 8.1 shows the images of bare graphite (a), first bilayer (b), third bilayer (c) and fifth bilayer (d). The roughness of bare graphite, first bilayer, third bilayer and fifth bilayer are , , and nm respectively. The increase in roughness confirmed the alternate adsorption of FG and AOX on Gr/PDDA. Figure 8.1: AFM images of (a) bare graphite, (b) first bilayer, (c) third bilayer and (d) fifth bilayers of FG and AOX modified Gr/PDDA. 131

5 Cyclic voltammetry Figure 8.2 shows the cyclic voltammetric behavior of bare graphite electrode and modified with FG and AOX were examined using Fe(CN)6 ^'''^' as an electrochemical probe. Cyclic voltammogram of bare graphite electrode in 1 mm Fe(CN)6 '^''^' shows a pair of quasireversible redox peaks with peak to peak separation (zlep) of 142 mv (curve a). When graphite electrode was modified with PDDA, the redox peak of Fe(CN)6 '^''^' was shifted to more negative potentials because the positive surface of PDDA could attract Fe(CN)6 '*"'^" redox couple, which results in the reduction of the overpotential and increase in peak current. Once FG layer is formed on PDDA/Gr electrode, zlep decreases to 98 mv and also there is enhancement in peak current which shows the electrocatalytic activity of FG. After immobilization of AOX enzyme, AEp increases to 104 mv along with decrease in peak current due to hindrance to electron transfer towards electrode surface c y:^^^^^^^^^ /^""V-^ ^s / -"'''''''^ 9 ^,_^^'^ _^^^,^ ^^IIIZI^^^^'^^^ ^"v y^ jy \y^// ^"^^J^ ' 1 ' 1 ' 1 ' I ' E/Vvs.SCE Figure 8.2: Cyclic voltammograms of (a) bare Gr, (b) Gr/PDDA, (c) Gr/PDDA-FG and (d) Gr/PDDA- [FG-AOXI electrodes in 1 mm Fe(CN)6 *"'^" phosphate buffer solution containing 0.1 M KCl (ph 7.0) at a scan rate of 0.1 V s"'. 132

6 EthanolSensor a ^ Electrochemical Impedance Spectroscopy EIS can give information on the impedance changes of the electrode surface during the modification process. EIS was carried out in the presence of 1 mm Fe(CN)6 ^''^' as a redox probe in the frequency range from 100 khz to 0.1 Hz with a amplitude of 5 mv. As shown in Figure 8.3, the charge transfer resistance (^ct) value for Gr/ PDDA- FG electrode is lesser than that of bare graphite electrode and Gr/ PDDA modified electrode, suggesting that very fast electron transfer occurs for the Fe(CN)6 '^''^' redox reaction on Gr/ PDDA- FG electrode. The ^ct for Gr/(PDDA-[FG - AOX]) electrode for single layer is more when compared to Gr/ (PDDA- FG) electrode, indicating that AOX immobilization hinders the electron transfer process for the redox couple Z /Q re Figure 8.3: Nyquist impedance plots of (a) bare Gr, (b) Gr/PDDA, (c) Gr/PDDA-FG and (d) Gr/PDDA- [FG-AOX] electrodes in 1 mm Fe(CN)6 "'^. Frequency range from 100 KHz to 0.1 Hz, amplitude 5mV Direct electrochemistry of alcohol oxidase in multilayer films Figure 8.4(A) shows the CVs of bare graphite (a), graphite/ PDDA-FG (b) and graphite/ PDDA-[FG-AOX] (c). No peaks were observed in curve (a) and (b), but after 133

7 EthanolSensor immobilizing AOX on the electrode surface, a pair of redox peaks with anodic peak potential (E pa) at V and cathodic peak potential (E pc) at V was observed (c). The peaks are attributed to the reduction and oxidation of FAD/FADH2 electro active centres of AOX enzymes by direct electron transfer process as per the equation. AOX (FAD) + 2e" + 2H^ ^ AOX (FADH2) 8.3 The redox potential * was obtained by taking the average of " pa and E pc, which is V, this value is close to the redox potential of V (vs. SCE) for FAD/FADH2 redox couple at ph 7.0 [15], suggesting that AOX molecule have retained bioactivity after immobilizing on FG surface. Thus DET of AOX in FG 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 Figure 8.4(B). Thus Gr/(PDDA-[FG-A0X]5) electrode was used for further studies. The surface coverage {P} was calculated for one layer of AOX according to equation r= Q/nFA, and was found to be 6x 10"^ mol cm'^, 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 and reached 1.0x10" mol cm' for Gr/(PDDA-[FG-A0X]5) electrode. A large amount of enzyme could be immobilized on FG, since FG have large surface area. 134

8 0, E/V vs.sce E/V vs.sce Figure 8.4: (A) Cyclic voltammograms of (a) bare Gr, (b) Gr/PDDA-FG and (c) Gr/PDDA-[FG-AOX] electrodes in 0.1 M Phosphate buffer containing 0.1 M KCl (ph 7.0). (B) Cyclic voltammograms of [FG-AOX]6 multilayer films assembled on Gr/PDDA electrode in phosphate buffer (ph 7.0). The number of multilayer of [FG-AOX] increasing from (a to f) at a scan rate of 0.1 V s ' Effect of scan rate at Gr/(PDDA-[FG- AOXJs) electrode The influence of scan rate on the vohammetric response of Gr/PDDA-[FG-A0X]5 in 0.1 M phosphate buffer solution at ph 7.0 was studied in the range of mv/s to determine the kinetics of electrode reactions. Figure 8.5 shows that with increase in scan rate, the anodic and the cathodic peak currents increased according to Randle-Sevcik equation, but the anodic peak potentials are slightly shifted in the positive potential direction and the cathodic peak potentials are shifted in the negative potential direction. 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 FG film is a quasireversible surface controlled electrochemical process. The linear regression equations are W) = x10'^ x10"^ v (Vs"'), (i?=0.9984) 8.4 V(A)= xio"^ xlo" v (Vs"'), (R = )

9 EthanolSensor <0.0<IOC - 0.0O ^,^^^y^^^^z^ /IL a f 1 1 ' 1 ' 1 ' 1 ' ' E/Vvs.SCE Figure 8.5: Cyclic voltammograms of Gr/(PDDA-[FG-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, and 0.15 V s"' (a-f). Inset shows the plot of peak current vs. scan rate Gr/(PDDA-[FG- AOXls) 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 the absence of ethanol, with an increased reduction peak current and decreased oxidation peak current. After addition of ethanol (3mM) into the buffer the reduction peak current decreased significantly (curve b). The reason is that the dissolved oxygen and AOX take part in oxidation of ethanol as shown in Figure 8.6. In the presence of oxygen, the reduced enzyme AOX (FADH2) is quickly oxidized to the oxidized form AOX (FAD).The electro catalytic regeneration of the reduced AOX (FADH2) enzyme through the reaction 3 causes the loss of reversibility and the increase in size of the cathodic peak current. As the substrate of AOX, the presence of ethanol may result an enzyme-catalyzed reaction which will decrease the concentration of the oxidized form of AOX on electrode surface thus lead to the decrease of reduction peak current. These results provide evidence that the AOX encapsulated on the electrode, still retains its biocatalytic activity towards ethanol oxidation. 136

10 E/Vvs.SCE Figure 8.6: Cyclic voltammetric measurements at Gr/PDDA-[FG-AOX]5 electrode in phosphate buffer (a) saturated with nitrogen saturation, (b) air saturation and (c) air saturation containing 3 mm ethanol in 0.1 M phosphate buffer (ph 7.0), at a scan rate of 0.1 V s"' Chronoamperometric biosensing of ethanol As shown in Figure 8.7(A), upon additions of ethanol aliqouts to static air saturated phosphate buffer solution of ph 7.0 the chronoamperometric curves showed decreasing response. The current quickly reached the steady value, the time of reaching 95% of the steady value was less than 10 s. The biosensing response decreased linearly in the ethanol concentration range from 250 xm to 1500 [im. The enzyme modified electrode displayed a correlation coefficient of and a slope of 31.9 na/mm. Since the Gr/(PDDA-[FG- AOXJs) electrode provided the advantage for detecting oxidase substrate at low potential (- 0.4 V), some common interference such as ascorbic acid, acetaminophen and glucose did not interfere as shown in Figure 8.7(B). The stability of electrodes in terms of repetitive uses was studied at 25 "C. The activity of the enzymes was found to be 75% after 20 days. The electrode was stored in 0.1 M phosphate buffer of ph 7.0, at 4 C when not in use. 137

11 (A ^ ^ ^ ^ I ^0,00012J \ V \» Ethanol coficentra(ion()iml A Time/s (B) 0.5 mm Ethanol 1.0 mm Ethanol mm Glucose 20 nm AA 20 (im ACT Time / s Figure 8.7: (A) Chronoamperograms of Gr/PDDA-[FG-AOX] 5 electrode in air saturated 0.1 M phosphate buffer solution containing 0.1 M KC 1 (ph 7.0) containing 0, 250, 500, 750,1000, 1250 and 1500 nm ethanol (a-g) Inset shows the calibration curve. (B).Effect of interfering species at Gr/PDDA- FG-AOX]5 electrode, applied potential V in a stirred 0.1 M phosphate buffer solution saturated with air containing 0.1 M KCl Effect of ph on the enzyme activity In the enzymatic reactions, protons are transferred from one chemical species to another. Hence the solution ph has a considerable effect on the performance of the prepared ethanol biosensor. Due to the denaturation of AOX in more acidic medium than 138

12 Ethanol Sensor i ph 4.0, the optimization study started from the range of ph at Gr/(PDDA-[FG- AOXJs) modified electrodes. Figure 8.8 shows the amperometric current response increased steeply with increase in ph values and reached maximum at ph 7.0. The response current decreased as the solution ph value was greater than 7.0 (alkaline side). This may be attributed to the denaturation of enzymes in the ph greater than ph found in intracellular fluids where the optimal ph is 7.4. Therefore, 0.1 M phosphate buffer solution of ph 7.0 is selected as a medium buffer for the determination of ethanol Figure 8.8: The effect of ph on the response of enzyme electrode in 0.1 M phosphate buffer solution containing 0.1 M KCl. PH Effect of temperature on the enzyme activity The effect of temperature on the activity of enzyme electrode was determined at ph 7.0. The temperature was increased from 30 C to 50 C and the response increased with temperature, due to faster enzyme reaction and analyte diffusion and then started to decrease due to denaturation of enzymes. Heat denaturation is known to be a continuous process and the rate of denaturation increases with rise in temperature. The temperature at 139

13 Ethanoi Sensor which enzyme electrode yielded a maximum current was found to be at 48 C as shown in Figure Temperature [ C] Figure 8.9: The effect of temperature on the response of enzyme electrode in 0.1 M phosphate buffer solution containing 0.1 m KCl Real sample analysis For the practical applications, the analysis of ethanoi concentration in beer samples was performed by amperometric technique. The beer samples were diluted in such away that, ethanoi concentrations should fall in the working range of above mentioned electrodes. The current response from each beer sample at an applied potential of-400 mv was measured and the results are given in table 8.1. It should be noted that the concentration of ethanoi declared by the manufacturer is 8.0% and our mean value was 8.21 %. 140

14 Table 8.1 Determination of Ethanol in beer sample (n=5) at Gr/(PDDA-[FG- AOXJs) electrode Trial no Labelled (%) Found(%) RSD Conclusion By using LBL method, multicomponent ultrathin film was constructed with alcohol oxidase and FG on PDDA modified graphite electrode. AFM measurement confirmed the formation of nanoscale multilayer architecture. The presence of FG leads to excellent catalytic activity of the nanoscale multilayer film to the reduction of dissolved oxygen, producing a sensitive ethanol biosensor. The nanoscale multilayer shows direct electron transfer of AOX and the modified electrode. The biosensor shows rapid and highly sensitive amperometric response to ethanol at a lower reduction potential without stirring. The reproducibility as well as the operational and storage stability was good. The interferents free, mediator less (reagent less) ethanol biosensor were fabricated. 141

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