The Investigation of Electrochemistry Behaviors of Tyrosinase Based on Directly-Electrodeposited Grapheneon Choline-Gold Nanoparticles

A novel catechol (CA) biosensor was developed by embedding tyrosinase (Tyr) onto in situ electrochemical reduction graphene (EGR) on choline-functionalized gold nanoparticle (AuNPs-Ch) film. The results of UV-Vis spectra indicated that Tyr retained its original structure in the film, and an electrochemical investigation of the biosensor showed a pair of well-defined, quasi-reversible redox peaks with Epa = −0.0744 V and Epc = −0.114 V (vs. SCE) in 0.1 M, pH 7.0 sodium phosphate-buffered saline at a scan rate of 100 mV/s. The transfer rate constant ks is 0.66 s−1. The Tyr-EGR/AuNPs-Ch showed a good electrochemical catalytic response for the reduction of CA, with the linear range from 0.2 to 270 μM and a detection limit of 0.1 μM (S/N = 3). The apparent Michaelis-Menten constant was estimated to be 109 μM.


Introduction
Phenolic compounds are a very important and widespread class of substances [1]. However, they are toxic and injure mammals, fishes and other aquatic organisms [2]. Due to their toxicity and persistency in the environment, they are on the priority pollutants list of the European Community and the Environmental Protection Agency of the United States [3]. The determination of phenols is of paramount importance in environmental analysis, and medical and food quality characterization. Spectrophotometry and chromatography are commonly-useful methods for the determination of phenols [4]. These techniques have a number of disadvantages, such as expense, time-consumption, and difficult in situ application [3], which limit their applications to laboratory settings and rapid analyses under field conditions. To overcome the defects mentioned above, a simple and effective analytical method for the determination of phenols is needed. An electrochemical method has been considered as the best choice for in situ monitoring of phenolic compounds by virtue of its high sensitivity, simple instrumentation, low production cost, and promising response speed [5]. The high applied voltage needed in direct electrochemistry of phenols [6] is also followed by an increase of the background current and noise level. Furthermore, direct electrochemical oxidation of phenols is coupled with fouling reactions [7]. Enzyme-based amperometric biosensors for the determination of phenols have been experimentally demonstrated as an efficient route to solve the obstacles mentioned above. Electrochemical biosensors based on enzymes have been used for the determination of phenolic compounds in vivo and in vitro with high selectivity, sensitivity, and rapid analysis rates in various biological species [8]. Tyrosinase (Tyr), known as polyphenol oxidase, is a copper monooxygenase that catalyzes the oxidation of phenolic compounds to their corresponding o-quinines [9]. It belongs to the
All electrochemical experiments were carried out on a CHI 660D electrochemical workstation (Shanghai CH Instrument Co., Ltd., Shanghai, China) using a three-electrode system. The working electrode was GCE or modified GCE. GCE of 3-mm diameter, before use, was first polished to a mirror-like surface with 1.0, 0.3 and 0.05 µm Al 2 O 3 slurry on a polish cloth, and rinsed with double-distilled water, then sonicated in ethanol and double-distilled water for 5 min, respectively. Then, the electrode was allowed to dry under nitrogen. A saturated calomel electrode (SCE) and a platinum electrode served as reference and counter electrodes, respectively. UV-Vis spectra were obtained on a UV-Vis spectrophotometer attached to an Elx800 absorbance microplate reader (BioTek, Winooski, VT, USA). Herein, ITO (Hebei LingxianGaoke Co., Ltd., Shi Jia Zhuang, China) was used as the substrate for the investigation of the morphology. Before use, it was cleaned by sonication sequentially for 20 min in acetone, 10% KOH in ethanol, and distilled water. Scanning electron microscopic (SEM) measurements were carried out on a JSM-6700F microscope (Japan Electron Company, Tokio, Japan) at 15 kV. All of the electrochemical experiments were conducted at room temperature (25 ± 2 • C).

Preparation and Modification of Nafion/Tyr/EGR-AuNPs-Ch/GCE
EG and ChCl (2:1) were gentle heated by continuous stirring until a homogeneous solution formed. Five-hundred microliters (500 µL) of HAuCl 4 solution (w/w, 1%) was added into 50 mL of the above solution. Then, 5 mL of sodium borohydride solution (w/w, 1%) was slowly added, while stirring vigorously. The wine red solution was kept stirring at 50 • C for another 30 min. Then, it was allowed it to cool and AuNPs-Ch was prepared and stored at 4 • C for use.
GO was synthesized from graphite powder by a modified Hummers method [45]. The fabrication process of the Nafion/Tyr/EGR-AuNPs-Ch/GCE was as follows: 10 µL of AuNPs-Ch homogeneous solution was casted onto the surface of the GCE by using a syringe to prepare AuNPs-Ch/GCE. The modified electrode dried at room temperature later. The preparation of electrolyte solution: 1 mg/mL GO was dispersed in 1/15 M, pH 9.18 PBS, and mixed via ultrasonication for several minutes to form a homogeneous solution. Prior to experiments, the solution was deoxygenated with high-purity nitrogen gas. The electrochemical deposition of EGR on the AuNPs-Ch/GCE was performed in the above eletrolyte solution from −1.5 to 0.5 V for 10 cycles at a scan rate of 10 mV/s. The treated substrate electrode was washed with distilled water.Ten microliters (10 µL) of Tyr (5 mg/mL in pH 7.0 PBS) was cast onto the electrode surface by using a syringe to prepare Tyr/EGR-AuNPs-Ch/GCE. Then, 3 µL of 0.5 wt % Nafion was dropped on it. Dried at room temperature later, the sensor was stored at 4 • C when not in use.

Fabrication and Characterization of the Nafion/Tyr/EGR-AuNPs-Ch/GCE
As shown Scheme 1A, electrostatic assembly of the positively-charged -N + (CH 3 ) 3 polar head group of Ch with AuCl 4 − . The surface modification of gold nanospheres with choline was prepared by the reduction process, and the superficial Ch has hydroxy groups, which could be covalently bound to the edge plane sites of the carbon surface through the oxygen atom [46]. Thus, a -C-O-C-bond was formed, which efficiently immobilized AuNPs-Ch onto the bare GCE. AuNPs-Ch was fixed on the GCE, as shown in Scheme 1B. The presence of oxygen-containing groups [27] controlling GO exists as a planar sheet that can be inlaid into AuNPs-Ch to form large-scale two-dimensional arrays. The further in situelectrochemically-synthesized GR nanosheets enhanced the attachment of AuNPs-Ch with the electrode surface, which could form a stable EGR-AuNPs-Ch composite. Additionally, the high surface area of GR was helpful for immobilizing more proteins or enzymes and the nanocomposite film could provide a microenvironment for proteins or enzymes to retain their native structure and activity, and to achieve a reversible direct electron transfer reaction at the electrode surface [47]. Tyr anchored onto EGR and attached firmly. The addition of Nafion as an immobilization matrix to entrap enzymes and proteins effectively prevented the leakage of Tyr at the outermost point. bond was formed, which efficiently immobilized AuNPs-Ch onto the bare GCE. AuNPs-Ch was fixed on the GCE, as shown in Scheme 1B. The presence of oxygen-containing groups [27] controlling GO exists as a planar sheet that can be inlaid into AuNPs-Ch to form large-scale two-dimensional arrays. The further in situelectrochemically-synthesized GR nanosheets enhanced the attachment of AuNPs-Ch with the electrode surface, which could form a stable EGR-AuNPs-Ch composite. Additionally, the high surface area of GR was helpful for immobilizing more proteins or enzymes and the nanocomposite film could provide a microenvironment for proteins or enzymes to retain their native structure and activity, and to achieve a reversible direct electron transfer reaction at the electrode surface [47]. Tyr anchored onto EGR and attached firmly. The addition of Nafion as an immobilization matrix to entrap enzymes and proteins effectively prevented the leakage of Tyr at the outermost point. As shown in Figure 1A, with the electrochemical process from −1.5 to 0.5 V for 10 cycles at a scan rate of 10 mV/s, the AuNPs-Chnanospheres with diameters of 70 to 150 nm were heterogeneously scatteredon the surface of the sample because of agglomerate (as shown in Figure  1B). The much smaller homogeneously-scattered AuNPs had meandiameters of 30 to 50 in matrices. However, EGR-AuNPs-Ch showed to be much more uniform with evenly-dispersed nanospheres ( Figure 1C). The EGR-AuNPs-Chshowed meandiameters of 50 to 120 nm (as shown in Figure 1D). EGR intercalated the structure of AuNPs-Ch, which restrained the GR layer overlap and AuNPs agglomeration, creating a favorable foundation for Tyr. As shown in Figure 1A, with the electrochemical process from −1.5 to 0.5 V for 10 cycles at a scan rate of 10 mV/s, the AuNPs-Chnanospheres with diameters of 70 to 150 nm were heterogeneously scatteredon the surface of the sample because of agglomerate (as shown in Figure 1B). The much smaller homogeneously-scattered AuNPs had meandiameters of 30 to 50 in matrices. However, EGR-AuNPs-Ch showed to be much more uniform with evenly-dispersed nanospheres ( Figure 1C). The EGR-AuNPs-Chshowed meandiameters of 50 to 120 nm (as shown in Figure 1D). EGR intercalated the structure of AuNPs-Ch, which restrained the GR layer overlap and AuNPs agglomeration, creating a favorable foundation for Tyr.
The   Electrochemical impedance spectroscopy (EIS) is an effective method for probing the features of surface modified electrode and can provide information on the impedance changes accompanying the stepwise electrode modification process. As shown in Figure 3, the semicircle of the AuNPs-Ch/GCE (curve b') and the EGR-AuNPs-Ch/GCE (curve d') were obviously smaller than that of the bare GCE (curve a'). The addition of Tyr blocked the electron transfer on the surface of GCE, resulted the increase of the impedance (curve c'), which was in good agreement with the results of the CVs.   Electrochemical impedance spectroscopy (EIS) is an effective method for probing the features of surface modified electrode and can provide information on the impedance changes accompanying the stepwise electrode modification process. As shown in Figure 3, the semicircle of the AuNPs-Ch/GCE (curve b') and the EGR-AuNPs-Ch/GCE (curve d') were obviously smaller than that of the bare GCE (curve a'). The addition of Tyr blocked the electron transfer on the surface of GCE, resulted the increase of the impedance (curve c'), which was in good agreement with the results of the CVs. Electrochemical impedance spectroscopy (EIS) is an effective method for probing the features of surface modified electrode and can provide information on the impedance changes accompanying the stepwise electrode modification process. As shown in Figure 3, the semicircle of the AuNPs-Ch/GCE (curve b ) and the EGR-AuNPs-Ch/GCE (curve d ) were obviously smaller than that of the bare GCE (curve a ). The addition of Tyr blocked the electron transfer on the surface of GCE, resulted the increase of the impedance (curve c ), which was in good agreement with the results of the CVs.

Direct Electrochemistry of Tyr on the Nafion/Tyr/EGR-AuNPs-Ch/GCE
The direct electrochemisty of Tyron the Nafion/Tyr/EGR-AuNPs-Ch/GCE was studied by cyclic voltammetry (CV). Cyclic voltammograms (CVs) of Tyrwith different scan rates were shown in Figure 4. The Nafion/Tyr/EGR-AuNPs-Ch/GCE showed a pair of well-defined, quasi-reversible redox peaks with Epa= −0.0744 V and Epc= −0.114 V (vs. SCE) in PBS (0.1 M, pH 7.0) with the formal potential E 0 ' = −0.0942 V. The value of E 0 ' corresponded with the active sites of Tyr from different sources were varied from 120 to 600 mV versus NHE [48]. The peak-to-peak separation ΔEp was 40 mV and about oneratio of cathodic to anodic current intensity at the scan rate of 0.1 V/s. The redox process of Tyr at the Nafion/Tyr/EGR-AuNPs-Ch/GCE gave roughly symmetric anodic and cathodic peaks at relative slow scan rates. When the scan rate increased, the redox potentials (Epa and Epc) of Tyr hardly shift. Meanwhile, the redox peak current increased linearly (inset, Figure 4): Ipa = −3.7 × 10 −1 − 1.4 × 10 −1 v, r = 0.9996; Ipc = 9.5 × 10 −1 + 1.2 × 10 1 v, r = 0.9996. The high electroactive area of EGR-AuNPs-Ch induces high capacitance [11] and high background currents varying proportionally with the scan rate. This indicated that the electron transfer process for Tyr at the Nafion/Tyr/EGR-AuNPs-Ch/GCE was a surface-confined mechanism in the abovementioned potential scope, manifesting the characteristics of the thin-layer surface-controlled electrochemical process.
The anodic and cathodic peak potentials were linearly dependent on the logarithm of the scan rates (n) with slopes of −2.3RT/anF and 2.3RT/(1−a)nF, respectively. Hence, the charge-transfer coefficient awas calculated to be 0.47. The heterogeneous electron transfer rate constant (ks) was further estimated according to the following equation [49]: where ɑ is the charge transfer coefficient. n is the number of electrons transferred. R, T, and F symbols have their conventional meanings. ΔEp is the peak-to-peak potential separation. The result was 0.66 s −1 , which was higher than 0.032 s −1 for Tyr-AuNPs/boron-doped diamond (BDD) [50] and 0.030 s −1 for Tyr/AgE [22]. Thus, Nafion/Tyr/EGR-AuNPs-Ch/GCE can provide a favorable microenvironment for Tyr to undergo a facile electron transfer reaction due to the structure of EGR-AuNPs-Ch, which is to the benefit of effective immobilization of enzymes, proteins, and other bioactive substances. EGR greatly increased the specific surface area and shortens the distance between the active centers of Tyr and the electrode surface. Furthermore, interlayers offered more binding sites for the immobilization of Tyr.

Direct Electrochemistry of Tyr on the Nafion/Tyr/EGR-AuNPs-Ch/GCE
The direct electrochemisty of Tyron the Nafion/Tyr/EGR-AuNPs-Ch/GCE was studied by cyclic voltammetry (CV). Cyclic voltammograms (CVs) of Tyrwith different scan rates were shown in Figure 4. The Nafion/Tyr/EGR-AuNPs-Ch/GCE showed a pair of well-defined, quasi-reversible redox peaks with E pa = −0.0744 V and E pc = −0.114 V (vs. SCE) in PBS (0.1 M, pH 7.0) with the formal potential E 0 = −0.0942 V. The value of E 0 corresponded with the active sites of Tyr from different sources were varied from 120 to 600 mV versus NHE [48]. The peak-to-peak separation ∆E p was 40 mV and about oneratio of cathodic to anodic current intensity at the scan rate of 0.1 V/s. The redox process of Tyr at the Nafion/Tyr/EGR-AuNPs-Ch/GCE gave roughly symmetric anodic and cathodic peaks at relative slow scan rates. When the scan rate increased, the redox potentials (E pa and E pc ) of Tyr hardly shift. Meanwhile, the redox peak current increased linearly (inset, Figure 4): I pa = −3.7 × 10 −1 − 1.4 × 10 −1 v, r = 0.9996; I pc = 9.5 × 10 −1 + 1.2 × 10 1 v, r = 0.9996. The high electroactive area of EGR-AuNPs-Ch induces high capacitance [11] and high background currents varying proportionally with the scan rate. This indicated that the electron transfer process for Tyr at the Nafion/Tyr/EGR-AuNPs-Ch/GCE was a surface-confined mechanism in the abovementioned potential scope, manifesting the characteristics of the thin-layer surface-controlled electrochemical process.
The anodic and cathodic peak potentials were linearly dependent on the logarithm of the scan rates (n) with slopes of −2.3RT/anF and 2.3RT/(1−a)nF, respectively. Hence, the charge-transfer coefficient a was calculated to be 0.47. The heterogeneous electron transfer rate constant (k s ) was further estimated according to the following equation [49]: where a is the charge transfer coefficient. n is the number of electrons transferred. R, T, and F symbols have their conventional meanings. ∆E p is the peak-to-peak potential separation. The result was 0.66 s −1 , which was higher than 0.032 s −1 for Tyr-AuNPs/boron-doped diamond (BDD) [50] and 0.030 s −1 for Tyr/AgE [22]. Thus, Nafion/Tyr/EGR-AuNPs-Ch/GCE can provide a favorable microenvironment for Tyr to undergo a facile electron transfer reaction due to the structure of EGR-AuNPs-Ch, which is to the benefit of effective immobilization of enzymes, proteins, and other bioactive substances. EGR greatly increased the specific surface area and shortens the distance between the active centers of Tyr and the electrode surface. Furthermore, interlayers offered more binding sites for the immobilization of Tyr.

Amperometric Response Towards CA
The possible mechanism of electrocatalytic reduction of CA at the Tyr-based enzyme electrode can be expressed as follows (Scheme 2): However, o-quinone is extremely unstable, an accompanying reaction followed [44]: Similar structural compounds, such as gallic acid, caffeic acid, p-coumaric acid, and ferulic acid (as shown in Scheme 3) were also determined by the Nafion/Tyr/EGR-AuNPs-Ch/GCE. As derivatives of CA, the caffeic acid current response towards the same concentration was only one fifth to one third that of CA. The quite similar structure of caffeic acid made it easily catalyze by Tyr. The carboxyl group and double bond contributed to the current drop. Gallic acid showed slight current change. The third hydroxyl severely hindered the catalytic process of Tyr due to steric effects. There were no remarkable current change of p-coumaric acid and ferulic acid. Monophenols are hydroxylated to a variety of diphenols and then catalyzed by Tyr, followed by subsequent oxidation to quinines [51]. EGR can interact with the double bond of p-coumaric acid and ferulic acid by the π-π attraction, which blocks the monophenols' hydroxylation, resulting in the difficulty of Tyr catalysis. The above results confirmed the mechanism of electrocatalytic reduction of CAwith Nafion/Tyr/EGR-AuNPs-Ch/GCE.   to n: 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 mV/s). Inset: the relationship between cathodic and anodic peak current with scan rate v.

Amperometric Response Towards CA
The possible mechanism of electrocatalytic reduction of CA at the Tyr-based enzyme electrode can be expressed as follows (Scheme 2): However, o-quinone is extremely unstable, an accompanying reaction followed [44]: Similar structural compounds, such as gallic acid, caffeic acid, p-coumaric acid, and ferulic acid (as shown in Scheme 3) were also determined by the Nafion/Tyr/EGR-AuNPs-Ch/GCE. As derivatives of CA, the caffeic acid current response towards the same concentration was only one fifth to one third that of CA. The quite similar structure of caffeic acid made it easily catalyze by Tyr. The carboxyl group and double bond contributed to the current drop. Gallic acid showed slight current change. The third hydroxyl severely hindered the catalytic process of Tyr due to steric effects. There were no remarkable current change of p-coumaric acid and ferulic acid. Monophenols are hydroxylated to a variety of diphenols and then catalyzed by Tyr, followed by subsequent oxidation to quinines [51]. EGR can interact with the double bond of p-coumaric acid and ferulic acid by the π-π attraction, which blocks the monophenols' hydroxylation, resulting in the difficulty of Tyr catalysis. The above results confirmed the mechanism of electrocatalytic reduction of CAwith Nafion/Tyr/EGR-AuNPs-Ch/GCE.

Amperometric Response Towards CA
The possible mechanism of electrocatalytic reduction of CA at the Tyr-based enzyme electrode can be expressed as follows (Scheme 2): However, o-quinone is extremely unstable, an accompanying reaction followed [44]: Similar structural compounds, such as gallic acid, caffeic acid, p-coumaric acid, and ferulic acid (as shown in Scheme 3) were also determined by the Nafion/Tyr/EGR-AuNPs-Ch/GCE. As derivatives of CA, the caffeic acid current response towards the same concentration was only one fifth to one third that of CA. The quite similar structure of caffeic acid made it easily catalyze by Tyr. The carboxyl group and double bond contributed to the current drop. Gallic acid showed slight current change. The third hydroxyl severely hindered the catalytic process of Tyr due to steric effects. There were no remarkable current change of p-coumaric acid and ferulic acid. Monophenols are hydroxylated to a variety of diphenols and then catalyzed by Tyr, followed by subsequent oxidation to quinines [51]. EGR can interact with the double bond of p-coumaric acid and ferulic acid by the π-π attraction, which blocks the monophenols' hydroxylation, resulting in the difficulty of Tyr catalysis. The above results confirmed the mechanism of electrocatalytic reduction of CAwith Nafion/Tyr/EGR-AuNPs-Ch/GCE. Scheme 2. Schematics of the possible mechanism of catalytic oxidization of Tyr towards catechol with Nafion/Tyr/EGR-AuNPs-Ch/GCE.

Scheme 2.
Schematics of the possible mechanism of catalytic oxidization of Tyr towards catechol with Nafion/Tyr/EGR-AuNPs-Ch/GCE. Chronoamperometry was also used for the investigation of electrocatalysis of CA with Nafion/Tyr/EGR-AuNPs-Ch/GCE. The effect of applied potential was investigated at different potentials ranging from 0.10 V to −0.30 V. At the applied potentials from 0.10 V to −0.10 V, the current increased and reached a peak plateau. From −0.10 V to −0.30 V, the response of the electrode gradually decreased. Moreover, the baseline current of the signal became unstable above −0.10 V. As a result, −0.10 V was finally chosen as the applied potential throughout all the amperometric measurements. Chronoamperometry was also used for the investigation of electrocatalysis of CA with Nafion/Tyr/EGR-AuNPs-Ch/GCE. The effect of applied potential was investigated at different potentials ranging from 0.10 V to −0.30 V. At the applied potentials from 0.10 V to −0.10 V, the current increased and reached a peak plateau. From −0.10 V to −0.30 V, the response of the electrode gradually decreased. Moreover, the baseline current of the signal became unstable above −0.10 V. As a result, −0.10 V was finally chosen as the applied potential throughout all the amperometric measurements.     Figure 6, inset A, obvious amperometric responses are shown. The 95% steady-state current can be obtained at 2-3 s, revealing the faster response of the sensor than that of previously-reported CA sensors [7,14]. The current response increased along with the CA concentration. The calibration curve at the biosensor showed linearity from 0.2 to 270 μM ( Figure 6, inset B). The linear regression equation was obtained as Iss (μA) = 9.11 × 10 −2 C (μM) + 5.13 × 10 −1 (r = 0.9962) with a detection limit of 0.1 μM (S/N = 3). Thesensitivity obtained from the slope of the calibration curve is 122A/M·cm −2 , Scheme 3. The structure ofgallic acid, caffeic acid, p-coumaric acid, and ferulic acid. Chronoamperometry was also used for the investigation of electrocatalysis of CA with Nafion/Tyr/EGR-AuNPs-Ch/GCE. The effect of applied potential was investigated at different potentials ranging from 0.10 V to −0.30 V. At the applied potentials from 0.10 V to −0.10 V, the current increased and reached a peak plateau. From −0.10 V to −0.30 V, the response of the electrode gradually decreased. Moreover, the baseline current of the signal became unstable above −0.10 V. As a result, −0.10 V was finally chosen as the applied potential throughout all the amperometric measurements.     Figure 6, inset A, obvious amperometric responses are shown. The 95% steady-state current can be obtained at 2-3 s, revealing the faster response of the sensor than that of previously-reported CA sensors [7,14]. The current response increased along with the CA concentration. The calibration curve at the biosensor showed linearity from 0.2 to 270 μM ( Figure 6, inset B). The linear regression equation was obtained as Iss (μA) = 9.11 × 10 −2 C (μM) + 5.13 × 10 −1 (r = 0.9962) with a detection limit of 0.1 μM (S/N = 3). Thesensitivity obtained from the slope of the calibration curve is 122A/M·cm −2 ,   Figure 6, inset A, obvious amperometric responses are shown. The 95% steady-state current can be obtained at 2-3 s, revealing the faster response of the sensor than that of previously-reported CA sensors [7,14]. The current response increased along with the CA concentration. The calibration curve at the biosensor showed linearity from 0.2 to 270 µM ( Figure 6, inset B). The linear regression equation was obtained as I ss (µA) = 9.11 × 10 −2 C (µM) + 5.13 × 10 −1 (r = 0.9962) with a detection limit of 0.1 µM (S/N = 3). The sensitivity obtained from the slope of the calibration curve is 122A/M·cm −2 , which was much higher than the listed CA sensors in the literature [7]. The biosensor had a much better catalytic response towards CA than that of the agarose-guar gum-entrapped Tyr [10] and PANI-polyphenol oxidase (PPO) film [14].

Repeatability and Stability of CA Biosensor
The stability and repeatability of the biosensor were studied. The relative standard deviation (RSD) was 2.4% for eight successive measurements of 10 μM CA in PBS, showing that the proposed biosensor possessed good repeatability. The cyclic voltammetric responses of the modified biosensor in PBS containing 10 μM CA showed no obvious change after 25 cycles, and then it decreased slowly with the increase of the cycle, indicating that the biosensor was stable. The storage stability the biosensor was further investigated. The peak currents of the Nafion/Tyr/EGR-AuNPs-Ch/GCE was measured using the same electrode and it retained above 95% of its initial response stored at 4 °C after three weeks. These results displayed that the biosensor based on the Nafion/Tyr/EGR-AuNPs-Ch/GCE had good stability.

Repeatability and Stability of CA Biosensor
The stability and repeatability of the biosensor were studied. The relative standard deviation (RSD) was 2.4% for eight successive measurements of 10 µM CA in PBS, showing that the proposed biosensor possessed good repeatability. The cyclic voltammetric responses of the modified biosensor in PBS containing 10 µM CA showed no obvious change after 25 cycles, and then it decreased slowly with the increase of the cycle, indicating that the biosensor was stable. The storage stability the biosensor was further investigated. The peak currents of the Nafion/Tyr/EGR-AuNPs-Ch/GCE was measured using the same electrode and it retained above 95% of its initial response stored at 4 • C after three weeks. These results displayed that the biosensor based on the Nafion/Tyr/EGR-AuNPs-Ch/GCE had good stability.

Conclusions
In the present work, GR was successfully prepared in situ on choline-functionalized gold nanoparticle-modified GCE. Based on the EGR-AuNPs-Ch/GCE platform, the CA biosensor exhibited a variety of good electrochemical characteristics, including a low detection limit, high catalytic ability, wide linearity, and a larger electron transfer rate constant of 0.66 s −1 . These advantages should be attributed to the following: (1) AuNPs-Ch can be efficiently immobilized on the bare GCE, and interlayer EGR provides more attachment sites for Tyr immobilization; (2) π-π electron transfer between EGR, plays an important role in facilitating the electron transfer between Tyr and the electrode surface; and (3) synergistic effects of AuNPs-Ch and EGR exhibited the signal amplification of nanosized materials.