Electrodeposition–Assisted Assembled Multilayer Films of Gold Nanoparticles and Glucose Oxidase onto Polypyrrole-Reduced Graphene Oxide Matrix and Their Electrocatalytic Activity toward Glucose

The study reports a facile and eco-friendly approach for nanomaterial synthesis and enzyme immobilization. A corresponding glucose biosensor was fabricated by immobilizing the gold nanoparticles (AuNPs) and glucose oxidase (GOD) multilayer films onto the polypyrrole (PPy)/reduced graphene oxide (RGO) modified glassy carbon electrode (GCE) via the electrodeposition and self-assembly. PPy and graphene oxide were first coated on the surface of a bare GCE by the electrodeposition. Then, AuNPs and GOD were alternately immobilized onto PPy-RGO/GCE electrode using the electrodeposition of AuNPs and self-assembly of GOD to obtain AuNPs-GOD multilayer films. The resulting PPy-RGO-(AuNPs-GOD)n/GCE biosensors were used to characterize and assess their electrocatalytic activity toward glucose using cyclic voltammetry and amperometry. The response current increased with the increased number of AuNPs-GOD layers, and the biosensor based on four layers of AuNPs-GOD showed the best performance. The PPy-RGO-(AuNPs-GOD)4/GCE electrode can detect glucose in a linear range from 0.2 mM to 8 mM with a good sensitivity of 0.89 μA/mM, and a detection limit of 5.6 μM (S/N = 3). This study presents a promising eco-friendly biosensor platform with advantages of electrodeposition and self-assembly, and would be helpful for the future design of more complex electrochemical detection systems.


Introduction
Diabetes is a growing public health problem and its complications, rather than the disease itself, are harmful for human body [1]. In medicine, diabetes is diagnosed mainly by detecting the concentration of glucose in the blood/urine [2]. Hence, glucose detection and quantification has received much attention. Until now, numerous techniques, such as chromatography [3,4], colorimetric [5,6], and electrochemistry [7,8] have been proposed for the determination of glucose. Although each glucose detection system has strengths and weaknesses, electrochemical methods have gained wide acceptance for analyses and are preferred over other techniques because of their simplicity, relatively low-costs, easy manufacturing and industrialization, and portable instruments [7][8][9][10]. Of the pyrrole monomer were purchased from J&K Scientific Ltd. (Beijing, China). Glucose, ascorbic acid, 4-acetamidophenol and uric acid were purchased from Aladdin Industrial Inc (Shanghai, China). Phosphate-buffered saline (0.1 M PBS, pH 7.0) buffer was employed as a supporting electrolyte. All electrochemical experiments were performed in PBS at room temperature, approximately 25 • C.

Apparatus and Measurements
Electrochemical measurements were carried out with CHI800C electrochemical analyzer (Chen Hua Instruments, Shanghai, China) with a conventional three-electrode system, in which a glassy carbon electrode (GCE), a saturated calomel electrode (SCE) and a platinum wire electrode served as the working, reference, and auxiliary electrodes, respectively. The surface morphological features of PPy-GO and PPy-RGO-AuNPs were characterized using ZEISS Ultra 55 scanning electron microscopy (Carl Zeiss, Oberkochen, Germany).

Configuration of the Amperometric Glucose Biosensor
PPy-RGO-(AuNPs-GOD) n /GCE was prepared according to the following steps: Step 1: The bare GCE electrode was polished with chamois leather containing 0.05 µm alumina slurry, rinsed thoroughly with water, sonicated in ethanol, and dried at room temperature.
Step 2: GO (0.5 mg/mL) were sonicated in pyrrole monomer solution (0.3 M), centrifuged at 10,000 rpm for 10 min, and collected the supernatant. Then, the cleaned bare GCE was immersed into the supernatant, and a continuous cyclic voltammetric sweep of 10 cycles with potential ranging from 0 V to 0.8 V versus SCE was performed at a scan rate of 25 mV/s for the electrooxidation of pyrrole monomer and simultaneous immobilization of polypyrrole (PPy) and GO onto the electrode surface. The as-obtained electrode was regard as PPy-GO/GCE.
Step 3: PPy-GO/GCE was immersed into PBS containing 6.5 mM HAuCl 4 ·4H 2 O. Then, a continuous cyclic voltammetric sweep of 6 cycles with potential ranging from 0 V to −1.5 V versus SCE was performed at a scan rate of 25 mV/s to ensure the simultaneous electroreduction of both GO and HAuCl 4 on the PPy-GO/GCE surface, which was regarded as PPy-RGO-AuNPs/GCE electrode.

Glucose Biosensor Based on Polypyrrole, Reduced Graphene Oxide, Nanogold and Glucose Oxidase
The fabrication process of the glucose biosensor based on the electrodeposition-assisted assembled multilayer films of AuNPs and glucose oxidase (AuNPs-GOD) n , and the polypyrrole/reduced graphene oxide film (PPy-RGO) modified GCE is shown in Figure 1. PPy and graphene oxide (GO) was first assembled on the bare GCE surface via the electrooxidation of pyrrole to obtain PPy-GO/GCE electrode. Second, AuNPs were electro-reductively deposited on the PPy-GO/GCE electrode surface with the electrochemical reduction of GO immobilized on the PPy-GO/GCE to obtain PPy-RGO-AuNPs/GCE. Third, GOD was self-assembled on PPy-RGO-AuNPs/GCE surface mainly via the interaction between AuNPs and GOD to obtain the PPy-RGO-(AuNPs-GOD) 1 /GCE electrode. It has been reported that AuNPs can bind strongly to the biomolecule surface through covalent bonds with the functional groups such as -NH 2 , SH, and -CN, and it can provide a suitable microenvironment for biomolecules, such as enzyme and nucleic acids [11,36]. Finally, different layers of AuNPs-GOD multilayer films were built up on the PPy-RGO/GCE surface by repeating the process of electrodeposition of AuNPs and self-assembly of GOD to obtain PPy-RGO-(AuNPs-GOD) n /GCE electrode. Herein, this proposed amperometric glucose biosensor, using the PPy-RGO and AuNPs-GOD multilayer films as the sensitive layer, was developed by the eco-friendly electrodeposition and self-assembly without redox reagent, separation procedure, crosslinking or adhesive agents. The possible working mechanisms may involve the following biochemical reactions: glucose biosensor, using the PPy-RGO and AuNPs-GOD multilayer films as the sensitive layer, was developed by the eco-friendly electrodeposition and self-assembly without redox reagent, separation procedure, crosslinking or adhesive agents. The possible working mechanisms may involve the following biochemical reactions: H2O2 → O2 + 2 H + + 2e − When GOD (FAD) catalyzes glucose oxidation, GOD catalyzes glucose to form gluconolactone, and GOD itself is reduced to GOD (FADH2). Then, GOD (FADH2) is oxidized to GOD (FAD), which can be achieved in two ways: by oxygen re-oxidation back to GOD (FAD) and producing hydrogen peroxide (H2O2) in the process (first generation biosensors), or via direct electron transfer (DET) between FADH2 and electrodes (third generation biosensors) [37,38]. Thus, either the electroactivity of H2O2 or the direct response current of FADH2 can be used as a measure of glucose in this study. The electrochemical procedure for the preparation of polypyrrole, reduced graphene oxide, and AuNPs was prepared using literature research [24], with some changes. Here the electrochemical oxidation and the electrochemically reduction are separately performed to avoid drop casting side effects. The electrochemically oxidation of pyrrole with a potential range of 0 V-0.8 V versus SCE and the morphology of PPy-GO film are shown in Figure 2. Cyclic voltammograms (Figure 2A) illustrated the continuous electrochemical oxidation of pyrrole, and PPy-GO presented a typical wrinkled and crumpled shape ( Figure 2B). As reported in the literature [39,40], pyrrole can be oxidized at 0.8 V. Higher than 0.8 V may potentially lead to the formation of overoxidized polypyrrole films with poor adherence at the electrode surface. The electrochemical When GOD (FAD) catalyzes glucose oxidation, GOD catalyzes glucose to form gluconolactone, and GOD itself is reduced to GOD (FADH 2 ). Then, GOD (FADH 2 ) is oxidized to GOD (FAD), which can be achieved in two ways: by oxygen re-oxidation back to GOD (FAD) and producing hydrogen peroxide (H 2 O 2 ) in the process (first generation biosensors), or via direct electron transfer (DET) between FADH 2 and electrodes (third generation biosensors) [37,38]. Thus, either the electroactivity of H 2 O 2 or the direct response current of FADH 2 can be used as a measure of glucose in this study.
The electrochemical procedure for the preparation of polypyrrole, reduced graphene oxide, and AuNPs was prepared using literature research [24], with some changes. Here the electrochemical oxidation and the electrochemically reduction are separately performed to avoid drop casting side effects. The electrochemically oxidation of pyrrole with a potential range of 0 V-0.8 V versus SCE and the morphology of PPy-GO film are shown in Figure 2. Cyclic voltammograms (Figure 2A) illustrated the continuous electrochemical oxidation of pyrrole, and PPy-GO presented a typical wrinkled and crumpled shape ( Figure 2B). As reported in the literature [39,40], pyrrole can be oxidized at 0.8 V. Higher than 0.8 V may potentially lead to the formation of overoxidized polypyrrole films with poor adherence at the electrode surface. The electrochemical reduction of HAuCl 4 and GO with a potential range of −1.5 V-0 V versus SCE, and the morphology of PPy-RGO-AuNPs, are shown in Figure 3. Cyclic voltammograms ( Figure 3A) illustrated the continuous electrochemical reduction of HAuCl 4 and the surface oxygen groups of GO as well as liberating AuNPs and reduced graphene oxide, which was in agreement with previous reports [21,41]. Clearly, the surface morphological feature of PPy-RGO-AuNPs film ( Figure 3B) was remarkably different from the PPy-GO film. Many AuNPs were electrochemically deposited in uniform manner, and only a few AuNPs formed larger clusters. The color of PPy-RGO-AuNPs/GCE was visible through a color change of PPy-GO/GCE from clear black to dark brown. The electrochemical deposition process is a potential method for nanomaterials modified electrodes of various active surfaces without redox reagent and separation procedures [42,43]. Thus, all electrochemically conducting surfaces can be covered by PPy, RGO and AuNPs in uniform manner, which can improve the performance of biosensors. reduction of HAuCl4 and GO with a potential range of −1.5 V-0 V versus SCE, and the morphology of PPy-RGO-AuNPs, are shown in Figure 3. Cyclic voltammograms ( Figure 3A) illustrated the continuous electrochemical reduction of HAuCl4 and the surface oxygen groups of GO as well as liberating AuNPs and reduced graphene oxide, which was in agreement with previous reports [21,41]. Clearly, the surface morphological feature of PPy-RGO-AuNPs film ( Figure 3B) was remarkably different from the PPy-GO film. Many AuNPs were electrochemically deposited in uniform manner, and only a few AuNPs formed larger clusters. The color of PPy-RGO-AuNPs/GCE was visible through a color change of PPy-GO/GCE from clear black to dark brown. The electrochemical deposition process is a potential method for nanomaterials modified electrodes of various active surfaces without redox reagent and separation procedures [42,43]. Thus, all electrochemically conducting surfaces can be covered by PPy, RGO and AuNPs in uniform manner, which can improve the performance of biosensors.
A B reduction of HAuCl4 and GO with a potential range of −1.5 V-0 V versus SCE, and the morphology of PPy-RGO-AuNPs, are shown in Figure 3. Cyclic voltammograms ( Figure 3A) illustrated the continuous electrochemical reduction of HAuCl4 and the surface oxygen groups of GO as well as liberating AuNPs and reduced graphene oxide, which was in agreement with previous reports [21,41]. Clearly, the surface morphological feature of PPy-RGO-AuNPs film ( Figure 3B) was remarkably different from the PPy-GO film. Many AuNPs were electrochemically deposited in uniform manner, and only a few AuNPs formed larger clusters. The color of PPy-RGO-AuNPs/GCE was visible through a color change of PPy-GO/GCE from clear black to dark brown. The electrochemical deposition process is a potential method for nanomaterials modified electrodes of various active surfaces without redox reagent and separation procedures [42,43]. Thus, all electrochemically conducting surfaces can be covered by PPy, RGO and AuNPs in uniform manner, which can improve the performance of biosensors.
A B

The Electrochemical Properties of PPy-RGO-AuNPs Nanocomposite
The electron transfer properties of various modified electrodes (bare GCE, PPy-GO/GCE, PPy-RGO-AuNPs/GCE, PPy-RGO-(AuNPs-GOD) 1 /GCE) were studied using cyclic voltammetry in the KCl solution containing [Fe(CN) 6 ] 3−/4− redox couple with a san rate of 50 mV/s. From the results shown in Figure 4A, the oxidation and reduction peaks were observed at bare GCE, which are related to the electrochemical conversions of Fe (II) to Fe (III) and vice versa. When the GCE electrode was modified with PPy-GO and AuNPs, the peak current of redox had a significant increase, while the peak separation had a decrease compared with bare GCE, confirming the contribution of PPy-GO, RGO and AuNPs. After GOD was adsorbed on PPy-RGO-AuNPs/GCE surface, the resulting PPy-RGO-(AuNPs-GOD) 1 /GCE electrode decreased peak current and increased peak separation, mainly due to the electrical insulating properties of GOD [44].
increase, while the peak separation had a decrease compared with bare GCE, confirming the contribution of PPy-GO, RGO and AuNPs. After GOD was adsorbed on PPy-RGO-AuNPs/GCE surface, the resulting PPy-RGO-(AuNPs-GOD)1/GCE electrode decreased peak current and increased peak separation, mainly due to the electrical insulating properties of GOD [44].
Direct electron transfer (DET) of GOD was investigated at a PPy-RGO-(AuNPs-GOD)1/GCE electrode in PBS using cyclic voltammetry with a san rate of 50 mV/s. As can be seen from Figure 4B, no GOD redox peak corresponding to the conversion between GOD (FADH2) and GOD (FAD) could be observed, indicating that reaction (2)/DET of GOD did not taking place in the present PPy-RGO-AuNPs-GOD-based glucose biosensor. This may be attributed to the fact that the active redox center (FAD/FADH2) of GOD is deeply embedded in a protective protein shell, resulting in the direct electron transfer between GOD and electrode (DET-GOD) being difficult to realize [45]. Although DET is important for the development of electrochemical biosensor, previous studies have reported DET-GOD will lose their native bioactivity owing to the great change in the GOD structure [46,47]. Wooten et al., reported GOD adsorbed on CNT yielded a pair of GOD redox peak, but the anodic peak did not increase in the presence of glucose in an O2-free solution [48]. It has also been demonstrated that DET-GOD is without value in certain mediator-free applications [49]. So the electroactivity of glucose oxidase product H2O2 (Reactions (3) and (4)) was still chosen as the measure of glucose for PPy-RGO-(AuNPs-GOD)n/GCE in the study. Meanwhile, the result further revealed that our proposed method could maintain the structural integrity of glucose oxidase.
A B

Electrochemical Performance of PPy-RGO-(AuNPs-GOD)n/GCE Electrode
The immobilization amount of GOD on the electrode has a significant effect on the biosensor response. PPy-RGO-(AuNPs-GOD)n/GCE electrodes based on the different layers of AuNPs-GOD multilayer films were fabricated by the combination of the electrodeposited AuNPs and self-assembled GOD, and were characterized by cyclic voltammogram in 10mM glucose. As shown in Figure 5, curves 1-5 displayed cyclic voltammograms of 1-5 layers of AuNPs-GOD multilayer films modified PPy-RGO/GCE electrode, respectively. The response current of the resulting glucose biosensors increased with the increase of the layer number of AuNPs-GOD films. From one to three layers, the current approximately uniformly increased with an increase of more than 10 μA, and then the current slowly increased after three layers. So the PPy-RGO-(AuNPs-GOD)4/GCE electrode was chosen for further amperometric investigation. Direct electron transfer (DET) of GOD was investigated at a PPy-RGO-(AuNPs-GOD) 1 /GCE electrode in PBS using cyclic voltammetry with a san rate of 50 mV/s. As can be seen from Figure 4B, no GOD redox peak corresponding to the conversion between GOD (FADH 2 ) and GOD (FAD) could be observed, indicating that reaction (2)/DET of GOD did not taking place in the present PPy-RGO-AuNPs-GOD-based glucose biosensor. This may be attributed to the fact that the active redox center (FAD/FADH 2 ) of GOD is deeply embedded in a protective protein shell, resulting in the direct electron transfer between GOD and electrode (DET-GOD) being difficult to realize [45]. Although DET is important for the development of electrochemical biosensor, previous studies have reported DET-GOD will lose their native bioactivity owing to the great change in the GOD structure [46,47]. Wooten et al., reported GOD adsorbed on CNT yielded a pair of GOD redox peak, but the anodic peak did not increase in the presence of glucose in an O 2 -free solution [48]. It has also been demonstrated that DET-GOD is without value in certain mediator-free applications [49]. So the electroactivity of glucose oxidase product H 2 O 2 (Reactions (3) and (4)) was still chosen as the measure of glucose for PPy-RGO-(AuNPs-GOD) n /GCE in the study. Meanwhile, the result further revealed that our proposed method could maintain the structural integrity of glucose oxidase.

Electrochemical Performance of PPy-RGO-(AuNPs-GOD) n /GCE Electrode
The immobilization amount of GOD on the electrode has a significant effect on the biosensor response. PPy-RGO-(AuNPs-GOD) n /GCE electrodes based on the different layers of AuNPs-GOD multilayer films were fabricated by the combination of the electrodeposited AuNPs and self-assembled GOD, and were characterized by cyclic voltammogram in 10mM glucose. As shown in Figure 5, curves 1-5 displayed cyclic voltammograms of 1-5 layers of AuNPs-GOD multilayer films modified PPy-RGO/GCE electrode, respectively. The response current of the resulting glucose biosensors increased with the increase of the layer number of AuNPs-GOD films. From one to three layers, the current approximately uniformly increased with an increase of more than 10 µA, and then the current slowly increased after three layers. So the PPy-RGO-(AuNPs-GOD) 4 /GCE electrode was chosen for further amperometric investigation.  Figure 6. The applied potential obviously influenced the response current, and the current increased with the increase of applied potential. By considering the sensitivity and selectivity, an applied potential of 0.5 V versus SCE was chosen as the working potential.  After recording a baseline current in a stirred solution, 0.2 mM glucose was first added five times, followed by the addition of 1 mM glucose 12 times. The glucose concentration is from 0.2 mM to 13 mM. As the glucose was added into the stirring buffer solution, the biosensor responded rapidly to the substrates, which achieved 95% of the steady-state current within 5 seconds. With the increase of glucose concentration, the response current increased. From the corresponding calibration curve (Figure 7 inset) to see, the current and glucose concentration The applied potential has an important influence over the electrochemical biosensor performance. The effect of applied potential on the response current of PPy-RGO-(AuNPs-GOD) 4 /GCE electrode was explored by varying the potential from 0.35 V to 0.55 V versus SCE in the 4 mM glucose. The obtain amperometric results are shown in Figure 6. The applied potential obviously influenced the response current, and the current increased with the increase of applied potential. By considering the sensitivity and selectivity, an applied potential of 0.5 V versus SCE was chosen as the working potential. The applied potential has an important influence over the electrochemical biosensor performance.
The effect of applied potential on the response current of PPy-RGO-(AuNPs-GOD)4/GCE electrode was explored by varying the potential from 0.35 V to 0.55 V versus SCE in the 4 mM glucose. The obtain amperometric results are shown in Figure 6. The applied potential obviously influenced the response current, and the current increased with the increase of applied potential. By considering the sensitivity and selectivity, an applied potential of 0.5 V versus SCE was chosen as the working potential.  After recording a baseline current in a stirred solution, 0.2 mM glucose was first added five times, followed by the addition of 1 mM glucose 12 times. The glucose concentration is from 0.2 mM to 13 mM. As the glucose was added into the stirring buffer solution, the biosensor responded rapidly to the substrates, which achieved 95% of the steady-state current within 5 seconds. With the increase of glucose concentration, the response current increased. From the corresponding calibration curve (Figure 7 inset) to see, the current and glucose concentration  added five times, followed by the addition of 1 mM glucose 12 times. The glucose concentration is from 0.2 mM to 13 mM. As the glucose was added into the stirring buffer solution, the biosensor responded rapidly to the substrates, which achieved 95% of the steady-state current within 5 seconds. With the increase of glucose concentration, the response current increased. From the corresponding calibration curve (Figure 7 inset) to see, the current and glucose concentration had a linear relationship where glucose concentration ranged from 0.2 mM to 8 mM (R 2 = 0.99). The linear equation was I (µA) = 0.24 + 0.89C (mM) with an acceptable relative standard deviation of less than 7%. The sensitivity calculated from the linear calibration was 0.89 µA/mM, and a low limit of detection was estimated to be 5.6 µM (S/N = 3). The anti-interference ability and stability were also examined. The effects of the presence of three possible interfering substances ascorbic acid (AA), uric acid (UA), and acetaminophen (AC) were examined to evaluate the selectivity of the proposed biosensor. The interfere current obtained for each interfering substance presented at a concentration of 0.1 mM was compared to that of 4 mM glucose. As shown in Figure 8, a large current reached after glucose addition, and no significant response signals were observed upon successive addition of AA, UA, and AC into the system. The small interfering current may be attributed to two aspects: First, negatively charged AuNPs and glucose oxidase can repulse the negatively charged substance. Second, hydrophobic polypyrrole and reduced graphene oxide can repulse the hydrophobic substance. Hence, there may be a repulsive effect between the PPy-RGO-(AuNPs-GOD)4 mulitlayer films and interfering substances, and it hinders the diffusion of interfering substances toward the electrode surface. On the contrary, H2O2 becomes an uncharged hydrophilic small molecule when compared to these interfering substances, and it can easy diffuse to the electrode surface. To assess stability, PPy-RGO-(AuNPs-GOD)4/GCE was stored at 4 °C after use and measured the current response for 4 mM glucose every two days. The electrode retained 90% of its original current response over two weeks, indicating a good shelf lifetime. This may be attributed to the good stability and biocompatibility of PPy, RGO and AuNPs nanocomposites, and the developed method for PPy, RGO and AuNPs synthesis and GOD immobilization with advantages of the electrodeposition and self-assembly. The anti-interference ability and stability were also examined. The effects of the presence of three possible interfering substances ascorbic acid (AA), uric acid (UA), and acetaminophen (AC) were examined to evaluate the selectivity of the proposed biosensor. The interfere current obtained for each interfering substance presented at a concentration of 0.1 mM was compared to that of 4 mM glucose. As shown in Figure 8, a large current reached after glucose addition, and no significant response signals were observed upon successive addition of AA, UA, and AC into the system. The small interfering current may be attributed to two aspects: First, negatively charged AuNPs and glucose oxidase can repulse the negatively charged substance. Second, hydrophobic polypyrrole and reduced graphene oxide can repulse the hydrophobic substance. Hence, there may be a repulsive effect between the PPy-RGO-(AuNPs-GOD) 4 mulitlayer films and interfering substances, and it hinders the diffusion of interfering substances toward the electrode surface. On the contrary, H 2 O 2 becomes an uncharged hydrophilic small molecule when compared to these interfering substances, and it can easy diffuse to the electrode surface. To assess stability, PPy-RGO-(AuNPs-GOD) 4 /GCE was stored at 4 • C after use and measured the current response for 4 mM glucose every two days. The electrode retained 90% of its original current response over two weeks, indicating a good shelf lifetime. This may be attributed to the good stability and biocompatibility of PPy, RGO and AuNPs nanocomposites, and the developed method for PPy, RGO and AuNPs synthesis and GOD immobilization with advantages of the electrodeposition and self-assembly.

Conclusions
We have developed a convenient and eco-friendly approach for the facile fabrication of the PPy/RGO/AuNPs and GOD immobilization using the electrodeposition and self-assembly. A corresponding glucose enzymatic biosensor based on the PPy-RGO film and AuNPs-GOD multilayer films was developed without redox reagent, separation procedure, crosslinking and adhesive agents. The proposed method is green and will not result in contamination of the enzyme immobilization matrix/active layer. The as-prepared PPy-RGO-(AuNPs-GOD)4/GCE exhibited a satisfied-performance, which would primarily be attributed to the use of PPy, RGO and AuNPs as GOD immobilization matrix, enzyme catalytic specificity and efficiency, and greatly benefit from the combination of the electrodeposition and self-assembly method. Although the biosensor may still suffer from some drawbacks, such as enzymes stability, the proposed method is sufficiently adaptable and scalable due to the above-noted advantages. The resulting biosensor can serve as a model enzymatic biosensing platform for the development of other electrochemical biosensors, which makes a promising application potential in more complex electrochemical detection systems at various electrode surfaces under environmentally friendly conditions.

Conclusions
We have developed a convenient and eco-friendly approach for the facile fabrication of the PPy/RGO/AuNPs and GOD immobilization using the electrodeposition and self-assembly. A corresponding glucose enzymatic biosensor based on the PPy-RGO film and AuNPs-GOD multilayer films was developed without redox reagent, separation procedure, crosslinking and adhesive agents. The proposed method is green and will not result in contamination of the enzyme immobilization matrix/active layer. The as-prepared PPy-RGO-(AuNPs-GOD) 4 /GCE exhibited a satisfied-performance, which would primarily be attributed to the use of PPy, RGO and AuNPs as GOD immobilization matrix, enzyme catalytic specificity and efficiency, and greatly benefit from the combination of the electrodeposition and self-assembly method. Although the biosensor may still suffer from some drawbacks, such as enzymes stability, the proposed method is sufficiently adaptable and scalable due to the above-noted advantages. The resulting biosensor can serve as a model enzymatic biosensing platform for the development of other electrochemical biosensors, which makes a promising application potential in more complex electrochemical detection systems at various electrode surfaces under environmentally friendly conditions.