YANG Jinghe ，YANG Duo，LI Yamin

（1.College of Chemistry and Chemical Engineering，Henan University，Kaifeng475004，Henan，China；2.College of Chemistry and Molecular Engineering，Peking University，Beijing100871，China；3.Xinlian College（Faculty of Science），Henan Normal University，Xinxiang453007，Henan，China）

As we all know，metal nanostructures have been widely applied in many different fields such as cataly－sis，electronics，adsorption，life sciences，medicine and aerospace owing to their unique properties compared with bulk metals［1］.Many traditional physical and chemical methods have been explored to synthesize nanostructures.However，these methods are energy intensive or not environmentally benign，which makes it imperative to develop cleaner and non－toxic technologies for synthesizing nanostructures［1］.Recently，the use of microbial biomass in preparing nanoparticles（denoted as NPs）has emerged as one of the research highlights in nanotechnology［2］.For example，actinomycetes，bacteria，fungi，and even bacteria have been used to prepare metal NPs such as Au［3－4］，Pd［5－6］and Ag［7－9］.It has been demonstrated that enzymes of live microorganisms can catalyze the reduction from metal ions to NPs［10］.However，the main drawback of enzyme－catalyzed reduction is that as－synthesized NPs are usually formed in the cell periplasm，even if some NPs exist on the cell surface，and the biological substrate has poor stability and conductivity.These would limit the application scope of the NPs，especially in catalysis and electrochemistry.Carbon material is a class of carrier with excellent stability and conductivity.More importantly，in the course of the synthesis of supported metal nanostructures，especially NPs，the dispersion of metal salt precursor on the support is very important.Volumetric impregnation is a widely used method.However，this method is time－consuming and unsuitable for the synthesis of large quantities of materials.

We anticipate that microbial biomass can be combined with calcining to prepare carbon supported metal NPs.Namely，microbial biomass can be adopted to prepare supported NPs from the dispersion of expected metal salt precursor，and then the carbon supported metal NPs can be prepared by calcination the microbial biomass precursor containing metal.Herein，we report a facile method for the synthesis of carbonsupported gold NPs（AuC）by 2hcalcination of yeast－biosorption－Au species（AuY）at 1 273Kin N2.The electrochemical performance of as－prepared AuC in the electro－oxidation and determination of dopamine（denoted as DA）is also reported.

1 Experimental

1.1 Reagents

Nafion（5%ethanol solution，mass fraction）was purchased from Alfa Aesar and diluted to 0.1%（mass fraction）with doubly distilled water in use.Chloroauric acid（HAuCl4·3H2O），yeast extract powder，glucose and other agents were purchased from Sinopharm Chemical Reagent Co.，Ltd.All stock solutions were prepared with deionized water（resistance not less than 18.2MΩ·cm）.

1.2 Synthesis of various target products

10g of yeast extract powder，10g of glucose and 10mL of chloroauric acid solution（10g·L－1）were dissolved in 300mL of deionized water in an Erlenmeyer flask.Then the flask was put into a shaker and shaked at 310Kfor 3dand 6d，respectively.AuY species were separated by filtration and dried in an air oven at 310Kovernight.AuY species obtained after 3dof reaction and 6dof reaction are denoted as AuY－3and AuY－6，respectively.

AuC was obtained by calcining AuY in a tubular furnace under nitrogen atmosphere.The temperature rose at the rate of 10K·min－1until 1 273Kand then held there for 2h.AuC samples obtained from AuY－3and AuY－6are marked as AuC－3and AuC－6，respectively.

1.3 Characterization

The morphologies of various products were observed with a scanning electron microscope（SEM，XL30S－FEG，5kV）and a transmission electron microscope（TEM，FEI Tecnai G2T20）.To－be－tested products were seperately dissolved in ethanol under ultrasonic irradiation，and then a drop of resultant solutions was put onto a Cu grid providing samples for TEM analysis.

1.4 Preparation of modified glassy carbon electrode（GCE）

The glassy carbonelectrodes（GCEs，3mm diameter，Tianjin Aida，Inc.）were polished withα－Al2O3powder（40nm），rinsed twice by deionized water and ethanol，and then dried at room temperature.1mg of as－ynthesized catalyst was dispersed in 1mL of 0.1%nafion solution.Then the mixture was dropped on a pre－treated GCE to fabricate a modified GCE.Finally the modified GCEs were dried under infrared light.

1.5 Electrochemical measurements

Electrochemical experiments were performed at 298Kwith a CHI660Delectrochemical workstation（CHI，Shanghai）equipped with a three－electrode setup.GCE or modified GCEs acted as working electrode；saturated calomel electrode（SCE）was used as reference electrode，and platinum wire as the counter electrode.

2 Results and discussion

AuC hybrid was developed by a facile two－step method（Fig.1A）.Firstly，yeast as the organism precursor vector for dispersing metal was mixed with glucose and HAuCl4aqueous solution for 3dand 6dat 310Kin a shaker，generating AuY－3and AuY－6，respectively.Then AuY－3and AuY－6were calcined at 1 273Kunder N2atmosphere affording AuC－3and AuC－6，respectively.At the beginning，HAuCl4would be slowly adsorbed onto yeast in the mixture.Some HAuCl4would be reduced by the catalysis of enzymes of yeast besides the direct reduction by glucose［1］.The yeast would grow and divide over time.The absorbed gold and HAuCl4would be dispersed with the growth of yeast and the glucose was slowly consumed.As a result，AuY－3is obtained（Fig.1B）.The size of yeast in AuY－3is not uniform because of the absorption and package of plenty of glucose and the presence of many yeast spores.After 6d，most of the glucose is consumed and the yeast is able to fully grow and multiplicate.Thus，the number and density of yeast are larger than that of AuY－3（Fig.1C）.The dispersion of gold would be also higher than that of AuY－3.When AuY is calcined at high temperature under a nitrogen atmosphere，the yeast is carburized and gold is completely reduced；and gold NPs have no significant accumulation because of the protection of yeast（Fig.2A）.Though AuC－3and AuC－6exhibit the same amount of gold，the gold NPs of AuY－6have better dispersion than those of AuY－3.In other words，the size of gold NPs of AuC－6is smaller than that of AuC－3，and the number of gold NPs of AuC－6is greater than that of AuC－3 （Fig.2Band 2C）.As shown in Fig.2D，the size of gold NPs of AuC－6is about 10nm.We suppose that such differences in the number and size of gold NPs should influence the electro－catalytic activity of relevant carbon－supported gold catalyst，which is to be discussed in the forthcoming section.

Fig.1 Scheme of preparation of AuC－3and AuC－6（A）as well as SEM images of AuY－3（B）and AuY－6（C）

Fig.2 TEM images of AuC－3（A）and AuC－6（B，C and D）

Fig.3Ashows the steady－state cyclic voltammograms（CVs）illustrating electro－oxidation of DA.It can be seen that the anodic peak current of DA at the bare／GCE shows a small anodic peak with a peak potential of 0.35V，whereas two times larger anodic peak current and a marked reduction of the anodic peak potential about 80mV compared to unmodified bare／GCE are observed for AuC－6／GCE.AuC－6／GCE also presents well－defined and quasi－reversible redox peaks corresponding to the electrochemical reaction of DA，which well corresponds to its good electro－catalytic activity.These phenomena are clear evidences of catalytic effect of the chemically modified electrode towards DA oxidation.In terms of the anodic peak current and anodic peak potential，AuC－6／GCE exhibits better electrochemical performance（Fig.3B）.The effect of AuC could have a relationship with the size and dispersion of gold NPs on AuC.CVs of DA over AuC－6／GCE are depicted in Fig.3Cwith the scan rate ranging from 20to 300mV／s.The relation between redox peak currents and the scan rates implies a hybrid kinetic mechanism.At lower scan rates from 20to 130 mV·s－1，the electrochemical redox behavior of DA over the AuC－6／GCE surface is a diffusion－controlled process，while at higher scan rates（＞ 160mV／s）it is a surface absorption－controlled process with the current peak being proportional to the scan rate.This means that the reaction occurs not only at reactive sites within the adsorbed assembly but also at the outer surface of the electrode［11－14］.In other words，Au and C constitute a stable composite and AuC－6is a good and sensitive sensor for DA.Square wave voltammogram （SWV）of DA was carried out with AuC－6／GCE （Fig.3D）.The SWV amperometric currents show a prefect linear relationship with the concentration of DA in the range of 5－50μmol·L－1and 60－100μ·L－1，with the detection limit being 1.5μmol·L－1at a signal－to－noise ratio of 3.This means that the AuC－6／GCE electrode exibits high effectiveness in accurate determination of DA.

Fig.3 CVs（A and B）in the presence of phosphate buffer（PBS）solution（0.2mol·L－1）containing 1.0×10－3 mol·L－1 DA at pH＝7.4and 50mV·s－1；CVs on AuC－6／GCE in a 0.2mol·L－1 PBS solution containing 1.0×10－3 mol·L－1 DA at pH ＝7.4and different sweep rates（C）.Linear relationship between peak current versus the square root of the scan rate（scan rate＜130mV·s－1）and the scan rate（scan rate＞160mV·s－1）（C，inset）；Square wave voltammogram （SWV）for AuC－6／GCE in 0.2mol·L－1 PBS solution containing the DA （concentrations from 5to 100 μmol·L－1 at pH ＝7.4）（D）；Linear calibration curve for the determination of DA（D，inset）

3 Conclusions

Carbon－supported gold NPs hybrids were prepared by calcination precursor AuY obtained through mixing and shaking of yeast，glucose and HAuCl4solution at a pre－set temperature for different durations.The feasibility of as－prepared AuC catalyst in the electrochemical detection of dopamine was investigated.It has been found that the present method is significant for the synthesis of carbon－supported metal NPs.As－prepared AuC catalyst exhibits excellent activity at low peak potential，showing potential in the investigation and diagnosis of dopamine－related diseases.

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