Nitrogen-doped carbon nanospheres derived from cocoon silk as metal-free electrocatalyst for glucose sensing

Author’s Accepted Manuscript Nitrogen-doped carbon nanospheres derived from cocoon silk as metal-free Electrocatalyst for glucose sensing Tongtong Li, Yahang Li, Chunyu Wang, Zhi-Da Gao, Yan-Yan Song www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(15)30222-8 http://dx.doi.org/10.1016/j.talanta.2015.08.005 TAL15865

To appear in: Talanta Received date: 13 May 2015 Revised date: 29 July 2015 Accepted date: 2 August 2015 Cite this article as: Tongtong Li, Yahang Li, Chunyu Wang, Zhi-Da Gao and Yan-Yan Song, Nitrogen-doped carbon nanospheres derived from cocoon silk as metal-free Electrocatalyst for glucose sensing, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.08.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nitrogen-Doped Carbon Nanospheres Derived from Cocoon Silk as Metal-Free Electrocatalyst for Glucose Sensing

Tongtong Li,a Yahang Li,a Chunyu Wang,a Zhi-Da Gao,a, b Yan-Yan Song*a

a

b

College of Sciences, Northeastern University, Shenyang 110004, China

Lab of Solid State Microstructures, Nanjing University, Nanjing 210093, China.

Abstract Nitrogen-doped carbon materials have attracted tremendous attention because of their high activity in electrocatalysis. In the present work, cocoon silk- a biomass material is used to prepare porous carbon fibers due to its abundant nitrogen content. The as-prepared carbon microfibers have been activated and disintegrated into carbon nanospheres (CNS) with a diameter of 20-60 nm by a simple nitric acid refluxing process. Considering their excellent electrocatalytic activity towards the reduction of oxygen, the CNS modified electrodes are further applied in the construction of glucose amperometric biosensor using glucose oxidase as a model. The proposed biosensor exhibits fast response, high sensitivity, good stability and selectivity for glucose detection with a wide linear range from 79.7 to 2038.9 M, and a detection limit of 39.1 M. The performance is comparable to leading literature results indicating a great potential for electrochemical sensing application. Keywords: carbonization; carbon nanospheres; oxygen reduction; glucose; biosensor 1

1. Introduction As one of the reactants, dissolved oxygen is usually consumed during the enzymatic reactions. Thus, its detection can be utilized for developing biosensors to determine the activity of oxidases and their substrates [1, 2]. Various nanomaterials based on noble metal such as silver [3], palladium [4], platinum [5, 6] and gold [7] have been used as catalysts for the electrochemical detection of dissolved oxygen. Meanwhile, enormous efforts have been devoted to explore metal-free materials modified electrodes for oxygen reduction [8, 9]. Carbon materials especially in their nano-forms

have

attracted

extensive

attentions

because

of

their

large

surface-to-volume ratio, excellent conductivity, and biocompatibility. The oxidation treatment of carbon nanotubes (CNTs), carbon nanofiber (CNF) and graphene using acid can produce a range of oxygen-rich groups on their surface, which lead to better dispersion and wettability of the materials. In addition, the edge sites produced from the acid treatment accelerate the electron transfer of electroactive analytes, which is the key in preparing effective electrodes [10-12]. Till now, a series of modified electrodes based on CNF [13], CNTs [14] and reduced graphene oxide [15] have been developed for detecting dissolved oxygen. Most of these carbon nanomaterials are expensive and require some special and complex synthesis processes. Thus, developing facile synthesis methods to prepare carbon materials by using low-cost precursors holds great promise in practical applications. Recently, low cost carbon materials derived from biomass materials such as banana fibers [16], waste coffee ground [17], bamboo fungus [18], bacillus subtilis 2

[19], bombyx mori silk [20], fungi [21] and surplus sludge [22] have been explored and studied in energy storage devices. The main advantage of these valuable carbonaceous materials is their precursors, which are easily available, environmental friendly and renewable. In addition, natural materials are typically rich in carbon and nitrogen, and are easy to obtain high surface areas by treating with pore-forming substances [23]. Owing to their high photoluminescence [24], large surface-to-volume ratio and satisfied electric conductivity [25, 26], the formed carbon nanomaterials have been applied in the construction of supercapacitors [27, 28], catalysts or their supports [29], and bioimaging probes [20]. But so far, no report has been focused on application of the carbon materials derived from biomass materials for the reduction of dissolved oxygen. As one of the abundant and environmental friendly biopolymer in nature, cocoon silk has also been used to prepare porous carbon materials by carbonization [30]. Interestingly, the prepared 1D carbon fibers exhibit high BET surface area [31]. Moreover, owning to their rich nitrogen content ~18%, the as-formed carbon microfibers have high doped nitrogen content [32]. In the present study, we have used the porous carbon microfibers derived from cocoon silk to prepare carbon nanospheres (CNS). After treating by hot reflux process in nitric acid, the porous carbon fibers were disintegrated, and resulted in CNS. Because of their intrinsic rich nitrogen content and plenty of oxygen-rich groups produced by refluxing in nitric acid, the obtained CNS show good dispersion, water-solubility and excellent electrocatalytic activity towards dissolved oxygen reduction at a lower operating potential. The modified electrodes are further applied in 3

the construction of glucose amperometric biosensor using glucose oxidase as a model. Considering that large quantities of waste silk are produced in the textile industry [33, 34], and the high catalytic activity towards O2 reduction without using any noble metallic species can be achieved, CNS from silk is a promising electrode material.

2. Experiment section 2.1. Materials and reagents Glucose, Glucose oxidase (GOx) from Aspergillus niger (Type X-S, 100-250 U mg-1 solid), and Nafion were purchased from Sigma-Aldrich Chemicals Co. Other reagents were of analytical reagent grade. All aqueous solutions were prepared with deionized (DI) water. Phosphate-buffered saline (PBS) (0.2 M, pH 7.0) was prepared for assay. The O2-saturated standard solution having 2.6×10-4 M of dissolved O2was produced by bubbling deionized water with pure O2 at room temperature for 1 h [35]. Different concentrations of glucose stock solutions were prepared by using PBS solution and stored at 4 ◦C before use. 2.2. Apparatus The morphologies were characterized by using a field-emission scanning electron microscope (Hitachi FE-SEM S4800) and transmission electron microscopy (TEM, JEOL 2000). X-ray photoelectron spectra (XPS) were recorded on a Perkin–Elmer Physical Electronics 5600 spectrometer. The Raman spectra were recorded by LabRAM XploRA (HORIBA JOBIN YVON S.A.S). Electrochemical measurements were carried out on a CHI730D electrochemical workstation (CH 4

Instrument Co. Shanghai) with a conventional three-electrode system. The glassy carbon electrode (GCE, diameter 3 mm) was used as the support for working electrode. A Pt wire and an Ag/AgCl electrode were used as the counter and reference electrode, respectively. All the potential of electrochemical measurements is here expressed against Ag/AgCl reference electrode. 2.3. Synthesis of soluble CNS Silk cocoons were cleaned using DI water, and treated them in boiling water for 30 min to get the silk. In a typical synthesis, the silk was mixed with ZnCl2 at a mass ratios of 5:1, and treated them in a tube furnace at different temperature (600, 700, 800 and 850 ◦C) for 4 h under Ar gas flow. Subsequently, 100 mg of the as-formed carbon materials was dispersed in 30 % HNO3 and refluxed them at 140 ◦C for 10 h. The resulted supernatant was adjusted to neutral using Na2CO3. The residual ions were detached through a dialysis membrane (MD44MM). The obtained CNS was concentrated by rotary evaporation. For comparison, the products without ZnCl2 activation were also prepared by the same method. 2.4. Preparation of CNS-modified electrode and biosensor of glucose The glassy carbon electrode (GCE, 3 mm diameter) was first polished to mirror finish using fine sandpapers and alumina (particle size of about 50 m)/water slurry. The electrode was thoroughly washed with DI water and then sonication in ethanol and DI water for 10 min. To prepare the CNS-modified GCE electrode, 5 mg of CNS was dispersed into 1 mL 1 % Nafion solution. For the electrochemical measurement, 4 l of CNS–Nafion mixture was dropped onto the surface of a GCE and left it to dry at 5

40 C. Enzyme solution was prepared by dissolving 20 mg GOx in 1.0 mL PBS (0.2 M pH 7.0). To prepare the CNS/GOx modified electrode, the GOx solution was mixed with the 5 mg mL-1 CNS solution at a volume ratio of 1:2. Then, 4 L of the mixture was dropped onto the surface of GCE and dry at room temperature. The as-prepared electrodes were thoroughly rinsed with PBS and stored at 4 C before use. 2.5. Electrochemical measurements All

electrochemical

measurements

were

carried

out

on

a

CHI660d

electrochemical workstation (CH Instruments Shanghai Co., Ltd., China). A three-electrode system was used for all electrochemical measurements: a modified GCE as the working electrode, a platinum foil as the counter electrode and a saturated Ag/AgCl electrode as the reference electrode. Cyclic voltammetric (CV) experiments were carried out in PBS (0.2 M, pH 7.0) with a potential sweep rate of 10 mV s-1. Amperometric measurements were carried out in a stirred PBS (0.2 M, pH 7.0) and the applied potentials for the detection of dissolved oxygen and glucose were -0.2 V and -0.4 V, respectively. Before the measurement, first high purity N2 was bubbled into the detection PBS solution for 30 min. Then, different volumes of O2-saturated solution were spiked into the N2-saturated PBS. For the measurement of glucose, different volumes of glucose solution were injected into the air-saturated PBS. Scheme. 1. 3. Results and discussion 3.1. Characterization of as-prepared carbon materials 6

Fig. 1. Fig. 2. The SEM image in Fig. 1A shows the morphology of untreated cocoon silk. It's a well-defined 1D structure with smooth surface (inset of Fig. 1A). Fig. 1B exhibits the carbon fiber obtained after the carbonization of silk at 800 ◦C. Clearly, the carbon fiber contains hierarchical pores, including micropores (<5 nm) and small mesopores (5−50 nm). After the reflux treatment in nitric acid at 140 ◦C as shown in Fig. 1C, the morphology exhibits obvious changes from fibers (Fig. 1B) to irregular nanospheres. The size of CNS is ranging from 20 to 60 nm. The TEM image in Fig. 1D and particle size analysis in Fig. 2 further confirm this result. The formation of CNS can be attributed to the disintegration of the porous carbon skeleton during the activation treatment in hot acid. Since the carbon atoms on the edges of the pores are more active, they are prone to be oxidized to oxygen-rich groups (i.e. -COOH) and carbon dioxide by refluxing in hot nitric acid for enough time [29]. Fig. 3. To investigate the elemental compositions of CNS, XPS measurements were performed on the CNS sample (prepared by carbonization at 800 C). The survey spectra in Fig. 3A show the presence of C, N, O, and Na (most likely comes from residual Na+ in neutralization process) elements. In Fig. 3B, the high-resolution XPS of C 1s spectra show evident peaks at 284.4, 285.7 and 287.9 eV corresponding to graphite-like carbon (C=C), the secondary carbon (C-N, C-O) and double bond (C=N, 7

C=O), respectively. A raised bump at 289.2 eV in the spectra can be assigned to the COOH and COOR. In Fig. 3C, the O1s signal shows three peakes at 531.2 eV, 532.7 eV, and 535.8 eV, which can be assigned to the O=C, O-C, and H-O-H, respectively [36]. Moreover, the oxygen–carbon atomic ratio of CNS surface was 1.12, suggesting that plenty of oxygen-rich groups are formed on the CNS after refluxing treatment. These hydrophilic oxygen-rich groups lead to the excellent dispersion and water-solubility of CNS. In Fig. 3D, the N 1s spectra exhibit four peaks located at 398.0, 399.0, 399.8 and 400.6 eV, which correspond to the pyridinic, pyrrolic, graphitic and oxidized nitrogen, demonstrating that the different C-N bonding structures are obtained in the sample. The atomic concentration of N is approximately 1.6 %. Comparing with the initial N concentration in cocoon silk [32], this obvious decrease in N concentration could be resulted from the carbonization process due to the denitrogenation, dehydrogenation, and aromatization taking place during the process [37]. Fig. 4. Further, Raman spectroscopy was employed to provide additional insight to the structural properties (Fig. 4). Two dominant peaks around 1590 and 1345 cm-1 corresponding to the G- and D-bands of graphite-like-carbon, are observed for the carbonized CNS samples prepared from 600 to 850 C. The G-band can be used to characterize the sp2 bond structure of carbon atoms, and the D-band is related to the breaking of the symmetry caused by structural disorder and defects [38]. The relative intensity ratio of the D-band to the G-band (ID/IG) is directly proportional to the 8

amount of structural disorder in carbon [39]. The ID/IG of CNS calcined at 800 ◦C is estimated to be 1.14. This value is lower than that of the CNS samples carbonized under other temperatures, i.e. 600, 700 and 850 C. This result implies that the CNS prepared at 800 ◦C have better graphitization degree and less amorphous carbon impurities.

3.2. Electrochemical reduction of dissolved oxygen Fig. 5. To evaluate the application of CNS in electrochemcial reduction of dissolved oxygen, the electrochamical responses of bare GCE and CNS (prepared at 800 C) decorated GCE were compared using their cyclic voltammograms (CVs). As shown in Fig. 5A, the reduction of dissolved oxygen begins at -0.2 V on bare GCE and no reductive peak is obtained at the potential ranging from +0.4 to -0.6 V. In contrast, the CNS modified GCE electrode exhibited the reduction of dissolved oxygen starting at 0.0 V, and reach a reduction peak of -13.5A at about -0.4 V (In Fig. 5B). Moreover, comparing with the graphene oxide (GO) modified GCE electrode (Fig. S1, please see the supporting information), the CNS modified electrode prepared by the proposed technique of the present work demonstrates better electrochemical performance for oxygen reduction. To explore the influence of calcination temperature and ZnCl2 activation on the electrocatalytical activity of CNS, the electrochemical current of different electrodes are compared and summarized in Table 1. It can be noticed that the CNS-modified electrodes show variant electrocatalytic activity for oxygen 9

reduction depending on their preparation conditions. Among all the modified electrodes, the electrode obtained from CNS prepared at 800 C with ZnCl2 activation shows the most positive onset potential for oxygen reduction. On the other hand, the oxygen reduction is found to start at a more negative value when ZnCl2 was not used in calcination process. Since ZnCl2 can promote carbonization with a porous framework, the number of holes on carbon materials increases, and thereby more unsaturated marginal carbon atoms are obtained. Due to the presence of these carbon defect sites, CNS possess more oxygen-rich groups as actively catalytic sites after nitric acid treatment. From these results, the modified electrodes used in the following studies were prepared by using the CNS obtained under 800 ◦C with ZnCl2 activation. Table 1 Table 2 Fig. 6 illustrates a typical current-time curve of the as-prepared CNS-modified GCE electrode at -0.20 V upon successive addition of different volumes of O2-saturated PBS solution into N2-saturated PBS solution. The applied potential was optimized according to the signal to noise ratio when spiking 100 L of O2-saturated solution into 10 mL of N2-saturated solution (listed in Table 2). The CNS-modified electrode achieves a well-defined and step-rise current within 4 s. The response exhibits a linear range from 5.1 to 64.8 M with a correlation coefficient of 0.997, a sensitivity of 82.4 nA cm-2 M-1, and a detection limit of 0.15 M (S/N ≥ 3). The relative standard deviation estimated from the slopes of the calibration plots at six independently CNS-modified electrodes is 3.5 %. This satisfactory reproducibility can 10

be attributed to the high water solubility and homogeneous dispersibility of CNS. It should be also noted that no obvious decrease in the response to oxygen was observed after more than one months of storage. Fig. 6.

3.3. Amperometric response of glucose biosensing Electrocatalytic activity of CNS for dissolved oxygen reduction makes itself attractive for constructing oxidase-based amperometric biosensors. Such biosensors are based on the detection of the reduction signal of dissolved oxygen [40], which is usually consumed when GOx take enzymatic reaction with glucose. Fig. 7. Table 3 Fig. 7A exhibits the amperometric response of the CNS/GOx modified electrode to the successive addition of different concentrations of glucose aliquots at -0.4 V (this detection potential is determined according to the amperometric response in Table 3). The currents respond very rapidly towards the changes in glucose concentration, and reach stable signals within 4 s. As plotted in inset of Fig. 7A, the calibration plot is linear over a broad concentration range of 79.7-2038.9 M glucose with a correlation coefficient of 0.999, a sensitivity of 7.31 nA cm-2 M-1, and a detection limit of 39.1 M (S/N ≥ 3). The performance was rather comparable to leading literature results, which are summarized in Table 4. This finding demonstrates that the present electrode has satisfied detection limit, linear range and sensitivity 11

compared to the earlier reports. For example, it displays better sensitivity than the glucose biosensor based on MWCNT [42, 43] and gold nanoparticles decorated MWCNT [41], and the linear range for the determination of glucose is even wider than CuNP-SWCNT-Nafion/GCE sensing platform [44]. In addition, the response time is better than those obtained for CNF-based glucose biosensor [8], PPy-GOx-MWCNT/GCE [45] and POAP-GOx/FePc-MWCNT [46]. Table 4

The reproducibility on electrode fabrication was investigated by examining six freshly-prepared CNS/GOx modified electrodes in the PBS solution containing 500 M glucose. The relative standard deviation of current signals was 2.2 %. The storage stability of CNS/GOx electrodes was investigated by comparing the amperometric responses before and after storing at 4 C for one month. However, no obvious decrease in catalytic current was observed for glucose sensing. The excellent stability can be ascribed to the good biocompatibility of CNS membrane on GCE for preserving the activity of the enzyme molecules, and also the contribution from Nafion film which can prevent GOx from leaking out of the electrode surface. In a practical determination of glucose, specificity is another important point that requires special attentions. Generally, many coexisting electroactive species in physiological conditions [e.g., ascorbic acid (AA), uric acid (UA) and other carbohydrates such as fructose] can also be oxidized at similar or even lower potentials. Therefore, their interference in glucose detection should be avoided or minimized. Fig. 7B displays 12

the amperometric response of the CNS/GOx modified electrode to the stepwise addition of 0.3 mM glucose in the presence of interfering species including fructose, AA and UA at an applied potential of -0.4 V. The as-prepared CNS/GOx electrode exhibits high selectivity. Using oxidase/CNS enzymatic systems, the detection principle switches from an electrochemical oxidation to a reduction process under such lower operating potential. Meanwhile, the Nafion film also acts as an effective perm-selective membrane for the electroactive interferences [47]. Thus, the selectivity of the device is improved considerably.

4. Conclusions In summary, the present work demonstrates an effective approach to prepare carbon nanospheres (CNS) from the cocoon silk -a kind of available biopolymer, via a calcination process, and a subsequent simple acid reflux treatment. The CNS prepared at 800 C have better graphitization degree and exhibit apparent electrocatalytic activities towards the reduction of dissolved oxygen, which make them as a very promising material that can be applied in constructing GOx-based biosensor for electrochemical glucose sensing. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21322504, 11174046, 21275026), the Fundamental Research Funds for the Central Universities (N140505001, N140504006), and the Program for Liaoning Excellent Talents in University (LJQ2013028). 13

References [1] W. Luo, M.E. Abbas, L. Zhu, W. Zhou, K. Li, H. Tang, S. Liu, W. Li, Analytica Chimica Acta, 640 (2009) 63-67. [2] R.d.C.S. Luz, F.S. Damos, A.A. Tanaka, L.T. Kubota, Sensors and Actuators B: Chemical, 114 (2006) 1019-1027. [3] T.H. Tsai, S. Thiagarajan, S.M. Chen, Electroanalysis, 22 (2010) 680-687. [4] X. Liu, G. Fu, Y. Chen, Y. Tang, P. She, T. Lu, Chemistry – A European Journal, 20 (2014) 585-590. [5] Z. Li, C. He, M. Cai, S. Kang, P.K. Shen, International Journal of Hydrogen Energy, 37 (2012) 14152-14160. [6] Z. Yan, M. Zhang, J. Xie, H. Wang, W. Wei, Journal of Power Sources, 243 (2013) 48-53. [7] G. Fu, Z. Liu, Y. Chen, J. Lin, Y. Tang, T. Lu, Nano Research, 7 (2014) 1205-1214. [8] L. Wu, X. Zhang, H. Ju, Biosensors & bioelectronics, 23 (2007) 479-484. [9] J.-S. Ye, Y. Wen, W. De Zhang, H.-F. Cui, L. Ming Gan, G. Qin Xu, F.-S. Sheu, Journal of Electroanalytical Chemistry, 562 (2004) 241-246. [10] S.-U. Kim, K.-H. Lee, Chemical Physics Letters, 400 (2004) 253-257. [11] L. Li, C.M. Lukehart, Chemistry of Materials, 18 (2005) 94-99. [12] E. Rand, A. Periyakaruppan, Z. Tanaka, D.A. Zhang, M.P. Marsh, R.J. Andrews, K.H. Lee, B. Chen, M. Meyyappan, J.E. Koehne, Biosensors and Bioelectronics, 42 (2013) 434-438. [13] S. Maldonado, K.J. Stevenson, The Journal of Physical Chemistry B, 109 (2005) 4707-4716. [14] Y. Liu, X. Qu, H. Guo, H. Chen, B. Liu, S. Dong, Biosensors and Bioelectronics, 21 (2006) 2195-2201. [15] S.G. Leonardi, D. Aloisio, M. Latino, N. Donato, G. Neri, Dissolved Oxygen Sensor Based on Reduced Graphene Oxide, in: C. Di Natale, V. Ferrari, A. Ponzoni, G. Sberveglieri, M. Ferrari (Eds.) Sensors and Microsystems, Springer International Publishing, 2014, pp. 89-93. [16] V. Subramanian, C. Luo, A.M. Stephan, K.S. Nahm, S. Thomas, B. Wei, The Journal of Physical Chemistry C, 111 (2007) 7527-7531. [17] T.E. Rufford, D. Hulicova-Jurcakova, E. Fiset, Z. Zhu, G.Q. Lu, Electrochemistry Communications, 11 (2009) 974-977. [18] S. Gao, H. Fan, S. Zhang, J. Mater. Chem. A, 2 (2014) 18263-18270. [19] H. Zhu, J. Yin, X. Wang, H. Wang, X. Yang, Advanced Functional Materials, 23 (2013) 1305-1312. [20] Z.L. Wu, P. Zhang, M.X. Gao, C.F. Liu, W. Wang, F. Leng, C.Z. Huang, Journal of Materials Chemistry B, 1 (2013) 2868. [21] H. Zhu, X. Wang, F. Yang, X. Yang, Adv Mater, 23 (2011) 2745-2748. [22] K. Zhou, W. Zhou, X. Liu, Y. Wang, J. Wan, S. Chen, ACS applied materials & interfaces, 6 (2014) 14911-14918. [23] D. Kalderis, D. Koutoulakis, P. Paraskeva, E. Diamadopoulos, E. Otal, J.O.d. Valle, C. Fernández-Pereira, Chemical Engineering Journal, 144 (2008) 42-50. [24] J. Qian, J. Chen, S. Ruan, S. Shen, Q. He, X. Jiang, J. Zhu, H. Gao, Journal of colloid and interface science, 429 (2014) 77-82. [25] D. Liu, X. Zhang, Z. Sun, T. You, Nanoscale, 5 (2013) 9528-9531. [26] G. Panomsuwan, S. Chiba, Y. Kaneko, N. Saito, T. Ishizaki, J. Mater. Chem. A, 2 (2014) 14

18677-18686. [27] T.E. Rufford, D. Hulicova-Jurcakova, K. Khosla, Z. Zhu, G.Q. Lu, Journal of Power Sources, 195 (2010) 912-918. [28] Y.S. Yun, S.Y. Cho, J. Shim, B.H. Kim, S.J. Chang, S.J. Baek, Y.S. Huh, Y. Tak, Y.W. Park, S. Park, H.J. Jin, Adv Mater, 25 (2013) 1993-1998. [29] H. Yang, H. Wang, S. Ji, Y. Ma, V. Linkov, R. Wang, Journal of Solid State Electrochemistry, 18 (2014) 1503-1512. [30] B. Zhang, M. Xiao, S. Wang, D. Han, S. Song, G. Chen, Y. Meng, ACS applied materials & interfaces, 6 (2014) 13174-13182. [31] Y. Liang, D. Wu, R. Fu, Scientific reports, 3 (2013) 1119. [32] Z. H. Xiang, in: Biology of Sericulture, China Forestry Publishing, Beijing, 2005. [33] T. Iwazaki, R. Obinata, W. Sugimoto, Y. Takasu, Electrochemistry Communications, 11 (2009) 376-378. [34] T. Iwazaki, H. Yang, R. Obinata, W. Sugimoto, Y. Takasu, Journal of Power Sources, 195 (2010) 5840-5847. [35] H.X. Ju, C.Z. Shen, Electroanalysis, 13 (2001) 789-793. [36] Z.-H. Sheng, L. Shao, J.-J. Chen, W.-J. Bao, F.-B. Wang, X.-H. Xia, ACS Nano, 5 (2011) 4350-4358. [37] F. Su, Z. Tian, C.K. Poh, Z. Wang, S.H. Lim, Z. Liu, J. Lin, Chemistry of Materials, 22 (2010) 832-839. [38] A.C. Ferrari, J. Robertson, Phys. Rev. B, 61 (2000) 14095-14107. [39] M.S. Dresselhaus, A. Jorio, M. Hofmann, G. Dresselhaus, R. Saito, Nano Letters, 10 (2010) 751-758. [40] Y. Lin, F. Lu, Y. Tu, Z. Ren, Nano Letters, 4 (2003) 191-195. [41] M. Gao, L. Dai, G.G. Wallace, Electroanalysis, 15 (2003) 1089-1094. [42] Y.-C. Tsai, S.-C. Li, J.-M. Chen, Langmuir, 21 (2005) 3653-3658. [43] A. Salimi, R.G. Compton, R. Hallaj, Analytical Biochemistry, 333 (2004) 49-56. [44] K.B. Male, S. Hrapovic, Y. Liu, D. Wang, J.H.T. Luong, Analytica Chimica Acta, 516 (2004) 35-41. [45] J. Wang, M. Musameh, Analytica Chimica Acta, 539 (2005) 209-213. [46] J.-S. Ye, Y. Wen, W. De Zhang, H.F. Cui, G.Q. Xu, F.-S. Sheu, Electroanalysis, 17 (2005) 89-96. [47] D.R.S. Jeykumari, S.S. Narayanan, Carbon, 47 (2009) 957-966.

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Figure captions Scheme. 1. Schematic illustration for preparing monodisperse CNS from cocoon silk. Fig. 1. SEM images of A) cocoon silk (inset: the amplified image), B) carbon fibers obtained from calcination of silk with ZnCl2 at 800 C, C) CNS obtained by refluxing carbon fibers in nitric acid), and D) TEM image of CNS. Fig. 2. Size distribution of CNS calcined at 800 C A) before and B) after nitric acid treatment. Fig. 3. A) XPS survey spectra of CNS prepared at 800 C, and the details of B) C 1s, C) O 1s and D) N 1s peaks. Fig. 4. Raman spectra of CNS prepared at different calcination temperatures. Fig. 5. Cyclic voltammograms of A) bare GCE and B) CNS-modified GCE electrode in N2-saturated PBS (curve a) and O2-saturated PBS (curve b) at a scan rate of 0.01 V s−1. Fig. 6. Amperometric response at the CNS-modified electrode for successive addition of different volumes of O2-saturated PBS solution into N2-saturated PBS solution at an applied potential of -0.20 V. Upper inset: time response of adding 120 L of O2-saturated solution. Lower inset: corresponding linear calibration curve. Fig. 7. A) Current-time response of the CNS/GOx-modified electrode upon successive addition of glucose into a stirring air-saturated PBS at -0.40 V. Upper inset: time response of adding 50 M glucose. Lower inset: corresponding linear calibration curve. B) Amperometric response of the CNS/GOx-modified electrode to the addition of 0.3 mM glucose, fructose, AA and UA at -0.40 V. 16

Scheme 1.

17

Fig. 1

18

A

B

Fig. 2 (black-and-white in print)

19

O 1s

A

Raw C=C C-O(N) C=N(O) COOR

B

Intensity / a.u.

Na 1s

Intensity / a.u.

Na Auger

C 1s N 1s

1000

800

600

400

200

294 292 290 288 286 284 282 280 278 276

Binding Energy / eV

D

Raw H-O-H O-C O=C

Intensity / a.u.

C

538

0

Binding Energy / eV

536

534

532

530

528

Raw Oxidic N Pyridinic N Pyrrolic N Graphitic N

Intensity / a.u.

1200

526

Binding Energy / eV

404

402

400

398

396

Binding Energy / eV

Fig. 3 (black-and-white in print)

20

394

Intensity / a.u.

o

ID/IG=1.46

850 C

ID/IG=1.14

800 C

ID/IG=1.46

700 C

ID/IG=1.44

600 C

o

1200

o

o

1300

1400

1500

1600 -1

Raman shift / cm

Fig. 4 (black-and-white in print)

21

1700

Current / A

0

-2

A a

-4

-6

b

-8 -0.6

-0.4

-0.2

0.0

0.2

0.4

Potential / V (vs.Ag/AgCl)

0

B

Current / A

a -4

-8

-12

b -0.6

-0.4

-0.2

0.0

0.2

Potential / V (vs.Ag/AgCl)

Fig. 5

22

0.4

-0.800

50 L

-0.816

80L

-0.7

within 4 s

-0.808

Current / A

20 L

-0.6

-0.824 -0.832

530

-0.6

-0.8

-0.9

-1.0

532

534

536

538

540

542

Times / s

200 L

-0.7

Current / A

Current / A

120 L -0.840

-0.8

-0.9

-1.0

-1.1 0

20

-1.1 0

40

O2 concentration / M

200

400

60

600

Times / s

Fig. 6 (black-and-white in print)

23

800

1000

-0.4 -0.6

-1.36 -1.38 -1.40 -1.42

-1.0

426

428

430

432

434

Times / s

436

100 M

-1.2 -0.8

-1.4

Current / A

Current / A

-0.8

within 4 s

-1.34

Current / A

A

50 M

-1.2

20 M

-1.6

-1.6 0

-1.8 0

200

400

500

1000

1500

Concentration / M

600

2000

800

1000

200

250

Times / s -0.4

B Current / A

-0.6

glucose

-0.8 fructose glucose

-1.0

AA

UA

-1.2

-1.4 0

50

100

150

Times / s Fig. 7 (black-and-white in print)

24

Calcination temperature (◦C)

600

700

Initial reduction potential (V)

-0.14

-0.12

800

800 (without ZnCl2)

850

0

-0.16

-0.12

Table 1. The onset reduction potentials of dissolved oxygen on CNS modified GCE. The CNS were calcined at different conditions.

25

a

Detection Potential (V)

-0.1

-0.15

-0.2

-0.25

-0.3

-0.4

S/N

/a

5.00

30.25

9.88

7.14

3.39

/: no obvious signal to noise ratio

Table 2. The signal to noise ratio of amperometric response when spiking 100 L of O2-saturated PBS into 10 mL N2-saturated PBS solution.

26

a

Detection Potential (V)

-0.1 V

-0.2 V

-0.3 V

-0.4 V

-0.5 V

S/N

/a

11.24

12.80

16.69

12.84

/: no obvious signal to noise ratio

Table 3. The signal to noise ratio of amperometric response when spiking 50 L of 0.1 M glucose to an air-saturated PBS solution.

27

Electrode

Sensitivity -1

(mM )

Linear range

Response time

(mM)

(s)

Reference

CuNP-SWCNT-Nafion/GCE

256 μA

0 – 0.5

10

44

PPy-GOx-MWCNT/GCE

2.33 nA

0.2 – 50

15

45

GOx-SGC/MWCNT/bppg

196 nA

0.2 – 20

<5

43

GOx-Nafion-MWCNT/GCE

330 nA

0 – 0.002

<3

42

PPy-GOx-MWCNT array/Gold

350 nA

2.5 – 20

-

41

POAP-GOx/FePc-MWCNT

735 nA

0.0005 – 4

<8

46

CNF-GOx/Nafion

2.56 μA

0.01 – 0.35

<10

8

CNS/GOx-Nafion

516 nA

0.08 – 2.04

4

Present work

Table 4．The electrochemical behaviours of different carbon nanomaterials modified electrodes in glucose sensing.

Highlights 

Cocoon silk was used as the biomass material to prepare carbon materials;



Porous carbon microfibers were disintegrated to Carbon nanospheres (CNS) by hot acid treatment;



The as-preared CNS exhibited highly electrocatalytic activity towards O2 reduction;



The CNS/GOx modified electrodes demonstrated as metal-free glucose sensors.

28

29