Ni@NiO核-壳纳米球的合成及其电化学生物传感应用研究

彭 娟 李 晔 季鹏宇 姜立萍 朱俊杰

(南京大学生命分析化学国家重点实验室，南京大学化学化工学院，南京 210093)

Ni@NiO核-壳纳米球的合成及其电化学生物传感应用研究

彭 娟 李 晔 季鹏宇 姜立萍＊朱俊杰

(南京大学生命分析化学国家重点实验室，南京大学化学化工学院，南京 210093)

利用无模板水热法合成了镍纳米球，并通过部分氧化制备了Ni@NiO核壳结构的纳米复合物。合成的镍球和Ni@NiO复合物的尺寸可以通过简单调节反应条件来控制。运用XRD、EDS、TEM和SEM等测试方法对合成样品的形貌和组成进行了表征。Ni和Ni@NiO复合材料均有较好的磁性，其磁性用磁滞回线进行了表征。此外，Ni@NiO纳米复合物可以和血红蛋白结合构建过氧化氢生物传感器，该生物传感器对过氧化氢表现出很好的生物电催化活性，且具有较低的检测限和较宽的线性响应范围。该复合材料对于血红蛋白催化还原过氧化氢具有米氏响应和较小的米氏常数，表明Ni@NiO能较好地保持血红蛋白原有的活性。

Ni@NiO；核壳结构；纳米球；血红蛋白；生物传感器

Nanometer-sized magnetic particles have attracted considerable attention because oftheir unusual properties and potentialapplicationsin magnetic resonance imaging,biomedicine,data storage,catalysis, bioseparation and so on[1-3].Nickel(Ni)is one of the magnetic metals that exhibits interesting properties and applications,such as magnetic storage media[4-5],fuel cell electrodes[6-8]and catalysis[9-10].However,Ni nano-materials which are easy to be oxidized to nickel oxide (NiO),rarely have been utilized in biochemistry independently due to the lack of biochemical activity.Meanwhile,NiO nanomaterials have received considerable attention in recent years due to their wide applications in catalyst,battery cathode,gas sensors,electrochromic films[11-13].Particularly,NiO nanostructures have potential applications in biosensor since they have high chemical stability,excellent electrocatalytic property, and high electron transfer capability[14].

Because of the wide application,the synthesis of Ni and NiO nanomaterials have draw more and more attention[15-18].Ni nanomaterials have also been synthesized through electrochemical reduction, high temperature organometallic decomposition,chemical reduction and microwave assisted method[19-22].Hydrothermal method has been proved to be a fast,simple and effective route for synthesis of nanostructured materials which depends on the solubility of minerals in hot water under high pressure.Furthermore,the shape and size of these nanomaterials could be controlled by simply adjusting the parameters such as temperature, the reaction time,the ratio of the reactants.Herein,the hydrothermal method was used to synthesize ferromagnetic Ni nanospheres with different sizes.

The Ni and NiO nanocomposies can combine the advantages of both ferromagnetism and good electrocatalytic activity together,which can open up the multiple functional applications of them.Currently,the synthesis of Ni@NiO core-shell nanomaterials were limited probably because of the difficulty in reducing the Ni2+into metallic nickel through a liquid chemical process using common reducing agents.Only a few literatures have reported the synthesis of Ni@NiO coreshell nanomaterial[23-24].For instance,Li et al.prepared Ni/NiO core-shell nanoparticles by complex precipitation of Ni precursors in glycerol and thermal decomposition of the dried precipitate[23].Lee et al.reported the synthesis of Ni@NiO core-shell nanoparticles and application in protein separation and purification[24].However,these works always involved complicated steps and pretreatment.Therefore,a facile way to make Ni@NiO nanocomposites is still a great challenge and highly desirable.

In this work,we reported a facile method to synthesize size-controlled Ni nanospheres and Ni@NiO core-shell nanocomposites through hydrothermal method.The Ni@NiO nanocomposites exhibited some improved properties such as excellent electrochemical bioactivity,ferromagnetic property,and good dispersity in water,which made them attractive in applications for bioseparation and biosensor.Herein,Hb was assembled on the prepared nanocomposites and was further employed to fabricate a novel biosensor for the determination of H2O2.

1 Experimental

1.1 Materials and apparatus

Nickel chloride,NaH2PO2·H2O and 30%H2O2solution were purchased from Sinopharm Chemical Reagent Co.,Ltd.All chemicals were of analytical grade and used without further purification.Phosphate buffer solution(PBS,0.1 mol·L-1pH 7.0)was prepared by mixing stock standard solutions of Na2HPO4and NaH2PO4.

XRD patterns were obtained with a Philips X′pert Pro X-ray diffractometer(Cu K radiation,λ=0.154 18 nm).EDS analysis was carried out by using a SEM equipped with an energy-dispersive X-ray detector (Shimadzu,SSX-550).SEM and TEM images were taken on a LEO-1530VP field-emission scanning electron microscope and a FEI Tecnai-12 microscope with an accelerating voltage of 120 kV,respectively.Magnetic measurements were performed on a Quantum Design MPMS SQUID magnetometer at room temperature.

1.2 Synthesis of Ni nanospheres and Ni@NiO core-shell nanospheres

0.1 mmol nickel chloride and 0.6 mmol KOH were dissolved separately in 5 mL of water and mixed together,then 5 mL NaH2PO2·H2O (～0.6 mmol)was added under the nitrogen atmosphere.The mixture was transferred into Teflon-lined autoclave of 15 mL capacity.The autoclave was sealed and maintained at 140℃for 40 min.After cooled to room temperature, the black precipitates were rinsed with deionized water and ethanolrespectively by magnetic separation. Finally,the Ni nanosphere was produced after dried at 60℃for 4 h.Ni/NiO nanocomposites could be obtained by heating the as-prepared Ni nanospheres at 400℃for 1 h in the air.

1.3 Immobilization of Hb and construction of biosensor

Electrochemical measurements were performed on a CHI 630 electrochemical workstation(Chenhua, Shanghai,China)with a conventional three-electrode system.A platinum wire was used as the auxiliary electrode,and a saturated calomel electrode(SCE)was the reference electrode.Electrochemical experiments were carried out at room temperature.

For the assembly of Hb,2 mL Hb(5 mg·mL-1)was mixed with 2 mL Ni@NiO nanocomposites(≈1.4wt%). Then the mixture was equilibrated for 24 h at room temperature,then rinsed and separated with an external magnet.The obtained Hb-Ni@NiO nanocomposites were used for further electrochemical test.Glass carbon electrode (GCE)was polished with 1.0,0.3,and 0.05 μm alumina powder successively,followed by successive sonication in acetone and water and dried at room temperature.The Hb-Ni@NiO nanocomposites were suspended in 1 mL water,and 10 μL of the suspension was dropped onto the surface of GCE and then dried in desiccator.Finally,the Hb-Ni@NiO modified electrode was stored at 4℃for the following test.

2 Results and discussion

2.1 Characterizations of Ni nanospheres and Ni@NiO nanocomposites

Fig.1 (A)XRD pattern of the Ni nanospheres(a),Ni@NiO nanocomposites(b)(B)EDS data for the Ni@NiO nanocomposits; SEM images of Ni nanospheres(C)and Ni@NiO nanocomposites(D)(insert:TEM image of Ni@NiO nanocomposites)

XRD was used to characterize the Ni nanospheres and Ni@NiO nanocomposites.First,Ni nanospheres wereprepared by hydrothermalmethod.Allthe reflection peaks of prepared Ni are assigned to Ni(PDF No.04-0850) (curve b in Fig.1A).After the partly oxidation process of Ni nanospheres,Ni@NiO nanocomposites were obtained.The newly-appeared diffraction peaks located at 37.28°,43.26°and 62.88°, corresponded to the (111),(200)and (220)lattice planes of NiO,respectively.This result agreed well with face-centered cubic NiO(PDF No.78-0643)(curve a in Fig.1A).The as-prepared Ni@NiO nanocomposites also have been characterized by EDS,which indicated that the products were of high purity(Fig.1B).

The hysteresis loops at room temperature was used to study the magnetic properties of the as-prepared Ni@NiO nanocomposites.The result indicated that all the particles had the symmetric hysteresis loops behavior of ferromagnetic materials(Fig.2A).Most of the hydrothermally synthesized Niand Ni@NiO nanospheres aggregated and precipitated within a few minutes,however,they can be redispersed in water by vigorous shaking or sonication to form a clear darkbrown-colored dispersion.Moreover,they can be easily separated by using an external magnet,as shown in Fig.2B.

Fig.2 (A)A hysteresis loop showing ferromagnetic properties of Ni and Ni@NiO nanospheres; (B)Pictures showing magnetic response of prepared Ni@NiO nanoparticles

2.2 Controlled synthesis of Ni@NiO nanocomposites

Since Ni@NiO nanocomposites were obtained by the oxidization of Ni nanospheres,the size of the nanocomposites depended on the size of Ni nanospheres.Among the widely used reducing agents for electroless Ni plating,NaH2PO2·H2O was found to be unique for the formation of Ni nanospheres in the present synthesis system.Ni nanospheres were obtained by deoxidized NiCl2using NaH2PO2as reducing agents in the present of KOH.The redox reaction during the hydrothermal process could be formulated as[25]

Table 1 Reaction parameters to prepare Ni nanospheres with different sizes

To investigate the influence of the reaction parameters and obtain the resulted nanostructures with different sizes,various reactions were carried out with differentconcentrations ofreagentand different reaction temperatures.Table 1 shows the different reaction conditions to synthesize different samples which were depicted in Fig.3.The concentration of the reductive agent had an obvious effect on the size of obtained Ni nanospheres.In general,increasing the concentration of the reducing agent improves the reduction rate of metal ions,leading to smaller metal nanoparticles[26],as shown in Fig.3.In addition, heightening the reaction temperature,larger sized, better dispersed and more uniform products could be obtained according to the SEM images from sample 4 to 6 in Fig.3,which was probably attributed to that the high temperature can accelerate the reaction velocity to form larger particles.

Fig.3 SEM images of different samples under reaction condition(see Table 1)

Fig.4 SEM images of the Ni nanospheres synthesized using hydrothermal method with the reducing agent concentration of 0.04 g·mL-1for different reaction time:(A)40 min,(B)1 h,(C)2 h,(d)10 h

In order to investigate the growth process of the Ni nanospheres,the evolution of the morphology in the hydrothermal synthesis was studied.The SEM images of these samples are shown in Fig.4.When the reaction time was 40～60 min,Ni nanospheres with uniform, perfect spherical shape and small sizes were obtained.Therefore,in the present system,the product with perfect morphology could be achieved under the reaction time of 40～60 min.When the reaction time was prolonged,the product grew larger and became abnormity(Fig.4C and 4D),which might be attributed to the Ostwald ripening process[27].The size of Ni nanospheres can be controlled by adjusting the reaction conditions such as the concentration of reducing agent and reactive temperature to obtain Ni nanospheres with different sizes.Besides,Ni@NiO core-shell nanocomposites were obtained by partly oxidation of Ni nanospheres.

The thickness of the nanocomposite shell could be adjusted by controlling the oxidizing time and temperature of Ni nanospheres.In this way,the controlled synthesis of ferromagnetic Ni nanospheres and Ni@NiO core-shell nanocomposites can be realized successfully.

2.3 Electrochemical application of Ni@NiO nanocomposites

2.3.1 Direct electrochemistry of Hb-Ni@NiO

The electrochemical behaviors of the Hb-Ni@NiO modified electrode in the absence and presence of H2O2were studied by cyclic voltammetry.No redox response was observed on Ni@NiO modified GCE for H2O2in the potential range from 0.1 V to-0.8 V (curve a in Fig.5A).After the assembly of Hb on the Ni@NiO modified electrode,a pair of stable redox peaks appeared in curve b Fig.5A.The anodic and cathodic peaks were located at-0.307 V and-0.413 V (vs SCE)respectively.As shown in Fig.5A (curve c),the reduction peak current was greatly enhanced after the addition of H2O2,which further indicateed that Hb was successfully immobilized and still retained their electrochemical activity.

The dependence of the peak currents on the scan rate was also investigated.As shown in Fig.5B(inset), the cathodic and anodic peak currents increased linearly with the scan rate from 50 to 300 mV·s-1, indicating that the Hb adsorbed on the surface underwentasurface-controlledelectrontransferprocess.According to Faradays law (Q=nFAΓ),where Q is the total amount of charge,n is the number of electron transferred,F is Faraday′s constant,and A is the electron area,the average Γ values of electroactive Hb was estimated to be 6.36×10-10mol·cm-2,which was much larger than the theoretical monolayer coverage of Hb (≈1.89×10-11mol·cm-2).This indicated that a multilayer of proteins participated in the electrontransfer process in the composites.The electron transfer rate constant (ks)between Hb and electrode was estimated to be (2.8±0.3)s-1according to Laviron′s method[28],indicating that the Ni@NiO nanocomposite was an excellent promoter for the electron transfer between Hb and the electrode.

2.3.2 Determination of hydrogen peroxide

Fig.5 (A)Cyclic voltammograms of Ni@NiO/GCE(a),Hb-Ni@NiO/GCE(b),Hb-Ni@NiO/GCE with 50 μmol·L-1H2O2(c) in 0.1 mol·L-1pH 7.0 PBS solution;(B)Cyclic voltammograms of Hb-Ni@NiO/GCE in PBS(0.1 mol·L-1,pH 7.0) at different scan rates(from inner to outer curve:50,100,150,200,250,300 mV·s-1),and (inset)plots of cathodic and anodic peak currents vs Scan rates

Fig.6A shows the cyclic voltammograms for Hb-Ni@NiO modified GCE in PBS(pH 7.0)in the present of H2O2with different concentration.The reduction peak at approximately-0.30 V was greatly enhanced, while the anodic peak decreased,suggesting that an electrocatalytic reduction of H2O2occurred.Moreover, the reduction current increased dramatically with the increasing concentration of H2O2,and the anodic peak led tothegradualdisappearancesimultaneously.However,this phenomenon was not observed on Ni@NiO modified GCE,therefore,the catalytic reduction of H2O2was only due to the presence of Hb.The peak current increased with the concentration of H2O2(Fig.6B),and the linear regression equation was I (μA)=0.0121C(μmol·L-1)+0.045,with a correlation coefficient of 0.997 and a detection range of 1.3～710 μmol·L-1.From the slope of 0.0121 μA·μmol-1·L,the detection limit of the biosensor towards hydrogen peroxide was estimated to be 0.4 μmol·L-1at 3σ.

When the concentration of H2O2was higher than 600 μmol·L-1,a platform emerged in the catalytic peak current,showing the characteristics of Michaelis-Menten kinetics.The apparentMichaelis-Menten constant (Kmapp),which gives an indication of the enzyme-substrate kinetics,can be obtained from the Lineweaver-Burk equation[29]:

Where,Issis the steady-state current after the addition of substrate,C is the bulk concentration of substrate and Imaxis the maximum current measured undersaturated substrate solution.Kmappcan be obtained by the analysis of slope and intercept of the plot of the reciprocals of the steadystate current versusH2O2concentration.The Michaelis-Menten constant of the system(Kmapp)was found to be 1.07 mmol ·L-1,which was smaller than the previous reports[23], implying that the prepared Ni@NiO nanocomposites had good biocompatibility and could retain the original enzymatic activity of Hb.The reproducibility of the biosensor was estimated by determining same concentration of H2O2for five replicate measurements with relative standard deviations (RSD)in the range from-4.2%to 4.8%.The result indicated that the biosensor show satisfactory reproducibility.The stability of the prepared biosensor was investigated.The modified electrode was stored in phosphate buffer solution at pH 7.0 in the refrigerator at 4℃ for a week and no obvious change was found.The biosensor retained 90% of itsoriginalresponseafterone month.

Fig.6 (A)Cyclic voltammpgrams of the Hb-Ni@NiO/GCE at scan rate of 0.1 V·s-1in 0.1 mol·L-1pH 7.0 PBS solution with(a)0,(b)187.0,(c)317.0,(d)497.0 μmol·L-1H2O2;(B)Plots of the electrocatalytic current(i)vs H2O2 concentration

3 Conclusions

Ni@NiO nanocomposites have been prepared by a simple template-free hydrothermal synthesis of Ni nanospheres and a subsequent oxidation process.The core-shell nanostructure showed good magnetic property,satisfactory biocompatibility and chemical stability.Hb was further assembled on the Ni@NiO composites to construct a novel H2O2biosensor with a wide linear range and a low detection limit. Additionally,Ni@NiO enhanced the direct electron transfer between Hb and GCE and retained the native activity of Hb.The proposed method provided a new and simple strategy towards the fabrication of Ni and Ni@NiO nanospheres.The prepared Ni@NiO core-shell nanostructure would probably provide a novel and promising platform forthe construction ofother biosensor in the future.

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Synthesis of Ni@NiO Core-Shell Nanospheres and Application for Fabrication of Electrochemical Biosensor

PENG Juan LI Ye JI Peng-Yu JIANG Li-Ping＊ZHU Jun-Jie
(State Key Laboratory of Analytical Chemistry for Life Science,School of Chemistry and Chemical Engineering,Nanjing University,Nanjing 210093,China)

Ni nanospheres have been successfully synthesized through a template-free hydrothermal method. Ni@NiO core-shell nanocomposites were obtained by a subsequent oxidation of Ni nanospheres.The size of the final product could be controlled via simply adjusting the experimental parameters.The morphology and structure of the Ni@NiO nanocomposites were confirmed by transmission electron microscopy (TEM),field emission scanning electron microscopy(FESEM),energy-dispersive X-ray spectrometry(EDS)and X-ray diffraction(XRD). The hysteresis loops were used to study the magnetic properties of Ni and Ni@NiO.Additionally,hemoglobin (Hb)was assembled on the Ni@NiO composites to construct a novel biosensor for the determination of H2O2.The prepared biosensor showed an excellent electrocatalytic activity towards H2O2with a wide linear range and a low detection limit.The lower Michaelis-Menten constant indicated that the Hb immobilized on the Ni@NiO nanocomposites could retain its native activity.

Ni@NiO;core-shell;nanospheres;hemoglobin;biosensor

We greatly appreciate the support of National Natural Science Foundation of China (No.21075061) and the Natural Science Foundation of Jiangsu Province of China (BK2010363).We also appreciate the support of Jinchuan Group Co.,LTD.

O613.71

A

1001-4861(2012)06-1251-08

2012-04-23。收修改稿日期：2012-05-07。

国家自然科学基金(No.21075061)和江苏省自然科学基金(No.BK2010363)资助项目。

＊通讯联系人。E-mail：jianglp@nju.edu.cn，Tel：025-83597204