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Conferring natural-derived porous microspheres with surface multifunctionality through facile coordination-enabled self-assembly process Pingping Han, Jiafu Shi, Teng Nie, Shaohua Zhang, Xueyan Wang, Pengfei Yang, Hong Wu, and Zhongyi Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00335 • Publication Date (Web): 10 Mar 2016 Downloaded from http://pubs.acs.org on March 14, 2016

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ACS Applied Materials & Interfaces

Conferring natural-derived porous microspheres with surface multifunctionality through facile coordination-enabled self-assembly process Pingping Han a,c,†, Jiafu Shi b,c,†, Teng Nie a, Shaohua Zhang a,c, Xueyan Wang a,c, Pengfei, Yang a, Hong Wu a,c,d*, Zhongyi Jiang a,c a

Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical

Engineering and Technology, Tianjin University, Tianjin 300072, China b c

School of Environment Science and Engineering, Tianjin University, Tianjin 300072, China

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),Tianjin 300072,

China d

Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University,

Tianjin 300072, China † *

Pingping Han and Jiafu Shi contributed equally and shared the first authorship. Corresponding author: Hong Wu, [email protected]

ACS Paragon Plus Environment


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Abstract In this study, multifunctional chitin microspheres are synthesized and utilized as a platform for multiple potential applications in enzyme immobilization, catalytic reduction and adsorption. Porous chitin microspheres with an average diameter of 111.5 µm and a porous architecture are fabricated through thermally induced phase separation method. Then, the porous chitin microspheres are conferred with surface multifunctionality through facile coordination-enabled self-assembly of tannic acid (TA) and titanium (TiIV) bis(ammonium lactate) dihydroxide (Ti-BALDH). The multipoint hydrogen bonds between TA and chitin microspheres confer the TA-TiIV coating with high adhesion capability to firmly adhere to the surface of the chitin microspheres. In view of the biocompatibility, porosity and surface activity, the multifunctional chitin microspheres are used as carriers for enzyme immobilization. The enzyme-conjugated multifunctional porous microspheres exhibit high catalytic performance (102.8 U·mg-1 yeast alcohol dehydrogenase). Besides, the multifunctional chitin microspheres also find potential applications in the catalytic reduction (e.g., reduction of silver ions to silver nanoparticles) and efficient adsorption of heavy metal ions (e.g., Pb2+) taking advantages of their porosity, reducing capability and chelation property.

Keywords: multifunctionality; chitin; porous microspheres; TA-TiIV coating; coordination-enabled self-assembly

1. Introduction Multifunctional, porous microspheres are becoming effective materials for the prospective application in energy storage, separation, cell/drug delivery, tissue regeneration and catalysis.1-6 The advantages of multifunctional, porous microspheres are primarily focused on two aspects, porosity and multifunctionality. The porous structure usually provides the microspheres with high specific surface area, high cargoes loading and superior mass transfer properties, whereas the multifunctional surface could endow the microspheres with unique physical and chemical characteristics for different purposes. Till now, a series of porous microspheres have been successfully prepared based on both natural polymers (collagen, cellulose, chitosan, alginate and chitin, etc.) and synthetic polymers (PLGA, PCL and PLLA etc.).4,

7-13

Compared to synthetic polymers, natural polymers are particularly

attractive as green materials for their biocompatibility, nontoxicity and biodegradability. Recently,


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one exciting example is conducted by Zhang's group who fabricated porous chitin microspheres with a nanofibrous architecture.13 Specifically, the chitin chains first self-assembled rapidly into nanofibers during the thermally induced phase separation (TIPS) process and then formed weaved porous microspheres. The as-prepared microspheres had a nanofibrous surface, large specific surface area, high porosity and good biocompatibility. However, the direct utilization of chitin microspheres seems to be less practical and inefficient6, 12, 14, 15 because of the lack of surface multifunctionalilty. Few investigations report the surface-functionalization of chitin microspheres.16-17 Therefore, exploring innovative and effective methods to surface-functionalize the microspheres is highly desired and confronts challenges.6, 18 The existing toolbox for surface functionalization includes Langmiur-Blodgett deposition, chemical vapor deposition, layer-by-layer assembly, functionalized silanes, self-assembled monolayer (SAM) formation and mussel-inspired surface chemistry.

19-25

Particularly, very recently,

polyphenol-inspired chemistry proves to be a potential and versatile method for surface functionalization. Polyphenol can exert strong coordination with metal ions in aqueous solution, adhere tightly to a variety of materials and is prone to react with biomolecules. Typically, inspired by polyphenol chemistry, Caruso et al. reported that tannic acid-iron (III) (TA-FeIII) coordination coating could firmly adhere to such solid substrates as polymers, metal and metal oxide.26 The thickness of the coating could be controlled down to ~20 nm and the surface functionalization process could be accomplished within only 1 min. Particularly, our group previously reported an ultrathin microcapsule fabricated by tannic acid-titanium ions coordination compound which showed a weak pH-responsive property.27 The strong coordination bond with a bonding energy of 200-400 kJ/mol between TA and TiIV conferred the microcapsules with high structural stability in a wide pH range of 3.0 to 11.0. Herein, we presented the first example of TA-TiIV coordination coating on chitin microspheres with a porous architecture to form multifunctional chitin microspheres for different purposes. The chitin microspheres fabricated by thermally induced phase separation method were immersed simply into tannic acid and titanium (IV) bis (ammonium lactate) dihydroxide aqueous solution successively under mild conditions. Considering their biocompatibility, porosity and surface activity, the multifunctional chitin microspheres were used as carriers for enzyme immobilization. Considering their reducing capability and porosity, the multifunctional chitin microspheres were used as a



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platform to grow and support silver nanoparticle. Considering their biodegradability, porosity and chelation property, the multifunctional chitin microspheres were used as adsorbents for heavy metal ions Pb2+.

2. Experimental 2.1 Materials Chitin powder was purchased from Gold-Shell Biochemical Co. Ltd. (Zhejiang, China). Tannic acid (TA, ACS reagent), titanium (IV) bis (ammonium lactate) dihydroxide (Ti-BALDH, 50 wt% aqueous solution) were purchased from Sigma-Aldrich. Sodium hydroxide (NaOH), urea, isooctane, Span 80, Tween 80 and silver nitrate were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Lead nitrate was purchased from Kermel Fine Chemical Research Institute (Tianjin, China). Yeast alcohol dehydrogenase (YADH, EC1.1.1.1, from Saccharomyces cerevisiae, Mw 14−15 kDa, pI 5.2−5.6), nicotinamide adenine dinucleotide (NADH, grade I, 98%) were purchased from Sigma-Aldrich. High-purity water with a resistivity of 15.0 MΩ was obtained from the Millipore Milli-Q purification system. 2.2 Fabrication of chitin microspheres The chitin powder was purified according to the procedure described in literature.28 Firstly, 10 g chitin powder was soaked in 40 g NaOH solution (5 wt%) for 10 h under stirring. The suspension was then filtered and washed with deionized water till the pH value reached 7.0. After that, the chitin powder was treated with 40 g hydrochloric acid aqueous solution (7% v/v) and 40 g NaOH solution (5 wt%) for 24 h, respectively. In order to remove the pigments, the chitin powder was treated with 40 g sodium chlorite (1.7 wt%) in 0.3 M sodium acetate buffer solution for 6 h at 80 oC. The purified chitin powder was obtained followed by washing with deionized water. The chitin microspheres were fabricated as follows. First, purified chitin powder (3~5 wt%) was dispersed into a mixed solution containing NaOH, urea and deionized water with a weight ratio of 8:4:88. Subsequently, the suspension was frozen at -20 oC for 10 h and then thawed. The freezing/thawing cycle was repeated to acquire a transparent solution. A well-mixed suspension containing 20 ml of isooctane and 0.22 g of Span 80 was dispersed in a reactor. The suspension was stirred at 500 rpm for 30 min, and then the chitin solution was dropped into the suspension. The mixture was kept stirring for 1 h at the same stirring speed at 0 oC. A solution containing 0.12 g of


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Tween 80 and 2 ml of isooctane was added to the mixture for another 1 h to obtain stable water-in-oil emulsion droplets. After that, the mixture was transferred to a 60 oC bath for 10 min to form the chitin microspheres. Subsequently, dilute hydrochloric acid (10%) was added to the suspension till the pH reached 7.0. Finally, the chitin microspheres in the substratum were filtered and rinsed with ethanol and deionized water. 2.3 Functionalization of chitin microspheres by TA-TiIV coating The preparation process of TA-Ti

IV

coating was described as follows. The as-prepared chitin

microspheres were dispersed into a specific amount of deionized water with 1 min stirring to ensure complete dispersion. Next, the mixture was stirred immediately for 1 min after the individual additions of TA and Ti-BALDH. Then the chitin microspheres with TA-Ti

IV

coating were washed

with deionized water to remove excess TA and Ti-BALDH. The final concentration of TA was 0.06-0.24 mM and the molar ratio of TA to Ti-BALDH was 1:10. 2.4 Enzyme immobilization Yeast alcohol dehydrogenase (YADH) was dissolved in Tris-HCl buffer solution (50 mM, pH 7.0) to get an enzyme solution (1 mg/ml). The multifunctional chitin microspheres or chitin microspheres were dispersed into the above enzyme solution for 2 h. The amount of immobilized YADH was measured using Bradford’s method.29 Specifically, the absorbance at 595 nm was measured after mixing 1 ml enzyme solution or supernatant solution with 5 ml Coomassie Brilliant reagent for 3 min. The immobilization efficiency and loading capacity were calculated by eq. (1) and eq. (2), Immobilization efficiency =

Loading capacity =

C0V0 − C1V1 × 100 % C0V0

the quantity of immobilized enzyme × 100 % the quantity of supports

(1)

(2)

where C0 (mg/ml), V0 (ml) were the initial concentration and volume of enzyme, respectively, C1 (mg/ml), V1 (ml) were the residual concentration and volume of enzyme in the supernatant solution, respectively. The enzyme activity of free and immobilized YADH was tested by monitoring the conversion reaction of formaldehyde into methanol with the aid of NADH as the cofactor. Briefly, the substrate HCHO (10 mM) and the cofactor NADH (133 µM) were dissolved in Tris-HCl buffer solution (50



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mM, pH 7.0) to prepare the substrate solution. Subsequently, 0.05 mg of free or immobilized YADH was added into 10 mL substrate solution for a certain time and the change of NADH concentration was recorded by measuring the absorbance of NADH at 340 nm by using an ultraviolet-visible spectrophotometer. One unit (U) of YADH was defined as the quality of YADH required to transform 1 µmol NADH per minute at pH 7.0 and 25 oC. And the initial reaction rate was defined by eq 3: Initial reaction rate (% s

-1

)=

C0 − C30 × 100 % C30 × 30

(3)

where C30 (mg/ml) was the concentration of NADH after reacting 30 s. 2.5 Enzyme stability For recycling stability, the immobilized YADH was recollected after each reaction batch and then implemented the next reaction cycle. The activity after each batch was measured and compared with the initial enzyme activity. For storage stability, the activity of immobilized YADH was determined after it was stored at 4 °C for a period of time. The initial activity for the first day was defined as 100% and the activity after storage was the relative value through comparison with the initial activity. For pH stability, the free and immobilized YADH were incubated in buffer solutions at different pH values (4.0-10.0) for 2 h, respectively. For thermal stability, the free and immobilized YADH were incubated at different temperature (20-70 oC) for 2 h, respectively. The activity of free and immobilized YADH were measured. The relative activity was defined as eq (4),

relative activity (%)=

YADH activity at specific pH (or temperature) × 100 % YADH activity at 30 oC, pH 7.0

(4)

The thermal denaturation constants (kd) were calculated using the first order experimental equation30 by eq. (5),

A=A0 exp (-kd t)

(5)

where A0 was the initial activity of YADH, A was the incubated activity of YADH and t was the incubation time. The half-time value (t1/2) for enzyme thermal denaturation was calculated by eq. (6), t1/2 =

ln 2 kd

(6)


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The enthalpy (∆H°, kJ mol−1) for YADH thermal denaturation was calculated by eq. (7),

ln kd = Ed / RT + ln C slope = - Ed / R ∆H ° = Ed - RT

(7)

where Ed was the activation energy, T was the corresponding absolute temperature and R was the gas constant (8.3145 J mol−1 K−1). 2.6 A platform for Ag+ reduction and Pb2+ adsorption To investigate the reduction capability of multifunctional chitin microspheres, the multifunctional chitin microspheres were used to reduction of Ag+ to silver nanoparticles. A certain amount of multifunctional chitin microspheres were dispersed into 7 ml of silver nitrate aqueous solution (100 mM). Then the mixture was stirred for different time periods (2 h, 4 h, 7 h, 10 h, 24 h). The SEM image and element analysis were taken to verify the reduction of Ag+. To investigate the adsorption capability of multifunctional chitin microspheres, the multifunctional chitin microspheres were used to adsorption of Pb2+, which was the most common heavy metal ion. Typically, 2 mg of chitin microspheres or multifunctional chitin microspheres were added into the lead nitrate aqueous solution (200 mg/L, 20 ml) in a breaker and shaken for 12 h. The concentration of Pb2+ after adsorption was determined by CCD Simultaneous ICP-OES. 2.7 Characterizations The optical images of the microspheres were obtained by an optical microscope. SEM images of chitin microspheres and the multifunctional chitin microspheres were recorded by scanning electron microscope (Nanosen 430). The energy dispersive spectroscope (EDS) attached to SEM was used to analyze the elemental composition of chitin microspheres and the multifunctional chitin microspheres. FTIR spectra of purified chitin powder, chitin microspheres and the multifunctional chitin microspheres were obtained on a Nicolet-6700 spectrometer. 32 scans were accumulated with a resolution of 4 cm-1 for each spectrum. The purified chitin powder, chitin microspheres and the multifunctional chitin microspheres were analyzed by an X-ray diffraction meter (XRD, D/MAX-2500). Nitrogen physisorption measurements were implemented at 77 K by using of a Micromeritics Tristar 3000. The samples were degassed at 105

o

C for 4h in vacuum.

Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halendar (BJH) analyses were performed by



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software. BET analysis was conducted for the relative pressures of 0.05-0.3 and BJH analysis was conducted from the desorption branch of the isotherms. X-ray photoelectron spectra (XPS) was recorded on a Kratos XSAM800 X-ray photoelectron spectrometer, using Mg Kα radiation as the excitation source. The size distribution of chitin microspheres was determined with a Mastersizer S (Malvern, UK). The interaction force between TA-Ti

IV

coating and chitin microspheres was

measured by the atomic force microscopy (AFM, Multimode 3, Bruker Co.) The concentration of metal ions (Pb2+) was determined by CCD Simultaneous ICP-OES.

3. Results and discussion 3.1 Preparation of multifunctional chitin microspheres

Fig. 1. Schematic preparation process of multifunctional chitin microspheres.

The preparation process of multifunctional chitin microspheres was schematically presented in Figure 1. Briefly, the chitin microspheres were prepared by the thermally induced phase separation method, and then coated by TA-TiIV layer through coordination-enabled self-assembly process. Specifically, the chitin dissolved by NaOH-urea aqueous solution was first emulsified into stable liquid microspheres in isooctane with the surfactants under stirring. Then, the mixture was transferred to a 60 oC water bath to induce the formation of chitin nanofibers. Porous microspheres


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were acquired after self-assembly of the chitin nanofibers. Finally, the TA-TiIV coating layer was formed on the surface of the chitin microspheres by simply soaking the chitin microspheres into the TA and Ti-BALDH solution, respectively.

Fig.2. SEM images of the pristine chitin microspheres (a, b) and the multifunctional chitin microspheres (c, d).

The morphology and surface structure of chitin microspheres and the multifunctional chitin microspheres were characterized. As shown in Figure 2a and 2b, the pristine chitin microspheres exhibited a well-distributed apparent porous architecture. After functionalization, the morphology of microspheres was little changed, while the pore size and porosity increased moderately (Figure 2c and 2d). The hydrogen-bond interaction between the chitin chains and TA-Ti

IV

coating layer would

interfere the original hydrogen-bond interaction between chitin chains13, resulting in an enlargement of pore size and porosity. The specific surface areas of chitin microspheres increased by 45.5% after TA-TiIV coating (see Figure S1a and S1b). In addition, the contact angle and ζ-potential of the pristine chitin microspheres were 48.98 o±2.41o and 0.0794 mV±3.48 mV. After modification by TA-TiIV coating, the contact angle and ζ-potential of the multifunctional chitin microspheres were 30.27o±1.56o and 26.1 mV±5.53 mV. The notable reduction in the contact angle and increase in ζ-potential suggested an enhanced surface polarity after modification, which would facilitate the adsorption of polar molecules.



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Fig.3. Optical photomicrograph and the size distribution of the pristine chitin microspheres (a, b). Optical photomicrograph and the size distribution of the multifunctional chitin microspheres (c, d).

The optical photomicrographs and the corresponding size distribution of chitin microspheres were recorded and measured. As shown in Figure 3a and 3b, the chitin microspheres exhibited a spherical shape and had an average diameter of 111.5 µm. The size of the microspheres was affected by the stirring speed, the surfactant amount and the oil/water ratio. It is well known that the size of microspheres decreased rapidly with increasing stirring speed, surfactant amount and the oil/water ratio. After functionalization by TA-TiIV coating, the color of the chitin microspheres turned yellow (Figure 3c), confirming the formation of TA-TiIV coating on the microspheres. The size distribution of the chitin microspheres with TA-TiIV coating (Figure 3d) showed no obvious change since the coating layer was rather thin26, 27. The appearance of Ti element from Ti-BALDH in EDS analysis (Figure 4a and Figure S2) and element mapping analysis (Figure 4b) further confirmed the successful formation of TA-TiIV coating. Moreover, the TA-TiIV coating uniformly covered the microspheres. On the FT-IR spectrum III (Figure 4c), the bands at 1716 cm-1 (carbonyl group), 1446 cm-1, 1035 cm-1, 871 cm-1,(benzene ring), 1317 cm-1 (phenolic hydroxyl group) and 1205 cm-1 (-C-O-C- group) were assigned to titanic acid31, 32, further demonstrating the successful formation of TA-Ti

IV

coating

on chitin microspheres. In order to clarify the formation mechanism of chitin microspheres, FT-IR spectra and XRD analysis were employed. As shown in FT-IR spectrum I and II (Figure 4c), the bands at around 3460


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and 3111 cm-1 were ascribed to -OH groups and –NH groups, while the bands at around 1663 and 1557 cm-1 were assigned to the acetamide groups.33-35 Compared to purified chitin powder, there was no apparent peak shifts in the spectra of chitin microspheres, indicating the chitin microspheres were assembled by the weaving of chitin fibrous. The hydrogen-bond interaction and hydrophobic interaction induced the formation of chitin microspheres. Meanwhile, the similar five peaks (2θ = 9.3o, 12.6o, 19.2o, 23.3o and 26.3o, indexed as (020), (021), (110), (130) and (013))34, 36 in the XRD patterns of purified chitin powder and chitin microspheres (Figure 4d) confirmed the intact crystalline structure of chitin powder in the chitin microspheres.

Fig.4. EDS analysis and elemental mapping of the multifunctional chitin microspheres (a, b); FTIR spectrum and XRD pattern of (I) purified chitin power, (II) chitin microspheres, (III) multifunctional chitin microspheres and (IV) enzyme conjugated multifunctional chitin microspheres (c, d).



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Fig. 5. Full-scale XPS spectra of (I) chitin microspheres, (II) the multifunctional chitin microspheres and (III) enzyme conjugated multifunctional chitin microspheres (a). High-resolution XPS spectra of Ti 2p (b), O1s (c), and N1s (d) elements.

XPS spectra were then conducted to clarify the interaction between TA and TiIV. As shown in Figure 5a, after functionalization, the appearance of Ti 2p also demonstrated the successful functionalization of chitin microspheres. The binding energy of Ti 2p2/3 and Ti 2p1/2 were located at 458.5 and 464.3 eV (Figure 5b), respectively, revealing that the Ti element was in an oxidation state of +4.37, 38 The O 1s XPS spectra of multifunctional chitin microspheres exhibited four peaks at 529.8, 530.3, 531.4, and 532.7 eV (Figure 5c), which could be assigned to Ti−O−Ti, N-C=O, Ti−O and C−O bond38. Compared to the O 1s peak of chitin microspheres (531.1 eV), the O 1s peak of multifunctional chitin microspheres shifted to a higher binding energy (531.4 eV), indicating the transformation of the electron from O to TiIV. As the coordination bond formed, numerous H+ in the phenolic hydroxyl groups would be substituted by TiIV to form coordination bond between TA and Ti (Figure S3).27


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Previously, atomic force microscopy (AFM) measurements were conducted to study the interfacial interaction.39-40 Therefore, TA-Ti

IV

coating modified Si3N4 AFM cantilevers were used to further

examine the interaction of TA-TiIV coating with chitin microspheres in depth. Figure 6 shows the representative force-extension curves for approach and retraction of a TA-TiIV coating modified cantilever from the chitin surface and Fe surface. The interaction force between the modified AFM tips and membrane surface can be read from Figure 6. The interaction force between TA-TiIV coated tip and chitin surface was about 4.53 nN, which was much lower than the coordination interaction between TA-Ti

IV

coated tip and Fe surface (9.61 nN). In addition, the DRUV spectrum of

Chitin-TA-Ti was the superposition of the DRUV spectra of TA-Ti and chitin-Ti (Figure 6c), indicating that there was no chemical bonding formed between TA-Ti coating and chitin. Therefore, the hydrogen interactions may contribute most to the formation of TA-Ti IV coating on the surface of chitin microspheres.

Fig. 6. Force-extension curves of TA-TiIV coated AFM tip interacting with a chitin surface (a) and a Fe surface (b). Diffuse Reflectance UV–visible spectra (c).

3.2 Multifunctional chitin microspheres for enzyme immobilization



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Considering their biocompatibility, porosity and surface activity, the multifunctional chitin microspheres were used as carriers for enzyme immobilization. YADH was selected as the model enzyme to test the immobilization efficiency and catalytic performance. As shown in Figure 4c (FT-IR spectrum III and IV), the bands at 1662 and 1552 cm-1 were slightly blue shifted after enzyme immobilization, suggesting the successful immobilization of YADH on the multifunctional chitin microspheres. Moreover, the appearance of S2p in the XPS fully scanned spectra (Figure 5a) also indicated the successful immobilization of YADH. Particularly, the highly active quinone derived from the oxidization of TA could further react with amino groups in enzyme to form C-N and C– NH3+ groups through Michael addition and/or Schiff base reaction. 41-42 When focusing on the spectra of N1s (Figure 5d), two peaks at 399.7 and 401.3 eV, assigned respectively to C-N and C–NH3+, were observed after YADH immobilization. Table 1 Summary of several immobilization parameters supports

Immobilization efficiency

Loading capacity

Specific activity

(%)

(mg/g)

(U·mg-1 enzyme)

1#*

4.8

48.7

31.6

2#*

40.8

412.5

45.8

3#*

8.7

84.2

35.4

4#*

50.9

514.8

102.8

5#*

50.8

462.0

58.1

6#*

48.1

301.8

43.2

7#*

47.1

276.6

48.7

*1# chitin microspheres prepared by chitin concentration of 5 wt%; 2# functionalization of 1# microspheres by 0.06 mM TA; 3# chitin microspheres prepared by chitin concentration of 3 wt%; 4#, 5#, 6#, 7# functionalization of 3# microspheres by 0.06 mM, 0.12 mM, 0.24 mM and 0.48 mM TA.


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Fig.7. SEM images of pristine chitin microspheres prepared by chitin concentration of 3 wt% (a) and 5 wt% (b). SEM images of multifunctional chitin microspheres (chitin concentration of 3 wt%) prepared by TA concentration of 0.06mM (c); 0.12 mM (d); 0.24mM (e); 0.48mM (f). Inset images were the corresponding optical images.

The morphology and the porous architecture of chitin microspheres as well as the catalytic performance of the immobilized enzyme thereon could be tuned by varying the chitin solution concentration. Compared to high concentration of chitin emulsion droplet, low concentration of chitin emulsion droplet resulted in a relatively loose surface. When the concentration of chitin was below 3 wt%, no chitin microspheres could be formed. At 3 wt% chitin concentration, the resultant microspheres showed a spherical shape with a looser structure (Figure 7a). As the concentration of chitin increased to 5 wt%, the microspheres became denser (Figure 7b). Compared to the denser microspheres, the immobilization efficiency, loading capacity and specific activity of the looser microspheres were all enhanced (Table 1) due to smaller mass transfer resistance and easier access to enzyme for substrates. Besides, the immobilization efficiency, loading capacity and specific activity of multifunctional chitin microspheres were much higher than those of chitin microspheres. The chitin microspheres containing only hydroxyl groups and acetamide groups lacked active groups for enzyme immobilization. The immobilization efficiency and loading capacity of pristine chitin



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microspheres were only 8.7% and 84.2 mg/g, respectively. After functionalized by TA-Ti

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IV

coating,

abundant active sites from TA were introduced into chitin microspheres, which is beneficial to combine enzymes. The effect of the concentration of TA on the properties of multifunctional microspheres was examined by fixing the concentration of chitin at 3 wt%. As the concentration of TA increased from 0.06 to 0.48 mM, the color of the microspheres changed from pale yellow to deep yellow (Figure 7c-f), the loading capacity decreased from 514.8 to 296.6 mg/g and the specific activity decreased from 102.8 to 48.7 U·mg-1 enzyme (Table 1). Specifically, the chitin microspheres modified by 0.06 mM TA showed the optimum performance with an immobilization efficiency of 50.9%, a loading capacity of 514.8 mg/g and a specific activity of 102.8 U·mg-1 enzyme. High concentration of TA lowered the specific area, pore volume and active sites of the chitin microspheres, resulting in decreased immobilization efficiency, loading capacity and specific activity.

Fig. 8. Methanol yield as a function of reaction time.

The methanol yield of multifunctional chitin microspheres for enzyme immobilization was illustrated in Figure 8. The transformation reaction reached equilibrium in 4.5 min for free YADH, while 11.5 min were required for YADH immobilized on multifunctional chitin microspheres. The equilibrium methanol yield for YADH immobilized on multifunctional chitin microspheres reached 98.0%, which was even higher than that for free YADH (87.2%). Compared to free YADH, the immobilized YADH showed a bit reduced reaction rate. The initial reaction rate (0.33 % s-1) was comparable and even higher than the previous report.43 The high reaction rate could be ascribed to the well-developed porous architecture of multifunctional chitin microspheres. The porous


Page 17 of 24

architecture enhanced the mass transfer of substrates/products (Figure S4), which was beneficial for the desirable high initial reaction rate.

b

100

Relative activity/%

Relative activity/%

a

80 60 40 Free YADH Immobilized YADH

20 0

100 80 60 40 Free YADH Immobilized YADH

20 0

20

30

40

50

60

70

4

5

Temperature/oC

6

7

8

9

10

pH value 120

100

d

Immobilized YADH

Relative activity/%

c Relative activity/%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40 20

100 80 60 40 20

Free YADH Immobilized YADH

0

0 0

1

2

3

4

5

6

7

8

9

10 11 12 13

0

5

10 15 20 25 30 35 40 45 50 55 60

Cycle numbers

Storage Time/ Days

Fig. 9. Thermal stability (a); pH stability (b); recycling stability (c); storage stability (d) of free enzyme and immobilized enzyme.

Considering the significance of the enzyme stability in potential industrial application, the stabilities including thermal stability, pH stability, recycling stability and storage stability were investigated (Figure 9). Compared to free enzyme, the immobilized enzyme had higher thermal stability, revealing that multifunctional chitin microspheres provided a more benign environment for immobilized enzymes, which prevented enzymes from thermal-induced denaturation. Compared with free ones, the increased half-life (t1/2), the decreased thermal denaturation constants (kd) and the increased ΔH° values for immobilized enzymes (Table S1 and Table S2) demonstrated that the multifunctional chitin microspheres were more thermally stable for enzymes. The multifunctional chitin microspheres with YADH also showed a superior pH stability in a broad range of pH values (Figure 9b). Under acidic condition and alkaline condition (pH 4.0 and pH 10.0), the multifunctional chitin microspheres with YADH maintained a relative activity of 52.8% and 32.1%, respectively. The



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obvious improvement of pH stability should be arisen from the weak pH-response of TA-Ti

Page 18 of 24

IV

coating, which hindered the leakage of enzymes.27 The coordination bond between polyphenol and Ti

IV

was strong enough against a wide range of pH fluctuations. As shown in Figure S3 (UV-vis

absorbance of the supernatant), the TA-TiIV coating would disassemble from the chitin microspheres under extreme alkaline condition (pH 13.0) but would not or only partially disassemble from the chitin microspheres at pH 4.0, 7.0 and 10.0.27 The recycling stability and storage stability of the multifunctional chitin microspheres containing YADH were also investigated (Figure 9c and 9d). The multifunctional chitin microspheres with YADH retained 53.2% of its initial activity after 12 reaction cycles and 66.8% of its initial activity after storage for 55 days. 3.4 Extended applications of the multifunctional chitin microspheres To explore the multifunctionality of the as-synthesized microspheres, exploratory experiments were conducted to test their catalytic reducing capability and adsorption property. In consideration of the catalytic reducing capability and porosity of multifunctional chitin microspheres, the multifunctional chitin microspheres were utilized to generate and grow Ag nanoparticles. In this process, the TA-Ti

IV

layer served as the reducing agents and the porous structure served as the

carriers for Ag nanoparticles. As shown in Figure 10a, the color of the solution containing AgNO3 and multifunctional chitin microspheres got deeper as the reaction proceeded. The existence of Ag nanoparticles was confirmed by DRUV analysis. As shown in Figure 10b, a wide adsorption band at around 480 nm was the characteristic of surface plasmon adsorption of Ag (0),44-45 indicating that Ag nanoparticles were formed and grew onto the surface of multifunctional chitin microspheres (Figure 10c). Besides their application in enzyme immobilization and catalytic reduction, the multifunctional chitin microspheres were also utilized to adsorb the heavy metal ions (Pb2+). After 12 h-absorption at neutral condition, the adsorption capacity for multifunctional chitin microspheres was 962.62 mg/g, much higher than that of pristine chitin microspheres (344.34 mg/g). Moreover, the adsorption capacity for multifunctional chitin microspheres outperformed many other adsorbents reported previously.46-47 The high adsorption capacity of multifunctional chitin microspheres could be ascribed to the strong chelation of polyphenol in the TA-Ti

IV

multifunctional chitin microspheres.


coating and the porosity of the

Page 19 of 24

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Fig. 10. Optical images of multifunctional chitin microspheres under various reaction times (a). Diffuse Reflectance UV–visible spectra of Ag nanoparticles (b). SEM images of multifunctional chitin microspheres after reducing Ag+ to Ag nanoparticles (c).

4. Conclusions A facile coordination-enabled self-assembly of tannic acid (TA) and titanium (IV) bis (ammonium lactate) dihydroxide (Ti-BALDH) was adopted to confer natural-derived porous chitin microspheres with surface multifunctionality. Based on coordination interaction and hydrogen bond interaction, the TA-TiIV coating forms and deposits on the porous chitin microspheres. In view of the biocompatibility, porosity and surface activity, the multifunctional chitin microspheres are used as carriers for enzyme immobilization. The enzyme-conjugated multifunctional porous microspheres exhibit high catalytic performance (102.8 U·mg-1 enzyme) and superior pH and thermal stability. In view of the reducing property and porosity, the multifunctional chitin microspheres are used as a platform for catalytic reduction, whereas in view of the biodegradability, porosity and chelation



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property, the multifunctional chitin microspheres are also used as adsorbents for heavy metal ions Pb2+. Through combining the unique characteristics of TA-TiIV coating and porous chitin microspheres, the multifunctional chitin microspheres can serve as a platform for various applications.

Acknowledgements The authors thank the financial support from Specialized Research Fund for the Doctoral Program of Higher Education (20130032110023), National Natural Science Funds of China (21576189, 21406163，91534126), Program of Introducing Talents of Discipline to Universities (B06006), and Program for New Century Excellent Talents in University (NCET-10-0623).

Supporting information Nitrogen adsorption-desorption isotherm and Pore size distribution curve of multifunctional chitin microspheres (Figure S1); EDS analysis of the pristine chitin microspheres (Figure S2); Probable structure of TA-TiIV coordination complex (Figure S3); Evolution of the mass percent of NADH in the solution diffused into the multifunctional (Figure S4); Thermal denaturation kinetic parameters of free and immobilized YADH (Table S1); The change in enthalpy (∆H°) for the thermal denaturation of free and immobilized YADH (Table S2); UV-vis spectra of the supernatant of the TA-Ti IV coating under various pH values. The inset was the optical images of the multifunctional chitin microspheres in aqueous solution at different pH values (Figure S5).

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