Dopamine-Modified Alginate Beads Reinforced by Cross-Linking via

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Dopamine-Modified Alginate Beads Reinforced by Cross-Linking via Titanium Coordination or Self-Polymerization and Its Application in Enzyme Immobilization Xiaoli Wang,† Zhongyi Jiang,†,‡ Jiafu Shi,§ Chunhong Zhang,† Wenyan Zhang,† and Hong Wu*,† †

Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China ‡ National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China § School of Environmental Science & Engineering, Tianjin University, Tianjin 300072, China ABSTRACT: Two novel kinds of dopamine-modified alginate beads were developed by using titanium(IV) coordination or selfpolymerization, besides the conventional Ca2+ ion cross-linking. Alginate was modified with dopamine (AlgDA) via EDC/NHS chemistry. Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) characterization confirmed that dopamine was covalently attached preferentially to the mannuronic acid residues of alginate; thus, gel-forming ability of the asprepared AlgDA was well retained. The titanium(IV) coordination-reinforced alginate beads [Ca−Ti(AlgDA)] were prepared by cross-linking with Ti4+ and Ca2+. Covalently cross-linking-reinforced alginate beads (Ca−AlgPDA) were also prepared by selfpolymerization of dopamine and cross-linking with Ca2+. The swelling of Ca−AlgPDA and Ca−Ti(AlgDA) were both obviously inhibited, and the mechanical properties were enhanced by 3 times compared to those of Ca−Alg beads. The as-prepared beads were utilized for immobilization of alcohol dehydrogenase (YADH). The immobilization efficiency of Ca−Ti(AlgDA) and Ca− AlgPDA reached up to 100% and 89%, respectively, both notably higher than that of Ca−Alg (67.4%). Stabilities of the immobilized YADH in Ca−AlgPDA and Ca−Ti(AlgDA) toward pH, storage, and recycling were all improved compared with those immobilized in Ca−Alg.

1. INTRODUCTION Immobilization of biological products is used for a myriad of applications, ranging from cell therapy1 and cell biosensors2 to immobilization of DNA3 and enzyme for catalytic reaction4 or drugs for targeted delivery.5 Hydrogels like calcium alginate (Ca−Alg), for example,6−8 are one of the most promising materials for immobilization due to their structural resemblance to the natural extracellular matrix of human tissues and high water content, resulting in good mass transport properties. It has been found recently that catechol-containing polymer networks are ubiquitous in nature9,10 and have fascinated many researchers due to their delicate structures and superior properties.11,12 The mussel adhesive plaques protein-enriched in catechol can deposit onto almost all material surfaces via the self-polymerization of catechol.13,14 What’s more, catechol coordinated with inorganic ions, notably Fe3+, endows the mussel cuticle with self-healing properties15 and a remarkable combination of high hardness (100−150 MPa) and high extensibility(>70% strain).16 Moreover, the polymerized catechol, a wholly organic material, endows some biological materials with superior mechanical properties, such as insect cuticles17 or squid beaks.9 The self-polymerization and coordination with metals of catechol were introduced to prepare two kinds of hydrogel beads in this work. Actually, several mussel-inspired hydrogels were developed via catechol polymerization,18,19 catechol−thiol reaction,20,21 catechol−boronic acid complexation,22,23 and catechol−ferric ion complexation.15 These hydrogels were designed for © 2013 American Chemical Society

different properties. Besides, the catechol can react with the primary amine groups through Michael addition and Schiff base reactions. Immobilization of a biomolecule assisted by catecholcontaining polymers has been developed, and bioactivity was well preserved.24−26 Due to the versatility of catechol, one can prepare hydrogels with desirable physical properties to meet the needs of specific applications in a facile and controllable way. The gelation of alginate is usually conducted by cross-linking with divalent or trivalent ions, like the most commonly used calcium ions. However, the resultant Ca−Alg beads always exhibit poor structural stability27 due to severe swelling, especially in aqueous solutions, thus prohibiting their practical applications where an aqueous environment is preferred. In this study, a mimic of mussel adhesive protein was synthesized by modifying alginate with dopamine via EDC/NHS chemistry. Similar to adhesive proteins, the as-prepared AlgDA can be cross-linked either by titanium(IV) coordination or by selfpolymerization of dopamine. Hence, two kinds of musselinspired alginate beads were developed and their structural stability was improved. The titanium(IV) coordinationreinforced alginate beads denoted as Ca−Ti(AlgDA) were prepared by dual cross-linking with Ti4+ and Ca2+. Covalently Received: Revised: Accepted: Published: 14828

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and Ca−Ti(AlgDA) beads according to the procedure described above. 2.2.4. Characterizations. Elemental compositions of the AlgDA polymers were analyzed by Elementar Vatio EL CUBE. Circular dichroism (CD) spectra of dilute Alg solution and AlgDA solution were recorded at 25 °C in a Jasco J-810 spectropolarimeter (Tokyo, Japan) with a scan rate of 20 nm min−1. Glass transition temperatures of the lyophilized Alg, AlgPDA, and Ti(AlgDA) were determined by differential scanning calorimetry (DSC, NETZSCH DSC 200F3 A) using sample mass of ∼5 mg in a covered aluminum sample holder. Alg: heated from 20 to 120 °C. AlgPDA and Ti(AlgDA): heated from 20 to 170 °C. The first heating rate of 8 °C/min, the cooling rate of 50 °C/min, and the second heating rate of 5 °C/min were performed under constant purging of N2 at 20 mL/min. Ultraviolet−visible spectroscopy was adopted to reveal the selfpolymerization of catechol units on AlgDA by UV−vis(Hitachi U-3010) spectrophotometer. The enzyme activity was determined by measuring the decrease of NADH absorbance at 340 nm using UV−vis (Hitachi U-3010) spectrophotometer. The surface properties of the lyophilized Ca−Ti(AlgDA) beads were characterized by XPS in a Perkin-Elmer PHI 1600 ESCA system with a monochromatic Mg Kα source and a charge neutralizer. 2.2.5. Swelling Degree. Fresh beads were weighed and denoted as the initial weight Wi. The beads were then immersed into Tris−HCl solution (50 mM, pH 7.0) at room temperature. The weight of the beads was monitored once every hour until a constant value was reached (Ws). The duration of the monitoring was 31 h. The swelling degree (Sw) was calculated as follows:

cross-linking-reinforced alginate beads (Ca−AlgPDA) were also prepared by self-polymerization of dopamine and cross-linking with Ca2+. The resultant modified alginate beads were utilized to anchor the model enzyme, alcohol dehydrogenase (YADH), through either physical immobilization or covalent linkage.

2. MATERIALS AND METHODS 2.1. Materials. Yeast alcohol dehydrogenase (YADH), nicotinamide adenine dinucleotide (NADH), Tris(hydroxymethyl)amiomethane (Tris), and titanium(IV) bis(ammonium lactato) dihydroxide (Ti-BALDH, 50 wt % aqueous solution) were purchased from Sigma Chemical Company. Sodium alginate (Alg) was purchased from Tianjin Jiang Tian Reagent Chemicals. Dopamine hydrochloride was purchased from Yuancheng Technology Development (Wuhan, China). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Medpep. The water used in all experiments was treated by the Millipore Milli-Q purification system. 2.2. Methods. 2.2.1. Synthesis of Dopamine-Modified Alginate (AlgDA). Sodium alginate (1 g, 5.05 mmol in terms of the repeating unit) was dissolved in 100 mL PBS buffer solution (50 mM, pH 5.5). EDC (968 mg, 5.05 mmol) and NHS (582 mg, 5.05 mmol) were added in the above solution. The reaction mixture was stirred at room temperature for 45 min to fully activate the carboxylic groups on alginate molecules. Then, 1.92 g of dopamine (10.1 mmol) was added into the above mixture and stirred for 12 h at room temperature under N2 protection. The product was precipitated with ethanol three times followed by lyophilization. The assynthesized dopamine-modified alginate was denoted as AlgDA. 2.2.2. Preparation of Dopamine-Modified Alginate Beads. Two kinds of dopamine-modified alginate beads reinforced either by titanium(IV) coordination or by self-polymerization of dopamine were prepared. AlgDA (150 mg) was dissolved in 10 mL of deionized water, and Ti-BALDH was added at a ratio of 3.0 equiv per catechol (calculated by the catechol ratio in the AlgDA). The above mixture (1.5% w/v) was added dropwise into 50 mL of 0.2 M CaCl2 solution through an injection needle. After 30 min incubation, the beads were filtered out, washed thoroughly with deionized water, and stored in the deionized water at 4 °C. The as-formed beads were denoted as Ca−Ti(AlgDA). For preparation of the covalently cross-linking-reinforced alginate beads, 150 mg of AlgDA was dissolved in 10 mL of alkaline Tris−HCl solution (50 mM, pH 8.0) to get a mixture of 1.5% (w/v), and the polymerization was conducted at room temperature for 24 h under stirring. Then, the mixture was added dropwise into 50 mL of 0.2 M CaCl2 solution through an injection needle. After 30 min incubation, the beads were filtered out, washed thoroughly with deionized water, and stored in the deionized water at 4 °C. The beads thus formed were denoted as Ca−AlgPDA. Conventional alginate beads (Ca−Alg) were also prepared for comparison by dissolving Alg 1.5% (w/v) in neutral Tris− HCl solution (50 mM, pH 7.0), followed by dropping into 0.2 M CaCl2 solution. 2.2.3. Immobilization of Enzyme in Ca−Alg, Ca−AlgPDA, and Ca−Ti(AlgDA) Beads. YADH was added in the Alg, AlgPDA, and Ti(AlgDA) solutions, respectively. The three stock solutions containing YADH were then added dropwise into CaCl2 solution to prepare YADH-containing Ca−Alg, Ca−AlgPDA,

Sw =

Ws − Wi × 100% Wi

(1)

2.2.6. Mechanical Strength. The mechanical strength of the capsules was examined using an Electronic Universal Testing Machine (CSS-44001, Changchun Research Institute for Testing Machines, China) at room temperature with a compressing rate of 0.1 mm/min until the bead ruptured. Five replicates were tested and averaged. 2.3. Assay of Immobilized Enzyme. 2.3.1. Immobilization Efficiency. The amount of YADH immobilized was determined by measuring the final concentrations of YADH in CaCl2 solution using Coomassie Brilliant Blue reagent, following the Bradford’s method.28 The immobilization efficiency was determined according to eq 2 E(%) =

(m‐C1V1) × 100% m

(2)

where E(%) represented the immobilization efficiency; m (in mg) was the amount of YADH added; C1(in mg/mL) and V1(in mL) were the final YADH concentration and the volume of CaCl2 solution, respectively. 2.3.2. Activity Assay. The enzyme activity was tested by conducting the reduction reaction of formaldehyde to methanol catalyzed by YADH in the presence of NADH as illustrated in eq 3. A specific amount of the immobilized YADH was added to Tris−HCl solution containing 10 mM HCHO and 133 μM NADH at 25 °C while stirring. The enzyme activity was determined spectrophotometrically by measuring the decrease in the absorbance of NADH at 340 nm after 20 min. One unit 14829

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AlgDA polymer was determined by elemental analysis on C, N, and H. The N/C ratio was found to be 4.49 × 10−2. The N element was from the amine groups on dopamine (C8H11NO2), and the C element was from both alginate (monomer C6H7NaO3) and dopamine. The graft ratio was determined to be 42.0% according to the N/C ratio, given by eq 4:

of enzymatic activity was defined as the quantity of YADH required to consume 1 μmol of NADH per hour. YADH

HCHO + NADH + H+ ←⎯⎯⎯→ CH3OH + NAD+

(3)

2.3.3. Stability Assay. The immobilized YADH was collected after each reaction batch and washed with Tris−HCl solution (50 mM, pH 7.0) to remove any residual substrate and then added to the next reaction cycle. The recycling stability was explored by measuring the enzyme activity in each successive reaction cycle and expressed by recycling efficiency. The effect of pH on the activity for immobilized YADH were determined by measuring the residual activity of enzyme after being exposed to varying pH (3.0−9.0) conditions for 2 h. Immobilized YADH were stored in Tris−HCl (50 mM, pH 7.0) at 4 °C. The storage stabilities were compared in terms of storage efficiency defined as the ratio of the residual activity to the initial activity.

Graft ratio =

6N /C 1 − 8N /C

(4)

Alginate is a linear copolymer consisting of guluronic acids (G) and mannuronic (M) acids that form regions of MM diads, GG diads, and alternating MG diads. The GG diads are the key structural elements responsible for the gelation with divalent cations like Ca2+. The M/G ratio is thus the critical factor affecting the physical properties of alginate and resultant hydrogels.29,30 FTIR spectra were recorded to investigate the conformation of Alg and AlgDA as shown in Figure 2. The peaks

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of DopamineModified Alginate (AlgDA). The synthesis and characterization of AlgDA polymer has been discussed in detail in our previous literature.4 Dopamine-modified alginate (AlgDA) was synthesized using EDC/NHS chemistry as illustrated in Figure 1. First, EDC reacted with the carboxylic groups on alginate to

Figure 2. FTIR spectra of Alg and AlgDA.

at 945 cm−1, 887 cm−1, and 810 cm−1 were attributed to the G residue and M residue, respectively.31−34 These characteristic peaks of alginate were also present in the AlgDA spectrum, but the peak intensity for M residue at 887 cm−1 and 810 cm−1 decreased notably, suggesting that the acylation reaction occurred principally between the M residue and dopamine. In addition, a new peak at 1080 cm−1 attributed to the C−N stretching vibration appeared after dopamine modification, which was a further indicator of the successful acylation. The C−O stretching vibration of alcoholic hydroxyl group at 1122 cm−1 and the C−O−C stretching vibration at 1024 cm−1 in the Alg spectrum35 did not change after dopamine modification. The peaks at 1591 cm−1 and 1405.8 cm−1 were assigned to the asymmetric and symmetric stretching of carboxyl groups.36 The peaks at 1595 cm−1 and 1406 cm−1 assigned to the N−H deformation and C−N stretching vibrations were remarkably strengthened after modification. The broad peak at 3197 cm−1 in Alg spectrum came from the −OH groups, and this peak shifted to 3246 cm−1 in AlgDA spectrum due to the overlapping of −OH and −NH stretching bands. It could be concluded from the FTIR spectra that dopamine had been successfully acylated preferentially to the M residues on alginate and the GG diads were not affected after dopamine modification. Therefore, the gel-forming ability of alginate was reasonably expected to be well maintained. Circular dichroism (CD) has been proposed as a rapid and nondestructive method to determine the monomer composition of alginates in solution.30,37 Hence, CD was used to

Figure 1. Schematic representation of the synthesis process of dopamine-modified alginate (AlgDA).

form an O-acylisourea intermediate, which was very unstable and susceptible to hydrolysis. The addition of NHS could stabilize the O-acylisourea intermediate by converting it to an amine-reactive NHS ester (succinimidyl ester). Then, the amine-reactive ester reacted with the amine groups on dopamine to produce the final dopamine-modified products (AlgDA). During the reaction, N2 was used as the protection gas to prohibit the self-polymerization of dopamine. The assynthesized AlgDA polymer was gray fibrous solid and became colorless when dissolved in water. The graft ratio increased with the increasing content of dopamine. In order to obtain a high graft ratio, the dopamine was two times excessive. The graft ratio of the as-synthesized 14830

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3.2. Beads Formation and Characterization. Two kinds of mussel-inspired alginate beads were developed on the basis of self-polymerization and coordination with titanium(IV) of the as-prepared AlgDA polymer. Before that, Ti(AlgDA) coordination complexes and the polymerized AlgPDA polymer were prepared. 3.2.1. Formation of Ti(AlgDA) Coordination Complexes and AlgPDA Polymer. Alginate can form strong and rigid ordered structures in the presence of divalent and trivalent metal ions but not tetravalent ions like titanium(IV).38 The high positive charge of titanium(IV) leads to a strong affinity toward the highly electronegative catechol groups.39 As we all know, catechol can coordinate with metal ions (Fe3+, Ti4+) and endows the catechol-containing polymer high hardness and high extensibility. Herein, titanium(IV) was introduced to reinforce the hydrogel structure by allowing for additional cross-linking of dopamine side chains besides the Ca2+ crosslinking of alginate matrix. Ti(AlgDA) coordination complexes was produced prior to contacting with Ca2+. Upon addition of Ti-BALDH to the AlgDA polymers solution, an immediate color change from transparent (Figure 4a) to yellow (Figure 4b) was observed.

examine the monomer composition of Alg and AlgDA in detail. The molar ellipticities in the CD spectrum for the MG and GG diads are both negative in the range 190−250 nm, whereas that for the MM diads is positive below 210 nm. The diadic frequencies of Alg and AlgDA were determined according to eqs 5−6 i [θ ]i rec (λ) = F i GG × [θ ]GG (λ) + FMM × [θ ]MM (λ)

+ F i MG + E × [θ ]MG + GM (λ)

(5)

1 = F i GG + F i MM + F i MG + GM

(6)

[θ]irec(λ) was the molar ellipticity in the AlgDA; FiGG, FiMM, and FiMG+GM fractions

where CD spectra for Alg or were the diadic frequencies of Alg or AlgDA corresponding to various residues; [θ]GG(λ), [θ]MM(λ), and [θ]MG+GM(λ) were the molar ellipticity of pure diads in the whole range. According to the literature,30 the [θ]GG(λ), [θ]MM(λ), and [θ]MG+GM(λ) (values all in (deg cm2)/dmol) of pure diads at 198 and 207 nm were listed in Table 1. Table 1. Molar Ellipticity of Alg Diads wavelength

[θ]MM

[θ]MG+GM

[θ]GG

198 nm 207 nm

1214.6 38.16

−879.8 −837.7

−1713.9 −1867.2

The CD spectra of Alg and AlgDA were shown in Figure 3. The values of [θ]irec(198) and [θ]irec(207) were −423.9 and Figure 4. Images of AlgDA (a), Ti(AlgDA) (b), and AlgPDA (c) solutions.

This change in color indicated the formation of the titanium(IV)−catecholate coordination complexes, in each of which three catecholate ligands bound with one titanium(IV).39 Furthermore, the high positive charge of titanium(IV) ions results in a high affinity toward ligands with a negative charge and hard oxygen donor groups. This high affinity causes the facile generation of titanium(IV) tris-catecholate.39 Similar to the natural adhesive proteins, the as-prepared AlgDA would polymerize with each other through an oxidative process under mild alkaline conditions to give the polymerized AlgPDA polymer. Ultraviolet−visible spectroscopy was adopted to reveal the oxidative behavior of the catechol units of AlgDA. Figure 5a showed the UV−vis spectra of a 0.6 mM solution of catechol (calculated by the catechol ratio in the AlgDA). The UV−vis spectrum of AlgDA (0 min) revealed a peak at 280 nm

Figure 3. CD spectra of Alg and AlgDA.

−845.9 (deg cm2)/dmol for Alg, and −742.3 and −1179.0 (deg cm2)/dmol for AlgDA, respectively. According to eqs 5−6, the FiGG, FiMM, and FiMG+GM for Alg and AlgDA were calculated and listed in Table 2. Calculations were based on the principle that Table 2. Diadic Frequencies of Alg and AlgDA sample

FiMM

FiMG+GM

FiGG

Alg AlgDA

0.335 0.299

0.372 0.115

0.293 0.586

modified residues showed no signals in the CD. After dopamine modification, FiMM and FiMG+GM for AlgDA were decreased, suggesting that the carboxylic groups on the MM and MG diads were more accessible for acylation. Moreover, the significant decrease in FiMG+GM after modification, which was 7 times larger than the decrease in FiMM, confirmed that the acylation reaction mainly occurred between dopamine and the carboxylic groups on the MG diads.

Figure 5. Time-dependent UV−vis spectra of AlgDA solution, where [catechol] = 0.6 mM with 2.5 equiv of NaOH per catechol (a) and the corresponding structural formulas of species produced (b). 14831

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Figure 6. Optical images and schematic structural formulas of Ca−Alg beads (a and d), Ca−Ti(AlgDA) beads (b and e) and Ca−AlgPDA beads (c and f).

Figure 7. XPS spectrum (a) and the high-resolution Ti 2p spectrum (b) of the Ca−Ti(AlgDA) beads.

corresponding to the catechol groups on AlgDA,19,33 showing little evidence of oxidized species (300 nm). The structural formula of AlgDA was shown in Figure 5b step 1. Oxidation was triggered by just adding NaOH to the pristine AlgDA solution. The UV−vis spectrum of AlgDA at 5 min after adding NaOH revealed a peak at 300 nm, suggesting that the catechol groups were converted into the o-quinone-type intermediate within 5 min. The structural formula of the o-quinone type intermediate was shown in Figure 5b step 2.40 The absorbance of this intermediate then decreased in intensity and shifted to lower wavelengths over time until 90 min, indicating the continuous consumption of the o-quinone-type intermediates during the self-polymerization of dopamine chains. After 90 min, the absorbance increased in intensity and further shifted to lower wavelengths, indicating the continuous production of the polymerized products, AlgPDA. The polymerized products, AlgPDA, revealed an absorbance peak at 280 nm,41 and its structural formula was shown in Figure 5b step 3. The UV−vis spectra no longer changed after 22 h, indicating the complete polymerization. During the polymerization process, the color of the solution became black and darkened over time (Figure 4c).

3.2.2. Formation of Ca−Alg, Ca−Ti(AlgDA), and Ca−AlgPDA Beads. The control Ca−Alg beads were obtained by crosslinking with Ca2+ ions. The beads produced had a mean diameter of 2 mm by controlling the needle gauge and distance from the CaCl2 solution (Figure 6a). The structure of Ca−Alg beads was expressed by the normal ‘‘egg-box junctions” model (Figure 6d). The Ca−Ti(AlgDA) beads were obtained by adding Ti(AlgDA) solution dropwise into 0.2 M CaCl2 solution. The Ca− Ti(AlgDA) beads were vivid yellow in color, as shown in Figure 6b. The N/Ti ratio of the Ca−Ti(AlgDA) beads calculated from the XPS spectrum (Figure 7a) was 3.0, which suggested the existence of only titanium(IV) tris-catecholate complexes. Moreover, the binding energies of Ti 2p3/2 and 2p1/2 centered at 458.5 and 464.2 eV, respectively, in the highresolution Ti 2p XPS spectrum revealed that the titanium was in an oxidation state of +4. Therefore, structure of the titanium(IV) coordination-reinforced alginate beads could be schematically shown in Figure 6e. The dopamine-modified alginate consisted of two parts: guluronic acid residues and dopamine-modified mannuronic acid residues. The former part cross-linked with Ca2+ ions to form the normal “egg-box 14832

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junctions”, and the latter part coordinated with Ti4+ ions to form the titanium(IV)-catecholate coordination network. After polymerization of the AlgDA polymer, the covalently cross-linking-reinforced alginate beads (Ca−AlgPDA) were obtained by further cross-linking with Ca2+ ions. Ca−AlgPDA beads were dark and were different in color with the Ca−Alg beads (white), as shown in Figure 6c. The structure of the Ca− AlgPDA beads was shown schematically in Figure 6f. Dopamine molecules modified on the mannuronic acid residues polymerized with their adjacent ones to form the covalent linkage network, and the guluronic acid interacted with Ca2+ ions to form the normal egg-box junctions. 3.2.3. Characterization of Ca−Alg, Ca−Ti(AlgDA), and Ca− AlgPDA Beads. The Tg of Alg, AlgPDA, and Ti(AlgDA) polymer were investigated by DSC (Figure 8). The higher the glass

degrees for Ca−Ti(AlgDA) (72.0%) and Ca−AlgPDA (84.8%) were both lower than that of Ca−Alg (136.6%). The selfpolymerization and coordination with titanium(IV) of AlgDA resulted in a more compact network structure, consequently inhibiting swelling. The swelling revealed two underlying molecular processes: penetration of water molecules to the void space in the network and subsequent relaxation of the network segments. The data of the process was fit to a rate equation

Sw = kt n

where k is the gel characteristics constant and n is the kinetic exponent. Equation 7 is a phenomenological law in which n is related to the type of sorption mechanisms of hydrogel beads and defines three situations: for Fickian kinetics, in which the rate of water diffusion is rate-limiting, n equals 0.5; when the rate of water diffusion is far greater than the chain stretching rate, the water uptake is proportional to the time, and thus, n equals 1.0; if a non-Fickian process occurs, n has a value between 0.5 and 1. To get n, log10 Sw (lgSw) was plotted versus log10 t (lgt). Linear fitting was conducted, and n was calculated as shown in Figure 9b. The values of n were 0.62, 0.95, and 1.04 for Ca− Alg, Ca−AlgPDA, and Ca−Ti(AlgDA) beads, respectively. These results indicated that the swelling of Ca−Alg beads followed the non-Fickian mechanism, suggesting a comparative rate between the water diffusion and chain stretching. The higher n for Ca− AlgPDA and Ca−Ti(AlgDA) beads was due to the lower chain stretching rate compared to that of Ca−Alg ones. Therefore, the chain stretching was inhibited by self-polymerization and coordination with titanium(IV). The result was consistent with the DSC measurements. 3.3. Immobilization of Enzyme in Ca−Alg, Ca−AlgPDA, and Ca−Ti(AlgDA) Beads. To demonstrate the feasibility of the dopamine-modified alginate beads as enzyme immobilization supports, YADH-containing Ca−Alg, Ca−AlgPDA, and Ca−Ti(AlgDA) beads were prepared. YADH was added to Alg, AlgPDA, and Ti(AlgDA) stock solutions, then the stock solutions were added dropwise into 0.2 M CaCl2 solution. The immobilization efficiency over a range of enzyme concentrations was investigated, and the result is shown in Figure 10a. The immobilization efficiency of YADH in the Ca− Ti(AlgDA) beads and the Ca−AlgPDA was 100% and 89.0%, respectively, which was higher than that in Ca−Alg beads (67.4%) at the same enzyme concentration of 0.2 mg/mL. The high yield of enzyme immobilization raised the question of whether the enzyme was physically entrapped within or covalently grafted with the dopamine-modified alginate beads. To investigate the existing state of immobilized enzyme, the enzyme molecules were released from Ca−AlgPDA and Ca− Ti(AlgDA) beads by incubating the beads in EDTA solution. EDTA can chelate with Ca2+ and, thus, collapse the beads. After incubation for 4 h, the Ca−AlgPDA collapsed into black flocculent materials; that is, the polymerized AlgDA (Figure 10b solution 1). After mixing with Coomassie Brilliant Blue, a dye for staining proteins, the black flocculent materials became blue (Figure 10b solution 2), confirming that the immobilized enzymes were covalently grafted with AlgPDA. The color of supernate (Figure 10b solution 3) slightly changed. UV−vis absorption suggested that about 8.0 wt % of YADH existed in the supernate of solution 1. This result suggested that most of enzymes were covalently grafted with AlgPDA, whereas a small amount of enzyme exited in its free state in the beads. Hence,

Figure 8. DSC curves of Alg, AlgPDA, and Ti(AlgDA).

transition temperatures (Tg) of the polymer, the higher the cohesive energy density and solubility parameter that have the relationship to the intermolecular forces.42 The Tg of Alg was estimated to be 80.6 °C. For AlgPDA and Ti(AlgDA), Tg was increased to 106.1 and 112.6 °C, respectively, indicating that the self-polymerization and coordination with titanium(IV) of AlgDA resulted in a more compact network structure with a decreased segmental mobility of polymers chains. The mechanical strength is an important factor for enzymecontaining beads, in regard to their practical application. The compression intensity, an index to represent the mechanical strength of the beads, was tested (Table 3). The compression Table 3. Compression Intensity of Ca−Alg, Ca−AlgPDA, and Ca−Ti(AlgDA) Beads sample

compression intensity (N)

Ca−Alg Ca−AlgPDA Ca−Ti(AlgDA)

4.7 ± 0.7 16.4 ± 1.02 14.2 ± 1.14

(7)

intensity of Ca−Alg beads was 4.7 N, which was close to that reported in the literature.43 The mechanical strength of Ca− AlgPDA and Ca−Ti(AlgDA) beads were remarkably enhanced by about 3 times compared to that of Ca−Alg beads because of the self-polymerization and coordination with titanium(IV). To study the swelling characteristics of enzyme-containing beads during the conversion reaction process, dried samples were immersed in Tris−HCl solution and the swelling degrees were measured, as shown in Figure 9a. It can be seen that all beads had a similar swelling tendency. The swelling degree increased quickly in the first 8 h, and afterward plateaued at a nearly constant swelling degree. The equilibrium swelling 14833

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Figure 9. Swelling degree of hydrogels in Tris−HCl butter solution (50 mM pH 7.0) (a), and the curves of lgSw as a function of lgt (b).

Ca−Alg beads (26.2 U mg−1 enzyme). This result was due to the appreciable compact network for Ca−AlgPDA, owing to the self-polymerization of catechol, which greatly increasing the mass transfer resistance. The activity of YADH immobilized in the Ca−Ti(AlgDA) beads was 25.8 U mg−1 enzyme, which was comparable to that of YADH loaded in Ca−Alg beads (26.2 U mg−1 enzyme). Recycling stability was very important for immobilized enzymes because of their potential industrial applications. The recycling stability was investigated by conducting successive batches of formaldehyde conversion and is shown in Figure 11a. After 6 batches, YADH immobilized in the Ca− Alg, Ca−AlgPDA, and Ca−Ti(AlgDA) beads retained about 50.6%, 60.1%, and 68.7%, respectively, of their initial activities. The decrease in activity was due to the loss of enzyme after swelling and enzyme inactivation during reaction.44 The swelling degrees of Ca−Ti(AlgDA) and Ca−AlgPDA were smaller than that of Ca−Alg, therefore resulting in better recycling stability. The Ca−Ti(AlgDA) beads exhibited the lowest swelling degree and consequently the highest recycling stability. The pH stability of the immobilized YADH was probed. YADH immobilized in the Ca−AlgPDA and Ca−Ti(AlgDA) beads showed higher activities over a wider pH range than that of YADH immobilized in the Ca−Alg beads, suggesting enhanced pH stability after reinforcement by titanium(IV) coordination and self-polymerization of AlgDA (Figure 11b). Upon incubation at high temperature, the decrease in activities of YADH immobilized in beads was caused by denaturation of enzyme molecules and increased mass transfer resistance after beads’ contraction. YADH immobilized in Ca− Alg, Ca−AlgPDA, and Ca−Ti(AlgDA) presented similar trends below 45 °C and lost about 23% of initial activities due to the same contraction rate (Figure 11c). When incubation was performed at 60 °C, YADH immobilized in Ca−AlgPDA and Ca−Ti(AlgDA) lost 27% and 55%, respectively, of their activity, which was lower than that of YADH immobilized in Ca−Alg (81%). The result suggested that YADH immobilized in Ca− AlgPDA experienced a good thermal stability because of the following two aspects: (1) The AlgPDA was biocompatibe and preserved the structure of enzyme molecules. (2) YADH was covalently attached to AlgPDA, and the interaction between YADH and AlgPDA prevented the inactivation of YADH when incubation at high temperature. The storage stability of the immobilized YADH in the Ca− AlgPDA beads and the Ca−Ti(AlgDA) beads was notably enhanced compared to that immobilized in the Ca−Alg beads owing to the reduced leakage and reduced inactivation effect during storage in the dopamine-modified alginate beads

Figure 10. The immobilization efficiency of Ca−Alg, Ca−Ti(AlgDA), and Ca−AlgPDA beads as a function of enzyme concentrations (a), and the images of solutions (b). (1) YADH-containing Ca−AlgPDA in EDTA solution; (2) precipitates in solution 1 with Coomassie Brilliant Blue; (3) supernate in solution 1 with Coomassie Brilliant Blue; (4) YADH-containing Ca−Ti(AlgDA) in EDTA solution; (5) solution 4 with Coomassie Brilliant Blue; (6) Coomassie Brilliant Blue reagent.

the high immobilization efficiency of Ca−AlgPDA beads was due to the covalent binding of enzymes with the catechol groups. The Ca−Ti(AlgDA) beads dissolved and became a transparent yellow solution (Figure 10b solution 4). After being mixed with Coomassie Brilliant Blue, the color of the solution became blue (Figure 10b solution 5), confirming that the enzyme was physically entrapped in the Ca−Ti(AlgDA) beads and leaked out when the beads collapsed. In addition, the content of YADH in the solution was identical with that immobilized in the Ca− Ti(AlgDA) beads as verified by the absorbence of solution 5 (data not shown). The high immobilization efficiency of Ca− Ti(AlgDA) beads was attributed to the electrostatic interaction between the positive titanium(IV) and the negatively charged YADH (pI 5.4) under immobilization conditions. The activities of YADH immobilized in the Ca−Alg, Ca− AlgPDA, and Ca−Ti(AlgDA) beads were compared. The activity of YADH immobilized in the Ca−AlgPDA beads (8.5 U mg−1 enzyme) was far lower than that of YADH immobilized in the 14834

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Figure 11. The recycling stability (a), pH stability (b), temperature stability (c), and storage stability (d) of the Ca−Alg, Ca−AlgPDA, and Ca− Ti(AlgDA) beads.

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ACKNOWLEDGMENTS The authors thank the financial support from the National Science Fund for Distinguished Young Scholars (21125627), National Basic Research Program of China (2009CB724705), the National Science Foundation of China (21076145), Program for New Century Excellent Talents in University (NCET-10-0623), and the Program of Introducing Talents of Discipline to Universities (B06006).

(Figure 11d). The YADH immobilized in the Ca−AlgPDA beads, particularly, retained 77% of its initial activity after storage for 50 days, showing a promising potential for practical application.

4. CONCLUSIONS Two novel kinds of dopamine-modified alginate gel beads reinforced by using titanium(IV) coordination and selfpolymerization of dopamine were fabricated. Alginate was modified with dopamine by EDC/NHS coupling chemistry. The gel-forming ability of alginate was well retained because the modification of dopamine preferentially occurred at the mannuronic acid residues. The swelling of modified alginate beads was inhibited and the mechanical properties were obviously enhanced compared the properties of the Ca−Alg beads because of the reinforcement by self-polymerization and coordination with titanium(IV). The YADH was covalently immobilized in the Ca−AlgPDA beads, leading to a high immobilization efficiency. The stability of the immobilized YADH in dopamine-modified alginate beads was significantly enhanced. The modification approach methods in this study may be extended to a wide variety of materials to give desirable physicochemical properties due to the wide applicability of catechol.

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REFERENCES

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Corresponding Author

*H. Wu. Fax: +86 022 2789 0882. Phone: +86 22 2350 0086. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 14835

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