Oriented Assembly of Zinc Oxide Mesocrystal in Chitosan and

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Oriented Assembly of Zinc Oxide Mesocrystal in Chitosan and Applications for Glucose Biosensors Shuyan Zhao, Bo You, and Linlei Jiang Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00337 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 11, 2016

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Crystal Growth & Design

(Cover Page) Oriented Assembly of Zinc Oxide Mesocrystal in Chitosan and Applications for Glucose Biosensors Shuyan Zhao, † Bo You , *,†Linlei Jiang*,‡ †

Department of Materials Science and the Advanced Coatings Research Center of the China

Educational Ministry, Fudan University, Shanghai 200433, PR China ‡

Shanghai Jiaotong University school of Agriculture and Biology and Shanghai Food Safety

Engineering Research Center, Shanghai 200240, PR China ABSTRACT. By imitating biomineralization, a facile synthetic method to fabricate oriented self-assembly zinc oxide mesocrystal in chitosan (CS-ZnO) with different morphology is presented. Chitosan acts as a structure-directing agent to guide the formation of ZnO nuclei and the self-assemble preferential growth from small building units to larger structures through Coulomb force and the adjustment of surface energy. The prepared CS-ZnO mesocrystals have been successfully used in the glucose sensors. The products obtained were characterized with FESEM, XRD, TEM, FTIR, TGA and EDX. By comparing the sensitivities of biosensor fabricated by different shape CS-ZnO structures, the starfish-like CS-ZnO mesocrystal with a grain size of 20-30 nm showed a better sensing performance due to its mesocrystal structure, good biocompatibility and specific architecture.

†

Name: Bo You, Address: 220 Han Dan Road, Shanghai, 200433, China Phone Number: 0086-21-55664033 Fax Number: 0086-21-55664033 E-mail: [email protected] Web address: http://www.fudan.edu.cn/

‡

Name: Linlei Jiang, Address: 800 Dong Chuan Road, Shanghai, 200240, China Phone Number: 0086-21-34206625 Fax Number: 0086-21-34206625 E-mail: [email protected] Web address: http://www.sjtu.edu.cn/

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Oriented Assembly of Zinc Oxide Mesocrystal in Chitosan and Applications for Glucose Biosensors Shuyan Zhao, † Bo You , *,†Linlei Jiang*,‡ †

Department of Materials Science and the Advanced Coatings Research Center of the China

Educational Ministry, Fudan University, Shanghai 200433, PR China. Email: [email protected] ‡

Shanghai Jiaotong University school of Agriculture and Biology and Shanghai Food Safety

Engineering Research Center, Shanghai 200240, PR China. Email: [email protected]


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ABSTRACT. By imitating biomineralization, a facile synthetic method to fabricate oriented self-assembly zinc oxide mesocrystal in chitosan (CS-ZnO) with different morphology is presented. Chitosan acts as a structure-directing agent to guide the formation of ZnO nuclei and the self-assemble preferential growth from small building units to larger structures through Coulomb force and the adjustment of surface energy. The prepared CS-ZnO mesocrystals have been successfully used in the glucose sensors. The products obtained were characterized with FESEM, XRD, TEM, FTIR, TGA and EDX. By comparing the sensitivities of biosensor fabricated by different shape CS-ZnO structures, the starfish-like CS-ZnO mesocrystal with a grain size of 20-30 nm showed a better sensing performance due to its mesocrystal structure, good biocompatibility and specific architecture.

Introduction Biological systems control the nucleation and growth of nanocrystals in exact shapes and sizes with high efficiency and accuracy via a biomineralization process.1 It is reasonable to make efforts to study and mimic the biomineralization process.2,3 Numerous kinds of organic matrix (polysaccharide,4 collagen,5 amino acids,6 etc.) were used as templates to simulate biomineralization. Chitosan is one important template in biomineralization process7-9 and owing to its nontoxicity and bio-compatibility, which was applied for biosensor, adsorbent, drug delivery, wound dressing and antimicrobial, etc.10 As a biomacromolecule with hydroxyl and amino groups, chitosan can control the morphology and crystal phase of produced mineral during biomineralization.11 With anticipation to combine the functions of zinc oxide and chitosan,


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herein we studied the formation process of zinc oxide mesocrystal in a chitosan matrix and their biosensing property. Mesocrystal is a superstructure of crystalline nanoparticles with external crystal faces which can be learned from the process of biomineralizition.12 In nature, mesocrystal is not only single crystals with intricate superstructure morphology but also made up of nanoparticle subunits interspaced by organic additives as defects.13,14 Mesocrystal has large surface area, large overall assembly size, high degree of crystallinity which makes it promising in many areas like energy storage, catalysts, optoelectronics, biomedical applications, etc.15, 16 Nowadays, most studies focus on the growth process of mesocrystal but the reason for the oriented assembly of nanoparticles is still lack of understanding. In addition, more applications of mesocrystal are also need to be explored Zinc oxide (ZnO) is a semiconducting material with good conductivity and biocompatibility.17 It has broad applications in solar cell, sensor, photocatalyst and antibacterial agent.18 ZnO has a high isoelectric point (IEP) of about 9.5 which can combine well with some enzymes with low IEP via electrostatic force in a neutral condition. In addition, ZnO has high electron transfer rate and good biocompatibility that makes it significant and promising in biosensors applications. At present, many kinds of ZnO structures like nanotubes19, nanofibers20 and forklike nanostructures21 have been used as enzyme immobilization materials in biosensors. However, few studies focused on the biosensor application of ZnO mesocrystal are reported to our knowledge. In this work, we report an oriented assembly of zinc oxide mesocrystals made up by building blocks with an average size of 25 nm in chitosan matrix and provide a possible mechanism of the


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preferential growth of chitosan/zinc oxide mesocrystal. By controlling the reaction conditions, zinc oxide mesocrystals in chitosan (CS-ZnO) with different morphology are synthesized. CSZnO with good biocompatibility and mesocrystal structure has a uniform and distinctive starfishlike shape, leading to a better sensing performance. Experimental Section Materials. Glucose oxidase (Aspergillus niger, 10 units/mg) was purchased from Sigma. Zinc acetate (Zn(Ac)2·2H2O), acetic acid (HAc, 99.5%), chitosan (deacetylation value≥90%), potassium hexacyanoferrate (III) (K3[Fe(CN)6]), potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6]·3H2O), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4·12H2O), sodium chloride (NaCl), potassium chloride (KCl), nano zinc oxide, sodium hydroxide (NaOH) were purchased from Guoyao Chemical Reagent Co. (China) Phosphate buffer solution (PBS, 0.01 M) was prepared by mixing 1.56 g of Na2HPO4·12H2O, 0.2 g of KH2PO4, 8 g of NaCl and 0.2 g of KCl in deionized water and then the pH was adjusted to 7.4. All chemicals are analytical-grade regents and all of them were used without further purification. The glassy carbon electrodes was purchased from Yancheng Jiaoyuan analytical instruments Co. (China) Synthesis of Chitosan/Zinc Oxide (CS-ZnO). In a typical synthesis of CS-ZnO, 0.03 g of chitosan was dissolved in 30 mL of deionized water containing 0.2 mL acetic acid. 0.082 g of zinc acetate [Zn(Ac)2·2H2O] as dissolved in 30 mL of deionized water and then the solution was added into the above chitosan solution under vigorous stirring. Then the mixed solution was adjusted by 5 mol/L sodium hydroxide solution (pH = 10.0) and a gel was obtained. The collected gel was transferred into a Teflon-lined autoclave of 100 mL capacity. The autoclave


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was heated to 100 °C for different time. Finally after being cooled to room temperature, the product was collected and washed several times with deionized water dried at 80 °C for 15 h. To obtain the calcinated CS-ZnO nanostructures, the above products were calcinated at 800 °C for 5 h to remove the excess chitosan. Preparation of GCE/CS-ZnO/GOx based glucose sensor. The glassy carbon electrodes (GCE 3 mm diameter) were polished by 0.5 and 0.05 µm aluminum slurries and then were dipped into 1:1 (V/V) aqueous solution of HNO3 and ethanol, deionized water respectively under ultrasonication. 1 mg of as-prepared CS-ZnO nanostructures was dispersed in 3 mL of ethanol under ultra-sonication for 1 min to obtain a uniformly dispersed CS-ZnO suspension. 10 µL of the above suspension was dropped onto the surface of GCE and dried at room temperature. The treated GCE was then modified by dropping 10 µL of GOx solutions (1 mg/mL in 0.01 M PBS) and dried at room temperature. Then 10 µL of 0.5 wt% chitosan solution dissolved in 0.05 M acetic acid buffer solution (pH = 5.0) was dropped onto the surface of modified GCE to avoid the leakage of the enzyme. Finally the prepared GCE/ CS-ZnO /GOx electrodes were dried and stored at 4 °C in a refrigerator when not in use. Results and Discussion Structure and Morphology. The X-ray diffraction (XRD) analysis of products obtained by hydrothermal process at different reaction time was shown in Figure 1. The marked diffraction peaks observed for all of the products after hydrothermal treatment matched bulk wurtzite hexagonal ZnO (JCPDS 36-1451). The characteristic peak of chitosan (2θ = 20°)22 was obvious before hydrothermal treatment. After hydrothermal treatment, the characteristic peak could also be observed in all CS-ZnO samples (for example, Inset in Figure 1) but was very weak compared


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to that of ZnO. No other characteristic peaks of impurities were observed. R(002)/(101) represented the ratio of diffraction density of (002) planes and (101) planes.23 The values of R(002)/(101) were 0.66, 0.80, 0.87, 0.91, 0.76 and 0.68 when reactions were carried out for 1, 3, 9, 18, 24 and 48 h. By comparing XRD patterns of different reaction time, the values of R(002)/(101) increased as the hydrothermal time increased before 18 h and the value of R(002)/(101) for the sample of 18 h was highest. This phenomenon might be due to the preferential growth of ZnO along the (001) plane during early period of hydrothermal process, but it needed to be justified in the following sections. After hydrothermal treatment for 18 h, the values of R(002)/(101) decreased probably owing to the collapse of CS-ZnO morphology. The average grain sizes of samples were calculated from XRD results. The grain sizes of 1, 3, 9, 18, 24 and hydrothermal treatment for 48 h CS-ZnO samples were 34.7, 26.3, 27.4, 27.4, 25.4 and 26.9 nm, respectively.

Figure 1. XRD patterns of chitosan and CS-ZnO samples obtained with different hydrothermal treatment time: (a) chitosan, (b) 0 h, (c) 1 h, (d) 3 h, (e) 9 h, (f) 18 h, (g) 24 h, (h) 48 h. The inset


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shows the amplified XRD patterns within the blue dotted line circle with regions from 15 ° to 25 °.

Figure 2 presented the effect of reaction time on the morphology of the CS-ZnO products. Before hydrothermal synthesis (Figure 2a), plenty of sheet precipitation was observed and some nanoparticles were dispersed on the precipitation. After hydrothermal treated for 1 h (Figure 2b), a large number of amorphous particles appeared and some spindle-like structures were find. When the reaction time was 3 h (Figure 2c), the amorphous particles disappeared and the size of spindle-like CS-ZnO structures increased. And there was a junction interface in the middle part. Then some small half-spindle petals began to grow at the junction interface after 9 h reaction (Figure 2d). As the reaction time increased to 18 h (Figure 2e), CS-ZnO spindles were completely disappeared, producing a great deal of starfish-like CS-ZnO structures which had exceedingly rough and particle-assembled surfaces. But after treated for 24 h (Figure 2f), the structures began to collapse and became loose. When the reaction time reached 48 h (Figure 2g), the size and number of starfish-like CS-ZnO structures decreased and lots of CS-ZnO spindles reappeared. All the FESEM images matched the changing trends of R(002)/(101) in XRD results and which implied preferential growth along (001) plane could lead to the spindle-like and finally the starfish-like CS-ZnO morphology. Figure 3h showed the high magnification SEM image of starfish-like CS-ZnO structures which had exceedingly rough and particle-assembled surfaces. From the morphological images of products at different stage, the growth process of CS-ZnO structures from nanoparticles to spindle to starfish-like morphology was observed. And it seemed that the nanoparticles serving as building blocks were tightly packed in spindle and starfish-like shape.


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Figure 2. FESEM images of samples obtained with different hydrothermal treatment time: (a) 0 h CS-ZnO, (b) 1 h CS-ZnO, (c) 3 h CS-ZnO, (d) 9 h CS-ZnO, (e) 18 h CS-ZnO, (f) 24 h CSZnO, (g) 48 h CS-ZnO, (h) high magnification of 18 h CS-ZnO.

Analysis of the Nanostructures Growth. The FTIR spectrum of the samples obtained at different condition was shown in Figure 3. For pure chitosan (Figure 3a), a broad absorption band attributed to the stretching vibration of –OH groups appeared at around 3430 cm-1 and the stretching vibration of C-H was at 2877 cm-1. The bands at 1094 and 1032 cm-1 were assigned to


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the stretching vibration of C3-O and C6-O bonds of chitosan.24 The characteristic bands of 1656 and 1599 cm-1 belonged to the stretching vibration of C=O and the scissoring vibration of –NH2, respectively.25 Compared with pure chitosan, the absorption band corresponding to the O-Zn-O group (470-500 cm-1) could be observed (Figure 3b, 3c, 3d, 3e) indicating the existence of ZnO. Besides, the relative absorbance integrals ratio AC-O/C-H of C-O (an integral of the C-O band in the range between 1140 and 1000 cm-1) and C-H (an integral of the C-O band in the range between 3000 and 2800 cm-1) represented as a criterion of C-O bond intensity. After adding zinc acetate (Figure 3b), the characteristic peak of C-O was shifted to a lower wavenumber of 1073 cm-1 and the bond intensity became stronger (AC-O/C-H increased from 0.58 to 0.69), which indicated the strong interaction between C-O group and Zinc ion. By hydrothermal method, typical peaks of chitosan could also be found in the FTIR spectrum (Figure 3c, 3d) and the peaks of C-O bonds were also shifted to lower wavenumber and were stronger (AC-O/C-H = 0.92 and 0.81, respectively) compared to that of pure chitosan (AC-O/C-H = 0.58). It could be concluded that CS-ZnO is hybrid chitosan-ZnO nanostructures and the reason of the formation was due to the interaction between C-O group and ZnO.


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Figure 3. FTIR spectra of: (a) chitosan, (b) CS-ZnO obtained before hydrothermal treatment, (c) 3 h CS-ZnO, (d) 18 h CS-ZnO, (e) calcinated 18 h CS-ZnO.

To further investigate the formation mechanism and the element composition of products, pure chitosan and CS-ZnO were analysed by TGA, XPS as shown in Figure 4. The TGA analysis performed under air was shown in Figure 4a. Below 200 °C, the loss of weight was due to the release of free water. For pure chitosan, a tremendous weight loss between 250 and 600 °C owing to the decomposition of chitosan appeared and the final weight loss was around 98 wt% within measuring range. For CS-ZnO composite, the weight loss rate was slower than pure chitosan owing to the interaction between chitosan and ZnO. The total weight loss at 800 °C was 47 wt%, so the content of ZnO in CS-ZnO was estimated to be about 50 wt%. The pure chitosan and products were tested by XPS as shown in Figure 4b, 4c and 4d. Figure 4b showed the general XPS of pure chitosan and CS-ZnO. There were new peaks of Zn in CSZnO structures and the atomic ratio of CS-ZnO sample was calculated from XPS spectra. The C/O/N/Zn atomic ratio was 70.88:24.90:2.15:2.08. The ZnO content in the CS-ZnO was estimated to be about 12% from XPS which was smaller than that analyzed by TGA. It was seen from the above results that the CS-ZnO was hybrid nanostructures. Moreover, the ZnO was more likely to assemble in the internal of the products and chitosan tended to distribute in the surface of the products. Figure 4c presented the C1s XPS of pure chitosan, CS-ZnO. For pure chitosan, a large C1s located at 284.6 eV was due to organic C-C, C-H bonds and the peaks at 286.6 eV could be attributed to C-O bonds.26,27 For CS-ZnO, a new peak at 288.5 eV appeared which could be


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assigned as the contributions from inorganic carbonate species (i.e. C-O and C=O bonds)28 which was probably due to the interaction between Zinc ion and C-O group. The O1s XPS was shown in Figure 4d. For pure chitosan, the peak at 532.0 eV could be attributed to the C-O-H bonds.29 For CS-ZnO, the peak of O1s shifted from 532.0 eV to 533.0 eV, indicating that the electron density of O atoms was less than that in chitosan. This implied that the coordination of O atoms to zinc ions which was consistent to the FTIR results. There was also a new peak at 530.9 eV due to the O2- ions in the ZnO lattice indicating the formation of ZnO.30

Figure 4. (a) TGA analysis (b) The general XPS spectra, (c) C 1s XPS spectra, (d) O 1s XPS spectra of chitosan and CS-ZnO.

The structures of CS-ZnO were analyzed by HRTEM as shown in Figure 5. Figure 5a showed the starfish-like shape of the 18 h CS-ZnO. Figure 5b revealed that the tip of the CS-ZnO structures consisted of smaller building blocks and had very rough surfaces, which supported the conclusion from FESEM images. Figure 5c showed the HRTEM image of the tip region of the


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CS-ZnO structures. There was one single nanoparticle building unit within a red dotted line circle in this image and the grain size was 20 – 30 nm which was consistent with the XRD results. The spacing between adjacent lattice planes was about 0.26 nm, which corresponded to the distance of (001) planes, indicating that [001] (c axis) was the growth direction of the CS-ZnO products. Figure 5d presented the corresponding SAED pattern of CS-ZnO. The diffraction pattern indicated that the CS-ZnO products was of single crystal and could be indexed as the hexagonal ZnO. It was suggested that the products consisted of nanoparticle building blocks had nearly the same crystallographic orientation, which could be attributed to the self-assembly of these building blocks. The phenomenon was similar to the reported mesocrystal.12

Figure 5. (a) TEM images of 18h CS-ZnO, (b) TEM images of the tips of CS-ZnO (c) highresolution TEM (HRTEM) lattice image of CS-ZnO nanostructures, (d) the selected area SAED pattern of (b). One single building block is within a red dotted line circle.


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Growth Mechanism. Based on the investigations and results above, a possible schematic illustration of the CS-ZnO structures self-assembly was presented in Figure 6. In aqueous solution without chitosan, the concentration of Zn2+ and OH- ions reached the critical value and ZnO nuclei could be formed. However, the insufficient quantity of the growth unit of [Zn(OH)4]2- limited the growth of ZnO and no additive restricted the preferential growth of ZnO, only the irregular ZnO nanoparticles (Supporting Information, Figure S1) could be seen. It is reported that chitosan form gels in alkaline solutions.11 In the presence of chitosan gels, ZnO nuclei could be adsorbed easily on these surfaces of long chain. As is known, crystal growth in gels occurs under quite high supersaturation which can lead to increased nucleation of small clusters – the building blocks for mesocrystals.12 Therefore the concentration of Zn2+ and OHions could easily exceed the critical value and large numbers of ZnO nuclei were formed and developed into many building blocks. In these experiments, there were large quantities of column-shaped building blocks in the structures from FESEM and TEM images. And then the O atoms in the C-O group of chitosan bound to the Zn2+ ions in ZnO via Coulomb force31 and ZnO were distributed preferentially on the connection sites (Figure 6b). It seemed that the chitosan might bond onto the side of the (001) plane, redistributed the surface energy of building units and directed the preferential growth of the (001) plane (Figure 6c). From the above, the columnshaped building blocks for CS-ZnO structures growing along the same 3D crystallographic orientation due to the strong binding linked selectively with chitosan could be observed. The diffusion of the mesocrystal building units was significantly lowered in gels,12 so the growth velocity could be controlled and the time-resolved structure formation process of CSZnO structures (Figure 6d) could be observed. As soon as the primary building units were formed, they assembled to develope larger and more stable structures. First, the column-shaped


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building blocks preferentially grew along the (001) plane owing to the surface energy redistribution effect of chitosan and formed half-spindle structures. Then there were some small building units assembled on the bottom of half-spindle structures and later they grew larger, made up spindle-like twinned structures and yielded a junction in the middle. Hence the redundant ZnO building blocks might be assembled on the center junction at which the defect density was highest32 and continued growing along the [001] direction to form other half-spindle petals. Finally, CS-ZnO mesocrystal nanostructures of starfish-like morphology appeared. However, the structures would collapse if the hydrothermal treatment time was too long due to the decrease of thermal stability of chitosan.


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Figure 6. Schematic illustration of the CS-ZnO structures self-assembly process. (a) Schematic graph of chitosan chains, (b) C-O groups of chitosan chains binding with zinc ions of ZnO nuclei via Coulomb forces, (c) the change of surface energy guiding the preferential formation of zinc oxide, (d) morphological evolution of the CS-ZnO structures. (Each scale bar in FESEM images is 200 nm)

Bio-sensing Properties. Prepared CS-ZnO structures were used for glucose biosensing assay. EDX chemical composition analysis of modified electrode was analyzed before and after the enzyme immobilization (Supporting Information, Figure S2). After enzyme immobilization, there was no Zn element but the content percentage of element N increased to 3.53 % and element Na, P, K, Cl also appeared which confirmed that GOx covered the surface of CS-ZnO structures and was successfully immobilized onto the modified electrode. The glucose biosensing assay of commercial ZnO (FESEM images was in Supporting Information, Figure S3) was tested as comparison with different prepared CS-ZnO structures. To compare the glucose biosensing properties of different materials, electrochemical impedance and cyclic voltammetry performances of different modified electrodes were measured. Figure 7 showed the electrochemical character of modified electrodes with different CS-ZnO nanostructures and commercial ZnO in PBS containing 5mM [Fe(CN)6]3-/4-. Three CS-ZnO structures and commercial ZnO were modified onto the GCE to produce three different modified electrodes, respectively. Compared to bare electrode, after CS-ZnO and ZnO modified onto the GCE, the semicircle diameter of electrochemical impendance spectrum (EIS), which equals the electron transfer resistance (Ret), increased implying the successful modification of materials


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and the corresponding Ret was different. From the figure 7, the order of Ret was: 3 h CS-ZnO (curve b) > commercial ZnO(curve e) > 48 h CS-ZnO (curve d) > 18 h CS-ZnO (curve c) electrode. The order indicated that the electron transfer ability was 18 h CS-ZnO > 48 h CS-ZnO (curve d) > commercial ZnO > 3 h CS-ZnO.

Figure 7. The electrochemical impedance of: (a) bare GCE, (b) 3 h CS-ZnO, (c) 18 h CS-ZnO, (d) 48 h CS-ZnO, (e) commercial ZnO modified GCE in 5 ×10-3 mol/L [Fe(CN)6]3-/4- + 0.01 mol/L PBS.

The electrochemical behavior of prepared glucose biosensor was measured by cyclic voltammetry over a range of glucose concentrations in PBS containing 5mM [Fe(CN)6]3-/4-. The following electrochemical reaction was occurred: GOD(FAD) + glucose → GOD(FADH2) + gluconolactone GOD(FADH2) + 2[Fe(CN)6]3- → GOD(FAD) + 2[Fe(CN)6]4-


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[Fe(CN)6]4- → [Fe(CN)6]3- + e-

Figure 8 showed the cyclic voltammetry results of biosensor using CS-ZnO and commercial ZnO with different reaction time. As the glucose concentration increased, the current increased linearly. In Figure 8b, 8d, 8f, and 8h, the linear equation of the relation between the anodic peak current and the glucose concentration was measured in order to evaluate the sensitivity of the glucose biosensor fabricated from CS-ZnO and commercial ZnO under different conditions. The sensitivities were 49.18, 314.0, 278.3, 94.39 µA/mM·cm2 (R2=0.9872, 0.9977, 0.9774, 0. 9473) for CS-ZnO of 3 h, 18 h, 48 h treatment and commercial ZnO, respectively, over a range of glucose concentration from 0.05 to 0.3 mM. The glucose biosensor of ZnO prepared by the same synthesis method but without chitosan was also investigated (Supporting Information, Figure S4). The glucose sensor made from pure ZnO without chitosan as control group had sensitivity of 144.5 µA/mM·cm2. But the correlation index of pure ZnO was quite low (R2=0.9254) which may be due to the saturation of glucose molecules before 0.3 mM or the limited amount of immobilized glucose oxidase. Higher sensitivity may be owing to faster electron transportation. And the above phenomenon exhibits the same order of electron transfer velocity with the EIS experiments results further support the above speculation.


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Figure 8. CV performances and calibration curves for the response current vs. glucose concentration of glucose sensors using different CS-ZnO of different hydrothermal treatment time: (a) (b) 3 h, (c) (d) 18 h, (e) (f) 48 h, and (g) (h) commercial ZnO.

From the results, it could be demonstrated that the self-assemble CS-ZnO starfish-like mesocrystal structures could improve the electron transfer more than other ZnO and spindle-like CS-ZnO structure. The self-assemble CS-ZnO structures made up of small subunits could provide more electron transfer passage than bulk commercial ZnO. And the highly oriented CSZnO mesocrystals which had less number of grain boundaries between adjacent subunits resulted


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in facile electronic conduction.33 Moreover, the single crystal-like nature and better crystallinity could facilitate fast electron transport34 which might result in the better biosensor performs of 48 h spindle-like CS-ZnO than that of 3 h spindle-like CS-ZnO. At the same time, the tips of starfish-like CS-ZnO connected to each other and the CS-ZnO structures formed a large network hierarchical structure, which could offer synergistic advantages to further increase the electron transfer channels. It made the electrochemical probe easier to arrive to the surface of the electrode. Hence, the starfish-like CS-ZnO structures performed better than spindle-like CS-ZnO structures. Compared to the previous reported glucose biosensors based on ZnO and doped ZnO nanocrystal with different morphology (in Table 1), the starfish-like CS-ZnO mesocrystal structure biosensor showed much higher sensitivity (314.0 µA/mM·cm2) than other ZnO based glucose biosensors (around 10-70 µA/mM·cm2 )35-38 in similar linear range. In addition, the sensitivity of mesocrystal structure biosensor in this work was also much higher than that of glucose biosensors using Co doped ZnO39 and C doped ZnO40 which contained conductive dopant. It should be attributed to the more electron transfer passage, as previous stated, provided by the highly oriented mesocrystal nature and starfish-like large network hierarchical structure. The results demonstrated that the starfish-like CS-ZnO mesocrystal was an attractive material for biosensing application.

Table 1. Comparison of perfomances between glucose biosensors based on ZnO and doped ZnO with different structures. Electrode

Sensitivities

Linearity

Ref


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material

(µA/mM·cm2)

(mM)

ZnO nanowires

26.3

0.001 0.76

35

ZnO-Co nanoclusters

13.3

0.2-4

39

C-ZnO nanowire array

35.3

0.01-1.6

40

Porous ZnO nanostructures

23.4

0.01–1

36

ZnO Nanowire

35.1

0.00660.38

37

ZnO nanorods

69.8

0–0.1

38

CS-ZnO mesocrystal

314.0

0.05-0.3

This work

The response time is also one important factor influencing the biosensing performance of CSZnO mesocrystal structures. In this work, the hierarchical structures with smaller submits may affect the electrochemical reaction because of different electron transport velocities. The measurement of the response time will provide evidence to prove it, which needs to be performed in the future. Conclusion In summary, by imitating biomineralization, a novel synthetic method to fabricate oriented assembly CS-ZnO mesocrystals with different morphology is described. The chitosan-zinc oxide hybrid materials in spindle and starfish-like shape are made up of small nanoparticles with an average size of 25 nm as building blocks; CS-ZnO mesocrystal is of single crystal and the nanoparticle building blocks have nearly the same crystallographic orientation. Chitosan acts as a structure-directing agent to guide the formation of ZnO nuclei and the self-assemble preferential


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growth from small building units to larger structures through Coulomb force and the adjustment of surface energy. The biosensing assays exhibited that starfish-like CS-ZnO exhibited excellent sensing performance toward glucose but more detailed tests are under way. The starfish-like CSZnO structure had good biocompatibility due to the chitosan matrix and faster electron transportation attributed to the mesocrystal structure and special architecture with high specific surface area. These findings may be particularly useful for the preparation of composite mesocrystals structures by biomineralization process and biosensing applications of mesocrystals. ASSOCIATED CONTENT Supporting Information. FESEM images of ZnO obtained by hydrothermal process without chitosan and commercial ZnO, EDX spectra of electrode before and after enzyme immobilization and results of glucose biosensing performance using ZnO without chitosan. This material is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author * Email: [email protected] * Email: [email protected] Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT Financial support of this research from the National Innovation Fund (1203H157000, 12C26213102007), the Baoshan Science and Technology Project of Shanghai (BKW2014126) is appreciated. REFERENCES (1) Nonoyama, T.; Kinoshita, T.; Higuchi, M.; Nagata, K.; Tanaka, M.; Sato, K.; Kato, K. J. Am. Chem. Soc. 2012, 134, 8841-8847. (2) Liang, K.; Ricco, R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby, N.; Mudie, S.; Haylock, D.; Hill, A. J.; Doonan, C. J.; Falcaro, P. Nat.Commun. 2015, 6, 7240. (3) Faivre, D.; Godec, T. U. Angew. Chem., Int. Ed. 2015, 54, 4728-4747. (4) Ehrlich, H. Int. Geol. Rev. 2010, 52, 661-699. (5) Liu, Y.; Li, N.; Qi, Y. P.; Dai, L.; Bryan, T. E.; Mao, J.; Pashley, D. H.; Tay, F. R. Adv. Mater. 2011, 23, 975-980. (6) Cai, Y.; Yao, J. Nanoscale 2010, 2, 1842-1848. (7) Xiao, J. W.; Zhu, Y. C.; Liu, Y. Y.; Liu, H. J.; Zeng, Y.; Xu, F. F.; Wang, L. Z. Cryst. Growth Des. 2008, 8, 2887-2891. (8) Wang, X.; Shi, J.; Li, Z.; Zhang, S.; Wu, H.; Jiang, Z.; Yang, C.; Tian, C. ACS Appl. Mater. Interfaces 2014, 6, 14522-14532. (9) Song, S.; You, B.; Zhu, Y.; Lin, Y.; Wu, Y.; Ge, X. Cryst. Growth Des. 2014, 14, 38-45.


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For Table of Contents Use Only Oriented Assembly of Zinc Oxide Mesocrystal in Chitosan and Applications for Glucose Biosensors Shuyan Zhao, Bo You* , Linlei Jiang*,

TOC Graphic.

Synopsis. By imitating biomineralization, chitosan-zinc oxide hybrid mesocrystal materials in spindle and starfish-like shape are synthesized. Chitosan acts as a structure-directing agent to guide the formation of ZnO nuclei and the self-assemble preferential growth from small building units to larger structures through Coulomb force and the adjustment of surface energy. The starfish-like CS-ZnO exhibits excellent biosensing performance.


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Oriented Assembly of Zinc Oxide Mesocrystal in Chitosan and

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