Amyloid-graphene oxide as immobilization platform of Au nanocatalysts and enzymes for improved glucose-sensing activity

Accepted Manuscript Amyloid-graphene oxide as immobilization platform of Au nanocatalysts and enzymes for improved glucose-sensing activity Xiaochen Wu, Mingjie Li, Zehui Li, Lili Lv, Yan Zhang, Chaoxu Li PII: DOI: Reference:

S0021-9797(16)30935-3 http://dx.doi.org/10.1016/j.jcis.2016.11.058 YJCIS 21786

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

17 October 2016 15 November 2016 15 November 2016

Please cite this article as: X. Wu, M. Li, Z. Li, L. Lv, Y. Zhang, C. Li, Amyloid-graphene oxide as immobilization platform of Au nanocatalysts and enzymes for improved glucose-sensing activity, Journal of Colloid and Interface Science (2016), doi: http://dx.doi.org/10.1016/j.jcis.2016.11.058

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Amyloid-graphene oxide as immobilization platform of Au

nanocatalysts

and

enzymes

for

improved

glucose-sensing activity Xiaochen Wu1, Mingjie Li1, Zehui Li2, Lili Lv1, Yan Zhang3 and Chaoxu Li1,* 1

CAS Key Laboratory of Bio-based materials, Qingdao Institute of Bioenergy and Bioprocess

Technology, Chinese Academy of Sciences, Songling Road 189, Qingdao 266101, P. R. China

2

Beijing Engineering Research Center of Process Pollution Control, Division of Environmental

Technology and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China 3

School of Materials Engineering, Shanghai University of Engineering Science, Longteng Road 333, Shanghai 201620, P. R. China

*Corresponding authors: C. Li ([email protected])

Abstract Two-dimensional GO nanosheets and one-dimensional lysozyme nanofibrils were hybridized through electrostatic interaction to get a novel amyloid-GO composite, which promised a biocompatible immobilization platform for Au nanocatalysts as well as enzymes. The immobilization platform could load a large and tunable amount of Au NPs while maintaining their high catalytic activity. The immobilized catalysts showed high electrochemical behaviors, being ideal as glucose sensing systems. Furthermore, enzymes could also be immobilized on the residual bare surfaces of amyloid-GO, and served by a colorimetric method for a sensitive and selective analytical glucose-detecting platform. The introduction of amyloid fibrils with super large aspect ratios (>103) on GO nanosheets offers an unprecedented possibility of designing and developing novel biomimetic catalysts for broad applications in biotechnology.

Keywords: amyloid nanofibrils; graphene oxide; Au nanoparticles; biocompatible immobilization platform; biosensing.

1. Introduction Catalysis activity and recovery have long been targeted in various industrial reactions for the accelerated reaction rate and possible lowest production cost. Among diverse catalysts, noble-metal nanoparticles (NPs) with high surface/volume ratios have been paid particular attention due to their super catalytic activity and catalytic stability.[1, 2] However, the catalytic behavior of noble-metal NPs is normally affected by their sizes and aggregation states.[3] For example, the highest activity appears with their diameters less than 10 nm.[3, 4] Also, metal NPs with high surface energy are apt to aggregate together and hereby give lower catalytic activities.[5] In addition, there exist specific technical hindrances to recover these nanocatalysts from their reaction medium. Thus, in order to pursue excellent catalytic activity and recovery efficiency, noble-metal NPs are normally required to anchor onto specific supports. In principle, an ideal catalyst-carrier system is able to load a large amount of metal NPs without blocking their catalytic surfaces. Recently, a variety of molecules (e.g. polymers, dendrimers and surfactants) and colloids (e.g. amyloid fibrils) have been tested as the carrier systems.[6-8] Also the two-dimensional (2D) nanomaterials have shown their abilities of loading different metal NPs.[9] In particular, graphene oxide (GO) nanosheets have incredibly large specific surface area (two accessible sides), abundant oxygen-containing functionalities (e.g epoxide, hydroxyl, and carboxylic groups) and high water solubility, endowing great promise for the immobilization of diverse molecule and nanomaterials.[10] In most cases, metal NPs were synthesized on GO surfaces by in situ reduction of metal ions.[11] However, the synthesized Au NPs usually had relative larger sizes (e.g. >20 nm)[11, 12] and thus relative lower surface/volume ratios. In order to load smaller Au NPs, pre-synthesized small Au NPs

were attempted to attach on GO nanosheets.[13] But the pre-synthesized Au NPs were required to be modified by synthetic macromolecules (e.g. polyvinyl alcohol) or positively charged surfactants to ensure noncovalent interactions with negatively charged GO.[14] The GO surfaces were also required to be modified in order to adhere Au NPs.[15] And thus both the requirements increased the difficulty of operation process. On the other hand, although the modification of Au NPs increased the stability of the Au-based catalyst upon recycling, sometimes the protective layer may block the contact between the active site of Au NPs and the reactant, which would lower the activity of catalyst.[16] Recently amyloid fibrils as a building block of functional materials have attracted great interests in biotechnology.[17] Amyloid fibrils are highly ordered, insoluble, self-assembling protein nanostructures often associated with protein misfolding diseases, such as Alzheimer’s disease, Parkinson’s disease and numerous others.[18] As a biocompatible one-dimensional (1D) material, due to their attractive features including ease of self-assembly synthesis, nanoscale dimensions, excellent mechanical properties and amino-acid surfaces, amyloid fibrils offer a natural playground to develop new building blocks.[19] Meanwhile, amyloid fibrils are thermally stable at high temperatures and different pHs, and contain multiple potential ion-binding sites within the amino acid sequences, which enable their usage in metallization reactions under relatively harsh conditions.[17, 20] Thus, amyloid fibrils have been successfully explored for a number of potential applications, such as nanowire production, hydrogels, solid functional organic films, fibrous cell scaffolds, biosensing and bioremediation.[21-23] In this study we show that amyloid GO could form by combining 1D protein nanofibrils and 2D GO nanosheets, and serve as a promising immobilization platform for Au nanocatalysts as well as enzymes. When electrostatically attaching on GO nanosheets, amyloid nanofibrils of lysozyme (with

super large aspect ratios) offered not only large positively charged regions on GO nanosheets, but also specific surface area for binding sites of negative charged Au NPs. An appropriate amount of attached nanofibrils were able to offer GO nanosheets with positive charged regions and yet without breaking their overall negatively charged states and initial properties. This biocompatible immobilization platform could load a large and tunable amount of small Au NPs (~ 6 nm) while maintaining their high catalytic activity. By measuring its electrochemical behaviors, we showed that this platform was highly promising as a high-performance glucose sensing system. Furthermore, considering that soluble enzymes have troubles in separation from reaction medium, horseradish peroxidase (HRP) as a model enzyme was also immobilized on amyloid GO and evaluated by a colorimetric method for a sensitive and selective analytical glucose-detecting platform. This immobilization platform was based on our previous study on functional hybridization of GO nanosheets and amyloid fibrils for shape-memory and enzyme sensing properties.[17, 24] The introduction of amyloid fibrils with super large aspect ratios (>103) on GO nanosheets offers additional possibility of their broad applications in biotechnology.

2. Experimental Section 2.1 Materials Lysozyme, glucose, ascorbic acid and tetrachloroauric acid (HAuCl4, 48% Au basis) were purchased from Sigma Aldrich. Horseradish peroxidase (HRP) was purchased from Majorbio Biotech Company, USA. Other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and used without any further purification. Ultrapure water (18.2 MΩ cm) produced by a Milli-Q system was used as the solvent throughout this work. 2.2 Preparation of immobilization platform with Au nanoparticles and HRP

Amyloid fibrillation: Briefly, lysozyme was dissolved in Milli-Q water and then adjusted to pH 2.0 and 2.0 wt%. Fibrillation was performed by incubating at 60 °C for 96 h under mildly stirring.[25] Synthesis of Au NPs with the diameter of ~6 nm: 0.2 mL HAuCl4 solution (0.025 M) and 10 μL sodium citrate (0.5 M) were added in 20 mL Milli-Q water and stirred at 25 °C for 15 min. The mixture was then transferred to ice-water bath and stirred for another 15 min. 0.6 mL NaBH4 solution (0.1 M) was injected under agitation and kept in ice-water bath for 1 h to get the solution of 6 nm Au NPs. Immobilization of Au nanoparticles: GO were prepared as described in our previous work.[1] 6 mL of GO solution (0.5 mg/mL) was dropped slowly into 4 mL of lysozyme fibril solution (1 mg/mL) under gently stirring at 25 °C, 100 mL of the as-prepared Au NPs solution was then dropped slowly into the mixture and stirred for 30 min. The mixture was then centrifuged at 3000 rmp for 5 min, washed by deionized water three times and redispersed in double distilled water to get the Au@amyloid-GO. Immobilization of HRP: 1 mL of 20 mM phosphate buffer (pH 7.0) that contained HRP (1 mg/mL) was added into 15 mL of Au@amyloid-GO (0.4 mg/mL). The mixture was then shook at 4 °C for 12 h, centrifuged at 3000 rpm for 4 min and washing three times with 20 mM phosphate buffer (pH 7.0) to get the HRP immobilized Au@amyloid-GO. 2.3 Electrochemical measurements Electrochemical measurements were carried out at room temperature and performed on a CHI 660E electrochemical workstation (CH Instrument, Shanghai, China) with a three-electrode cell, in which a saturated calomel electrode was the reference electrode and platinum slice (10 × 10 × 0.1 mm) serves as the counter electrode. A modified glassy carbon electrode (GCE) was prepared by a simple casting procedure as the working electrode: The bare GCE was polished with 0.3 and 0.05 µm aluminum oxide slurries respectively and ultrasonically cleaned with ethanol and double distilled water for 10 min to

remove any contamination. The immobilization system was solution-casted on the surface of GCE and air-dried for 4 h. The cyclic voltammograms were obtained in N2-saturated 0.1 M NaOH at a scanning rate of 50 mV/s in a potential range from −0.6 V to 0.6 V. 2.4 Colorimetric detection of glucose The colorimetric procedure follows the literature procedures with modifications.[26, 27] 0.2 mL of the HRP immobilized Au@amyloid-GO (0.4 mg/mL) was first mixed with 0.7 mL NaAc-NaOH buffer (20 mM, pH 8.5) containing desired amount of glucose (2 mM ~ 80 mM) as substrates. After incubating at 40 °C for 60 min, 60 μL sodium acetate solution (15 wt%) and 40 μL 3,3',5,5'-tetramethylbenzidine (TMB, 0.1 M) were added to the mixture (final pH ~5) and reacted for another 3 min at 37 °C. A UV-visible spectrophotometer was used to detect the reaction kinetics. 2.5 Characterization Field emission scanning electron microscopy (FESEM) measurements were carried out with a JEOL 7401 instrument (JEOL, Japan) operated at 10 kV. Transmission electron microscope (TEM) images were obtained using a JEM-2010 transmission electron microscope (JEOL, Japan) operated at 200 kV. The samples were prepared by mounting a drop of solution on a carbon-coated Cu grid and allowed to dry in the air. Atomic force microscope (AFM) images were taken with an Agilent 5400 AFM. AFM cantilever tips with a force constant of ~ 48 N/m and a resonance vibration frequency of ~ 330 kHz were used and the scanning rate was set at 1 Hz. FT-IR analysis was performed by a Nicolet 6700 FT-IR spectrometer (American) using the KBr pellets method. The UV-vis spectrophotometric analysis was performed on DU800 UV-vis spectrophotometer at 25 °C. All the solutions were diluted to appropriate concentrations and scanned in 1 cm path length quarts cuvettes with the wavelength scan mode between 800-200 nm. The scan speed was 400 nm/min and the sample interval was 1.0 nm.

3. Results and Discussion Morphologies of the as-used GO, lysozyme fibrils and Au NPs were shown in Fig. 1. The uniform contrast of the tapping mode AFM image of GO nanosheets (Fig. 1a) implies that they possess a homogeneous thickness and could well dispersed in H2O. The thickness of GO sheets is about 1.0 nm measured from the crossing section profile curve (the inset of Fig. 1a), revealing the single layered motif. The lysozyme nanofibrils showed a high aspect ratio with the typical length over 2 μm and the width of ~12 nm. Lysozyme nanofibrils were chosen as the model nanofibrils due to their non-branched feature, high aspect ratios, large contour lengths and biocompatibility.[25] Au NPs decorated by sodium citrate showed the diameter of ~ 6 nm with a narrow size distribution (Fig. 1c). Due to the easy routine synthesis and intrinsic catalytic activities, citrate-decorated Au NPs have been extensively studied to catalyze the aerobic oxidation of glucose and produce gluconate and hydrogen peroxide (H2O2).[27] This catalytic reaction was analogous to the reaction catalyzed by glucose oxidase (Gox), indicating that Au NPs could serve as a mimic for Gox.[28] GO has abundant oxygen-containing groups on the surfaces and thus is negatively charged at pH ~7. Nevertheless, lysozyme has the isoelectric point around 11 and is positively charged at pH ~7.[29] Generally, the immobilization platform of amyloid GO could be produced by attaching lysozyme fibrils to GO through electrostatic interactions. An appropriate amount of attached nanofibrils were able to offer GO nanosheets with positive charged regions and yet without breaking their overall negatively charged states and the initial properties, which might be a perfect platform for immobilizing enzymes (Fig. 2a).[10] The amyloid GO platform may immobilize a large and tunable amount of negatively charged Au NPs through electrostatic interactions (labeled as Au@amyloid-GO). As shown in Fig. 2b, Au NPs

were loaded homogeneously along the lysozyme fibrils on GO nanosheets, while not on bare GO surfaces. The loading amount depends on the amounts of attached lysozyme fibrils and Au NPs added. In sharp contrast, lysozyme nanofibrils could load a large amount of Au NPs (labeled as Au@ amyloid). But without the presence of GO, the addition of Au NPs led to the formation of large aggregates of Au NPs (Fig. 2c), indicating the combination of amyloid fibrils and GO offers a promising immobilization platform for Au NPs. In addition, the bare regions on the amyloid GO platform could further immobilize other molecules (e.g. HRP) to afford additional functions. The UV-vis absorption of amyloid-GO showed the typical absorbance of lysozyme at 280 nm and GO at 220 nm (Fig. 3a), and the colloidal Au NPs showed absorbance due to surface plasmon resonance (SPR) around 520 nm, while the formation of Au@amyloid-GO presented the absorbance of both amyloid-GO and Au NPs. Meanwhile, the SPR absorbance of Au@amyloid without GO showed an obvious red shift (Fig. S1), resulting from the aggregation of nanoscale Au NPs, which was in accordance with TEM images shown in Fig. 2. FT-IR was performed to probe the chemical structures of Au@amyloid-GO. As shown in Fig. 3b, upon the combination with GO, the peak intensity of the symmetric N-H stretching band of lysozyme at 1534 cm-1 decreased, while the C-O (alkoxy in GO) stretching peak at 1111 cm-1 increased dramatically.[30] After the immobilization of Au NPs, the peak centered at 1111 cm-1 broadened due to the presence of oxygen containing groups on the surface of Au NPs. In order to evaluate the catalytic activity of Au@amyloid-GO, their cyclic voltammograms (CVs) were first analyzed in N2-staturated NaOH (0.1 M) by adding different concentrations of H2O2 (0.15~2.4 mM). Upon adding H2O2, the abrupt change of CV curves (Fig. S2) suggests that the immobilized Au NPs have intense voltammetric response to H2O2. Moreover, the cathodic current

increased linearly with the H2O2 concentration, indicating that the Au@amyloid-GO have good electrocatalytic activity. In order to sense glucose, the CVs of Au@amyloid-GO were evaluated in N2-staturated NaOH (0.1 M) with the presence of 15 mM glucose, where both the modified glassy carbon electrode (GCE) and Au@amyloid (see Fig. 2c) were given as the control. NaOH was chosen as the electrolyte, as the current response from glucose oxidation was proved to be dependent on both glucose and NaOH concentration.[31] Chemisorbtion of hydroxide ions facilitates the adsorption of glucose to Au NPs, reducing the activation energy for the oxidation of glucose and neutralizing the protons generated during the dehydrogenation steps of the reaction.[32] In Fig. 4a, electron transfer seemed to appear by depositing both Au@amyloid-GO and Au@amyloid on GCE, in contrast to the lack of redox peaks for bare GCE. For both Au@amyloid and Au@amyloid-GO sensors, an oxidation peak appeared at 0.15 V with a shoulder peak at about -0.4 V when potential initially sweeps from -0.6 V to 0.6 V in the presence of glucose. The shoulder peak (-0.4 V) was due to the direct electrochemical oxidation of glucose to gluconolactone, and peak at 0.15 V is attributed to the further oxidation of gluconolactone.[32] In the negative scan, the presence of glucose lead to a peak at 0.02 V, and the current intensity of the reduction peak is smaller than that of the oxidation peak, revealing that the oxidation/reduction of glucose here is a quasi-reversible electrochemical process.[31] Moreover, Au@amyloid-GO has more pronounced redox peaks than Au@amyloid at the same composition of Au NPs, indicating that Au NPs maintained higher catalytic activities on amyloid GO. This can be attributed not only to the better dispersibility of Au NPs on amyloid GO, but also to the excellent electrochemical response of amyloid GO, which thus promoted effective surface areas of the electrode as well as electron transfer rates.[33, 34]

The CVs of Au@amyloid-GO were further evaluated by varying the glucose concentration in Fig. 4b. Due to the super biocatalytic activities of Au@amyloid-GO towards the glucose oxidation, a linear relationship of reduction peak current vs. glucose concentration was revealed in Fig. 4c with the glucose concentration between 0.3~30 mM. In order to get further insight of the catalytic properties of Au@amyloid-GO, differential pulse voltammetries (DPVs) of Au@amyloid-GO were measured with successive addition of glucose up to 15 mM. As shown in Fig 5a, an obvious peak at 0.08 V increased tremendously with the addition of glucose. And the peak current (Ip) showed a good linear relationship with the glucose concentration in the wide range of 0.3~15 mM (Fig. 5b). The corresponding linear regression equations is expressed in the following equation: Ip= 0.045C+0.386, with statistically significant correlation coefficient of 0.994, where Ip is current intensity of the oxidation peak in the DPVs (Fig. 5a) in μA and C is the concentration of glucose in the unit of mM. Moreover, the Au@amyloid-GO sensor was comparable to those previously reported data involving graphene- or Au-related electrodes (Table S1),[31, 32, 35-38] suggesting Au@amyloid-GO could be a promising biosensor to detect glucose. Different potential scan rates and base concentrations were further used to evaluate the electrocatalytic behaviors of this Au@amyloid-GO biosensor (Fig. 6). As shown in Fig. 6a and 6b, both the cathodic and anodic peak currents increased linearly with the scan rate from 10 to 100 mV/s, implying a surface adsorption-controlled process.[39] In Fig. 6c & 6d, the current response from glucose oxidation was dependent on the concentration of NaOH, both the anodic and cathodic peaks decreased proportionally with the base concentration, implying the participation of proton in electrode reaction.[40] OH− could neutralize the protons, the rate to form glucose oxidation intermediate become faster with the increasing of solution alkalinity, so glucose could be easily oxidized with increased

solution alkalinity at relatively low potentials and then current response enhanced, which lead to the peak shift corresponding to NaOH concentration. Besides electrochemical sensing, Au@amyloid-GO could then sense glucose through a colorimetric method with the assistance of HRP and 3,3,5,5-tetramethylbenzidine (TMB). During the sensing process, the Au NPs on Au@amyloid-GO act as nanocatalysts with oxidase-like activity under pH 8.5, catalyzed the glucose oxidation similar to Gox by the co-substrate oxygen (O2) in solution, yielding gluconic acid and H2O2. Amyloid fibrils were the bond between Au NPs and GO, GO was a platform for enzyme immobilization, also offered its mimic activity and large aromatic basal plan structure to accelerate the following TMB oxidation reaction[26]. Then the TMB oxidation was catalyzed by enzymes (e.g. HRP) or GO with H2O2 generated in the reaction under acid environment (pH ~5), resulting in the formation of a characteristic blue product of oxTMB which showed a maximum absorbance at 650 nm and could be monitored in real-time by UV-vis spectroscopy. As show in Fig. 7a, the two steps oxidation procedure occurred with the presence of Au@amyloid-GO alone was not satisfied. Although the peroxidase-like property of GO to directly catalyze the oxidation of H2O2 has been shown in previous works, this mimic activity is relatively low without additional carboxy groups modification or combination with other nanoparticles.[26] To solve this, HRP was brought into the system. Also shown in the Fig. 7a, in the presence of both free HRP and Au@amyloid-GO, the combination of the catalytic activity of HRP and GO lead to a much better result, a linear increasing of UV-Vis absorbance at 650 nm was found with the glucose concentration. Moreover, with better dispersity of Au NPs on amyloid-GO and the synergistic effect with GO, Au@amyloid-GO offered a much higher UV-vis absorbance at 650 nm than GO, Au NPs and Au@amyloid (Fig. 7b), proving its good catalytic activity.

In addition, GO was proved to be an ideal candidate as an enzyme carrier[10], thus the amyloid-GO could serve as a promising immobilization platform for Au nanocatalysts as well as enzymes, because the presence of amyloid fibrils on GO nanosheets did not broke their overall negatively charged states and initial properties for enzyme immobilization. In this way, considering the high cost and poor stability of natural enzymes, the free HRP used in the last step was immobilized on Au@amyloid-GO to form a novel hybrid catalyst to detect glucose through a one-step colorimetric method (see Fig. 2a). As shown in Fig. 7c & 7d, this hybrid catalyst enabled a clear colorimetric response towards glucose, where the UV-Vis absorbance at 650 nm increased linearly with the glucose concentration of 2~80 mM. Besides HRP, other enzymes could be immobilized on Au@amyloid-GO to enzyme-based biosensors as well. Their catalytic activity, stability and reusability rendered them promising bio-catalysts for a variety of applications in simple, robust, cost-effective and easy-to-make biosensors. This catalytic platform of amyloid GO could also inspire to combine different nanocomponents together into organized functional systems.

4. Conclusion In summary, the 2D GO nanosheets and 1D lysozyme nanofibrils were hybridized through electrostatic interaction to get amyloid GO, and constructed a biocompatible immobilization platform for Au nanocatalysts and enzymes without interfering their catalytic activity. The Au@amyloid-GO showed high electrochemical performance in glucose sensing a wide range of 0.3 to 15 mM. Furthermore, HRP as a model enzyme was immobilized on Au@amyloid-GO and built a sensitive and selective colorimetric method for glucose-detecting platform. The good catalytic activity, stability and reusability rendered Au@amyloid-GO a promising bio-catalyst for a variety of

applications in simple, robust, cost-effective and easy-to-make biosensors.

Acknowledgements

Chinese “1000 youth Talent Program”, National Natural Science Foundation of China (No. 21474125 and No. 51602192), Shanghai “Sailing Program” (No. 14YF1409500) and Shandong Collaborative Innovation Center for marine biomass fiber materials and textiles are kindly acknowledged for financial support. We also wish to thank Professor Daoyong Yu of the China University of Petroleum for assistance with the TEM measurements.

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Figure Captions Fig.1 (a) AFM image with height profile of GO sheets; (b) TEM image of lysozyme nanofibrils; (c) TEM image of Au nanoparticles (~6 nm), the insets show magnification of TEM images.

Fig.2 (a) Sschematic representation of the amyloid GO platform fabrication and the immobilization of Au NPs and enzymes; TEM images of (b) Au@amyloid-GO and (c) Au@amyloid.

Fig.3 The (a) UV-vis and (b) FT-IR spectra of Au NPs, amyloid, amyloid-GO and Au@amyloid-GO.

Fig.4 (a) CVs of bare GCE, Au@amyloid/GCE and Au@amyloid-GO/GCE in N2-saturated 0.1 M NaOH in the presence of 15 mM glucose with a scan rate of 100 mV/s; (b) CVs of

Au@amyloid-GO/GCE for successive addition of glucose in N2-saturated 0.1 M NaOH at scan rate of 50 mV/s; (c) Plots of peak currents as a function of concentration of glucose.

Fig.5 (a) DPVs of Au@amyloid-GO/GCE for successive addition of glucose in N2-saturated 0.1 M NaOH at scan rate of 50 mV/s; (b) Plots of peak currents as a function of concentration of glucose.

Fig.6 (a) CVs of Au@amyloid-GO/GCE in N2-saturated 0.1 M NaOH with different scan rates; (b) Plots of cathodic and anodic peak current versus scan rate; (c) CVs of Au@amyloid-GO/GCE in different concentration of NaOH at scan rate of 50 mV/s; (d) Anodic and cathodic peak potentials versus pH.

Fig.7 (a) Dose-response curve for glucose detection using Au@amyloid-GO with or without the help of free HRP; (b) Dose-response curve for glucose detection in the presence of GO, Au, Au@amyloid and Au@amyloid-GO with HRP; (c) Typical glucose concentration response curves using Au@amyloid-GO immobilized HRP; (d) The linear calibration plot for glucose. The inset shows the optical photograph of color change upon the adding of different concentration of glucose.

F .1 ((a) AF Fig. FM imaagee wiith heiightt prrofiile of o GO GO shheetts; ((b) TE EM im magee off ly ysozzym me nnannofibrills; (c) T M imaage of A TEM Au nannoppartticlees ((~6 nm m), tthe inssetss shhow w maagnnificcatiion of TE EM imaagees.

F .2 (a) Fig. ( Ssc S chem mattic repr r reseentatioon of o the am mylooid G GO O platfoorm m faabricationn an nd thhe imm i mobbilizzatiionn of A NP Au Ps and a enzzym mes; TE EM M im magges of (b) ( Au u@aamyyloid-G GO O an nd (c) ( Au A @aamy yloiid.

F .3 T Fig. Thee (a)) UV-v U vis andd (bb) FTF IR speectrra of o A Au NPs N s, am myyloidd, amy a yloiid-G GO annd Au@ A @am mylloidd-G GO.

F .4 (a) Fig. ( CV Vs of o bbaree GCE G E, Au@ A @amyyloidd/G GCE E annd Auu@aamyyloid-G GO O/GCE E inn N2-saaturrateed 0.1 0 M N OH inn the NaO t prreseence of o 155 mM mM gluc g cosee with w h a scan s n rratee of o 100 0 mV mV/s; (bb) CV Vs of A @am Au@ myyloid d-G GO//GC CE for f succcessivve add a ditio on of o gluc g cosee inn N2-saaturrateed 0.1 0 M NaO N OH H at scaan rate r of 5 mV 50 mV/s; (c) Ploots of peaak curr c rentts aas a funnctiionn of connceentrratio on of o ggluccosse.

F .5 (a) Fig. ( DP PVss off Auu@ @am mylooid--GO O/G GCE E fo or ssucccessivee addiitionn of o gluccosee inn N2-saaturrateed 0.1 0 M N OH at scaan rate of 50 mV NaO V/s; (bb) Plots P s off peeak currrennts as a fu uncctionn of o coonccenttrattionn off gluuco ose.

F .6 (a) Fig. ( CV Vs of o Au@ A @am myloid d-G GO//GC CE in N2-sat - turaatedd 0.1 M N NaO OH wiith diff d fereent scaan rate r es; ((b) P ts of Plot o ccath hoddic andd annoddic peaak currrennt vers v sus scaan ratee; (c) ( CV Vs oof Au A @aamyyloiid-G GO/GC CE in d ferent con diffe c ncenntraatioon of o N NaO OH at scaan ratee of 50 mV/ m /s; (d) Annoddic andd cathodiic peak p k ppoteentials v sus pH vers H.

F .7 ((a) D Fig. Dose-rrespponnse currve forr gllucoose detecttionn ussingg A Au@ @am myloidd-GO O with w h orr w withoout thee heelp o free of fr HR RP;; (b) Dose D e-reespoonsse ccurvve for f gglu ucosse ddeteection in the t preesenncee off GO O, A Au,, Auu@ @am mylooid a and A @am Au@ mylloidd-G GO wiith HR RP; (c) ( Ty ypiccal gllucoosee conccenttrattionn resp r ponse cuurvees usiing A @am Au@ myyloid d-G GO imm moobiliizedd HRP H P; (dd) The T e linneaar caalibbrattion n pllot for f gluucose. Thhe innseet shhow ws the t o icall phhoto opti ograaphh off color chaangge uupon thhe add a dingg off diffferrentt coonceenttratiion of gluucosse.

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