Performance comparison of immobilized enzyme on the titanate nanotube surfaces modified by poly(dopamine) and poly(norepinephrine)

Abstract

Novel hybrid carriers for enzyme immobilization, titanate nanotubes (TNTs) coated by poly(dopamine) (pDA) and poly(norepinephrine) (pNE), have been fabricated by a facile bio-inspired approach under mild conditions in this study. Catalase (CAT) has been immobilized on the TNT surface modified by pDA and pNE as a model enzyme, and their activity and stability have been compared. The immobilization capacities of TNT-pDA and TNT-pNE for CAT are 311.7 and 245.6 mg of g per support, respectively; while their corresponding relative activities are 52% and 56% of native CAT, respectively. It is found that the surface roughness play an important role for the performance difference of immobilized CAT. With the increase of surface roughness, the enzyme loading increases; however, the ratio of active proteins to adsorbed total proteins decreases, witnessing a reduction in the accessibility of enzyme active sites.

---

1. Introduction

Nanobiocatalysis, including the biocatalysis by nanomaterials-immobilized enzymes, becomes a rapidly growing research field in recent years, since nanomaterials possess many merits as enzyme immobilization carriers/scaffolds, such as high specific surface area, controlled size at the nanometer scale, uniform size distribution and similarity in size with enzyme molecules.^1–4 So far, the common immobilization strategy for enzyme on the nanomaterial surface is the covalent binding, which often needs chemically modify the enzyme by using the toxic chemical reagent.^5–7 Furthermore, the modification process can distort the native conformation of enzyme molecules in most cases, and then result in a decrease in enzyme activity or other catalytic performance. Therefore, a green efficient strategy for enzyme immobilization on the nanomaterial surface under mild conditions is urgently desired nowadays.

Recently, mussels have attracted much interest of researchers in the fields including materials science, enzyme immobilization and biomineralization etc., because of their rapid, strong and tough adhesive ability to virtually any type of solid surface, so-called bioadhesion.⁸ An extensive repeat of 3,4-dihydroxy-L-phenylalanine (DOPA) and lysine residues, which was found in the mussel adhesive protein Mefp-5 (Mytilus edulis foot protein-5) located at the top of the adhesive pad, plays a crucial role in bioadhesion. Catecholamines have been studied as ‘minimalist mimics’ of Mefp-5, in which the catechol is derived from a side chain of DOPA, and the amine is a side chain of the basic amino acid lysine.⁹ Catecholamines and their polymers have been widely applied for the functionalization of materials surface under ambient conditions, because of good biocompatibility, excellent environmental stability and high hydrophilicity.¹⁰ Among various catecholamine molecules, dopamine (DA) and norepinephrine (NE) have been extensively utilized as the surface modification reagent to achieve functional surfaces with bio-inert,⁸ bio-active,^11,12 and cell-adhesive properties successfully.^13–15 Furthermore, the catechol and quinone groups in the poly(dopamine) (pDA) or poly(norepinephrine) (pNE) coating are chemically active to various thiol- or amine-containing molecules, thus making it easily bond enzyme without additional coupling reagent. Therefore, it may be a feasible approach to immobilize enzyme on the nanomaterial surface modified by catecholamines or their polymers.

Compared with commonly-used inorganic nanoparticles, titanate nanotubes (TNTs), as a typical one-dimension (1D) nanostructure, can afford unique benefits as an immobilized carrier of enzyme in view of the following features.^16,17 First, its nanotubular structure is expected to have a high surface-to-volume ratio and well confine with the attached enzyme molecules. Second, its surface possesses many hydrophilic Ti–OH groups, which renders TNT good biocompatibility and facile modification. Third, it can be easily recycled from a solution by precipitation or filtration owing to its 1D nanostructure and high mechanical stability. Thus far, TNT-based materials have been applied in various fields, such as dye-sensitized solar cell,¹⁸ photocatalysis,¹⁹ biosensing,²⁰ and drug delivery.²¹ In our previous work,²² catalase (CAT) was pre-modified by 3-(3,4-dihydroxyphenyl) propionic acid (3,4-diHPP) via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry, and then covalently immobilized on the TNT surface by the chelation of catechol groups with Ti⁴⁺ ions. The immobilized catalase exhibits excellent catalytic performance, however, the immobilization procedure is tedious and not environmentally benign. Therefore, it should be of significant interest to investigate the enzyme immobilization on the TNT surface modified with catecholamine inspired by bioadhesion.

In this study, TNTs pre-coated by pDA and pNE were employed as the nano-carrier for pursuing the green efficient enzyme immobilization under mild conditions. TNT was synthesized by a facile and cost-effective alkaline hydrothermal method, and then modified by pDA and pNE coatings to obtain the surfaces with different structures. CAT was selected as the model enzyme to be immobilized on these two surfaces because of its well-defined structure and properties, as well as its widespread applications.^23–25 Moreover, the enzyme loading efficiency, relative catalytic activity and thermal, storage and recycling stability of two immobilized CATs were compared.

2. Experimental

2.1 Materials

Catalase (EC 1.11.1.6 from bovine liver, 2.48 × 10⁴ U mg⁻¹), tris(hydroxymethyl) aminomethane (Tris) and norepinephrine were supplied by Sigma-Aldrich Chemical Co. Ltd. (USA). Dopamine was purchased from Yuan Cheng Technology Development Co. Ltd. (Wuhan, China). Rutile TiO₂ powder (99.8%, 60 nm) was purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). Other reagents were analytical grade, and used without further purification. The water used in the experiments was purified by a three-stage Millipore Milli-Q Plus system with a resistivity higher than 15.0 MΩ cm.

2.2 Preparation of TNT-pDA and TNT-pNE

TNT was prepared by a facile hydrothermal method as previously reported.²² In a typical procedure, 2 g nano-sized rutile TiO₂ powders were firstly dispersed in 85 mL of NaOH solution (10 mol L⁻¹). Then, the suspension was transferred into a sealed Teflon-lined container, and statically heated for 72 h at 130 °C. The white precipitate was obtained after centrifuged, washed with excess deionized water, and soaked in abundant HCl solution (0.1 mol L⁻¹) for 10 h, followed by washing with deionized water until pH 7.0. Finally, alcohol was used to disperse the white precipitate in order to displace water on the TNT surface and enable the formation of aggregation-free nanotubes.

The preparation process of TNT-pNE is as follows: TNT (50 mg) was suspended in 20 mL of Tris–HCl buffer solution (50 mmol L⁻¹, pH 8.5) and ultrasonically treated for 15 min. Afterwards, 40 mg of norepinephrine powder was added into the TNT suspension, and the whole mixture was stirred at room temperature for 24 h. During this incubation step, norepinephrine monomers were polymerized to generate adhesive pNE layer on the TNT surface. Finally, TNT coated by pNE (TNT-pNE) was obtained after separated by centrifugation and washed with distilled water for several times to remove barely adsorbed pNE residues. Similarly, TNT coated by pDA (TNT-pDA) was prepared by the same approach except substituting norepinephrine for dopamine.

2.3 Immobilization of catalase on TNT-pDA and TNT-pNE

Catalase was selected as a model enzyme to be immobilized onto the TNT-pDA and TNT-pNE surface, respectively. At first, the as-prepared TNT-pDA or TNT-pNE was added in the CAT solution (20 mL, 2 mg mL⁻¹, pH = 7 Tris buffer solution) and gently stirred with magnetic stirrer for 4 h at room temperature. Then, the immobilized CAT was separated by centrifugation and washed several times with pH = 7 Tris buffer solution to remove the free and loosely bound CAT. The supernatant after immobilization was collected to determine the amount of CAT bound to the TNT-pDA or TNT-pNE surface. The wet immobilized CAT was stored at 4 °C for use afterwards. The amount of CAT loaded on the TNT-pDA or TNT-pNE was calculated by the difference of protein concentrations in the solution before and after immobilization. Bradford method was applied for measuring the protein concentration with bovine serum albumin (BSA) as the standard. The amount of enzyme immobilized on the TNT-pDA or TNT-pNE was calculated by mass balance with the following equation:In eqn (1), C₀ is the initial enzyme concentration (mg mL⁻¹), C₁ is the final enzyme concentration (mg mL⁻¹), V is the volume of enzyme solution (mL), and W_s is the support dose added (g).

2.4 Characterization

The topological morphology of TNT-pDA and TNT-pNE was observed by a high-resolution transmission electron microscopy (HRTEM, IEM-100CX II) instrument. The FTIR spectra were obtained by using a Nicolet-6700 Fourier transform infrared spectrometer (Nicole NEXUS 670, USA), and thirty-two scans were accumulated with a resolution of 4 cm⁻¹ for each spectrum. Thermal gravimetric analysis (TGA) was carried out with a Perkin-Elmer thermos-gravimetric analyses apparatus. Nanoscale study of the structure and surface roughness of pDA or pNE-coated quartz plate was investigated by using a Keysight-5500 atomic force microscopy (AFM, Keysight-5500, Germany). A Hitachi U3010 UV-vis spectrophotometer (Hitachi U3010) was used to analyze the concentration and activity of CAT.

2.5 Activity assay of immobilized catalase

The activities of free and immobilized CAT were measured using hydrogen peroxide as substrate in aqueous medium (pH = 7.0) at room temperature. Briefly, 0.1 mL of free or as-prepared immobilized CAT solution (1 mg mL⁻¹) was mixed with 20 mL of H₂O₂ solution (20 mmol L⁻¹) prepared by a Tris–HCl buffer solution (50 mmol L⁻¹, pH = 7.0). Then, the system was kept stirring for 3 min at room temperature, and the decomposition of H₂O₂ by CAT was observed by measuring the absorbance decrease at 240 nm, a characteristic absorption peak of H₂O₂. One unit of catalytic activity is defined as the enzyme amount decomposing 1 μmol H₂O₂ per minute under the assay conditions (25 °C, pH = 7.0). The CAT concentration was determined by Bradford method, and the specific activity of free and immobilized enzyme was given as U mg of per protein. Each result was obtained by averaging three individual experiments.

The kinetic parameters of free and immobilized CAT were investigated by using the classical Michaelis–Menten equation (eqn (2)):

In eqn (2), V (mmol (L min)⁻¹) is the initial reactive rate; V_max (mmol (L min)⁻¹) is the maximal velocity of the reaction; [S] (mmol L⁻¹) is the initial substrate concentration; and K_m (mmol L⁻¹) is the Michaelis–Menten constant. The activities of free and immobilized CAT were measured by using H₂O₂ solution with different concentrations (5–35 mmol L⁻¹, 50 mmol L⁻¹ Tris–HCl buffer solution, and pH = 7.0) as the substrate. V_max and K_m were calculated by the Lineweaver–Burk plot.

2.6 Stability of immobilized catalase

The stability experiments of immobilized CAT include three parts, i.e. the thermal, storage, and recycling stability. Briefly, the thermal stability was investigated by measuring the residual activities of free and immobilized CAT after incubation in a 50 mmol L⁻¹ of Tris–HCl buffer (pH = 7.0) with different temperatures (30–60 °C) for 2 h. The storage stability was determined through selectively measuring the residual activities of free and immobilized CAT after stored for 3, 7, 11, 16, 28, 40, 50, and 60 days at 4 °C, respectively. The recycling stability was determined by measuring the residual activity of immobilized CAT after each reaction cycle at room temperature and neutral pH. Simply, 0.1 g of immobilized CAT was dispersed in a 20 mL H₂O₂ solution at 25 °C and pH = 7.0, and their activity was tested as described above. Subsequently, the immobilized CAT were collected by centrifugation accompanied by thoroughly being rinsed with Tris–HCl buffer solution (50 mmol L⁻¹, pH = 7.0) and then reused in a fresh reaction medium for the next reaction cycle. In all the stability experiments, the initial activity of immobilized CAT is assumed as 100%, while other activities are the relative values in comparison with the initial activity. Each result was obtained by averaging three individual experiments.

3. Results and discussion

3.1 Bio-inspired adhesion immobilization of CAT on TNT-pDA and TNT-pNE

As shown in Scheme 1, the bio-inspired adhesion immobilization procedure of CAT on the TNT-pDA and TNT-pNE surface is divided into two steps. At first, TNT is immersed in an alkaline aqueous solution of DA or NE for several hours to form an adherent pDA or pNE coating on the TNT surface. The oxidation of DA yields an intermediate called 5,6-dihydroxyindole (DHI),²⁶ which acts as a monomer for subsequent polymerization. In the polymerization process of DA, unavoidable nano/microparticulate aggregates of pDA or oligo-DA in solution simultaneously attach and grow directly from the functionalized surface, resulting in a significant increase of surface roughness. Whereas, the oxidative polymerization of NE produces a different intermediate, 3,4-dihydroxybenzaldehyde (DHBA), which may react with NE via a reversible Schiff-base formation. The produced DHBA-NE plays an important role in decreasing the roughness of pNE-functionalized surfaces at the nanometer scale.²³ In the second step, the prepared TNT-pDA or TNT-pNE is used as support to immobilize CAT. The residual quinone groups on the pDA or pNE coating are reactive toward nucleophilic groups, which can couple covalently with CAT through Michael addition and/or Schiff-base formation.²⁷

Scheme 1 Illustration of CAT immobilization on the TNT-pNE and TNT-pDA surface by the bio-inspired adhesion strategy.

3.2 Comparison of TNT-pDA with TNT-pNE

Fig. 1 illustrates typical TEM images of the pristine TNT, TNT-pDA and TNT-pNE prepared in this work. The image of TNT (Fig. 1a) demonstrates that TNT possesses a hollow tubular structure with the inside and outside diameter around 4 and 10 nm, respectively. After modified by pDA or pNE, TNT can keep the nanotubular morphology very well, however, the thickness and morphology of polymer layers are different obviously (Fig. 1b and c). As exhibited in the higher resolution images of single TNT-pDA (inset in Fig. 1b), the pDA layer becomes a rough surface about 4–6 nm in thickness. Comparatively, the pNE coating (inset in Fig. 1c) is a well-controlled, nearly smooth surface about 3 nm in thickness.

Fig. 1 TEM images of TNT (a), TNT-pDA (b) and TNT-pNE (c). The insets in (a and b) and (c) are high-resolution images of single TNT, TNT-pDA and TNT-pNE, respectively.

Since it is difficult to determine the topological structure of TNT-pDA and TNT-pNE surface directly using the AFM technology, the quartz plates modified by pDA and pNE were prepared to be utilized as the model. As illustrated in Fig. 2, the pDA coating on the quartz plate has some protuberances about 100–200 nm in size with a standard deviation roughness value (rms roughness) of 9.6 nm. As for the pNE coating, the surface protuberance is less than that of pDA coating, and the standard deviation roughness value is about 4.3 nm. This result further confirms that the roughness of pDA surface is larger than that of pNE surface under the same conditions, in coordination with the literatures.²⁸ After the surfaces of pDA and pNE coated quartz plates load the CAT, their roughnesses increase to be about 31.6 and 14.1 nm, respectively, indicating that the pDA surface can load more enzyme than the pNE surface. Water contact angle can provide the information on the wettability and surface energy of the substrate, which has been widely used to track and evaluate the effectiveness of surface modification protocols. The water contact angles of the pDA and pNE coating on the quartz plate are 47.6° and 34.8°, respectively, suggesting that the pNE coating has higher wettability than the pDA coating. As shown in Scheme 1, a number of hydrophilic moieties, such as hydroxyl and quinonyl groups, exist in the molecular structure of pDA and pNE, which can obviously enhance the surface wettability of TNT. Compared with DA, NE owns another hydroxyl group that links to the aliphatic carbon atom beside the catechol, which enhances the hydrophilicity of functionalized surfaces. Therefore, the surface wettability of pNE-coated substrate is higher than that of pDA-coated substrate. The relatively stable water contact angle suggests that this novel modification method efficiently increases the wettability of TNT, which is benefited for manipulating the nanoscale environment of enzyme molecule, and thus holding their biological function and stability.

Fig. 2 Three-dimension (a) and topological (b) AFM images and height profiles (c) of quartz plates coated by pDA, pNE, pDA-CAT and pNE-CAT.

In order to identify the composition of TNT-pDA and TNT-pNE, their FTIR spectra were conducted. As exhibited in Fig. 3a, the FTIR spectrum of TNT has two absorption bands at 1623 and 467 cm⁻¹, which are assigned to H–O–H bending for water on the TNT surface and O–Ti–O stretching vibration, respectively.²⁹ Besides these two bands, FTIR spectra of TNT-pDA and TNT-pNE exhibit four same bands at 1613, 1484, 1261, and 1058 cm⁻¹, which are ascribed to C=C stretching vibration of aromatic ring, N–H bending vibration, C–OH stretching vibration of phenolic, and C–O stretching vibration of carbonyl group in pDA or pNE, respectively.^30,31 Compared with TNT-pDA, TNT-pNE shows a new band at 1401 cm⁻¹, which can be attributed to O–H bending vibration, in accordance with the pNE structure. These results further confirm that pDA and pNE coat on the TNT surface successfully.

Fig. 3 FTIR spectra (a) and TGA scans (b) of TNT, TNT-pDA and TNT-pNE.

Thermogravimetric analysis (TGA) of TNT, TNT-pDA and TNT-pNE samples was carried out to quantify the amount of pNE and pDA modified on the TNT surface. As shown in Fig. 3b, the TGA curve of pristine TNT reveals a three-step weight loss: room temperature–260 °C (4.8 wt%), 260–520 °C (21.7 wt%) and 520–700 °C (0.5 wt%). The first step can be attributed to the removal of adsorbed water; while the second step can be assigned to the removal of surface –OH groups and TiO₂ crystallization. When the temperature is higher than 520 °C, the sample weight hardly changes further. The TGA curves of TNT-pDA and TNT-pNE exhibit the similar weight-loss procedure to that of TNT, in which the pDA and pNE should be thermally decomposed in the second step. Therefore, the amount of pDA or pNE coating on the TNT surface can be evaluated by comparing the weight loss of TNT-pDA or TNT-pNE with that of TNT during 260–520 °C. It is calculated that TNT-pDA and TNT-pNE contain 27.8 and 4.7 wt% pDA and pNE, respectively. The amount of pDA coating on the TNT surface is much higher than that of pNE, which is similar to the result reported by Lee et al.³²

3.3 Comparison of TNT-pDA-CAT with TNT-pNE-CAT

Under alkaline conditions, the pDA and pNE coatings are expected to retain residual quinone groups that are reactive toward biomolecules containing amine and thiol groups. Thus, their facile conjugation with proteins can be easily achieved due to the presence of N-terminal and lysine amine in proteins. The immobilization capacity of TNT-pDA and TNT-pNE were evaluated by the CAT loading. The loading ratios of TNT-pDA-CAT and TNT-pNE-CAT are 311.7 and 245.6 mg of g per support (Table 1), respectively; while their corresponding relative activities are 52% and 56% of native CAT, respectively. This result indicates that as the surface roughness increases, the enzyme amount immobilized on the carrier enhances; while the relative enzyme activity decreases. The rough surface possesses higher surface area and more reaction sites than the smooth surface, thus leading to higher enzyme loading. However, the uneven surface can increase the steric hindrance of immobilized enzyme, thereby impeding the accessibility of the substrate with enzyme active center (Scheme 1).

Table 1 Immobilization efficiency and kinetic parameters of free CAT, TNT-pDA-CAT and TNT-pNE-CAT

	Loading ratio (mg g^−1)	Relative activity (%)	Kinetic parameters
			Km/mmol L^−1	Vmax/μmol H2O2 (mg protein min)^−1
Free CAT	—	—	43.6	43.4
TNT-pDA-CAT	311.7	52.0	11.6	28.5
TNT-pNE-CAT	245.6	56.0	18.5	32.3

---

The apparent Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max) were determined by measuring the initial rates of free and immobilized CAT with different substrate concentrations ranging from 5 to 35 (mmol L⁻¹) at 25 °C and then calculating from the Lineweaver–Burk plots (Table 1). Comparing to free enzyme, the V_max of TNT-pDA-CAT decreases by 34.3%; while the V_max of TNT-pNE-CAT decreases by about 25.6%. The V_max decrease for immobilized enzyme is a common phenomenon in the enzyme immobilization, which may be caused by either the conformation change of enzyme during the immobilization procedure, or a less accessibility to the active site of immobilized enzyme due to the steric hindrance of substrate.^33,34 The K_m values of TNT-pDA-CAT and TNT-pNE-CAT are 11.6 and 18.5 mmol L⁻¹, respectively, which are much lower than that of free CAT (43.6 mmol L⁻¹). The K_m decrease indicates a stronger affinity between immobilized CAT and substrate. There are mainly two reasons to explain this phenomenon: on one hand, the modification with pDA and pNE can improve the surface property of TNT, thus allowing the adsorbed enzyme to maintain its structural integrity and activity; on the other hand, the strongly hydrophilic surface can enhance the enrichment of enzyme molecules on the carrier, thus shortening the mass transfer distance and well keeping the enzyme's flexibility after immobilization.³⁵

Thermal stability experiments were conducted for free and two kinds of immobilized enzymes, which were incubated in water bath at 4–60 °C for 2 h. As can be seen from Fig. 4a, the highest activity for TNT-pDA-CAT and TNT-pNE-CAT is obtained at 30 °C, in consistence with free CAT. With the increase of incubation temperature, the activity of free CAT decreases more sharply than that of immobilized CAT. Approximately 47.7% activity of free CAT is retained after incubation at 50 °C for 2 h; while TNT-pDA-CAT and TNT-pNE-CAT retain about 59.5% and 52.0% of their activities, respectively. Catalase is a tetramer, which consists of four equal subunits with a 60 kDa molecular weight. These subunits can afford multiple anchoring sites with pDA/pNE coated surfaces, thereby enhancing the overall rigidity and thermal stability of CAT.^36–38 Nevertheless, the immobilization on the nanomaterial outside surface is not a good strategy to improve the thermal stability of enzyme, due to the enzyme can be directly affected by the change of external environment. The storage stability of free and immobilized CATs was examined in Tris–HCl buffer (50 mmol L⁻¹, pH 7.0) at 4 °C for 60 days by measuring the residual activity. As exhibited in Fig. 4b, the activity of immobilized CATs decreases slowly compared with free CAT. After 60 days' storage, free CAT only keeps 40% of its initial activity under the same conditions; while TNT-pDA-CAT and TNT-pNE-CAT retain approximately 95% and 92% of the initial activity, respectively. The enhanced storage stability of immobilized enzyme can be ascribed to the strong covalent coupling between pDA/pNE and enzyme molecules, as well as the moderate microenvironment provided by TNT-pDA or TNT-pNE. The operational stability is an important performance in the practical application of immobilized enzyme. In Fig. 4c, it can be seen that TNT-pNE-CAT exhibits slightly higher residual activity than TNT-pDA-CAT. After 10 consecutive operations for TNT-pDA-CAT and TNT-pNE-CAT, their residual activities are 48.7% and 45.0% of their initial activities, respectively.

Fig. 4 Thermal (a), storage (b), and recycling (c) stability of free and immobilized enzymes.

4. Conclusions

In summary, the catalase is immobilized on the TNT surface modified with pDA and pNE coatings by a bio-inspired adhesion method, respectively, and their catalytic performances are compared. The immobilization capacities of TNT-pDA and TNT-pNE for CAT are 311.7 and 245.6 mg of g per support, respectively; while their corresponding relative activities are 52% and 56% of native CAT, respectively. It is found that the surface roughness play an important role for the performance difference of immobilized CAT. As the surface roughness increases, the enzyme amount immobilized on the modified TNT surface increases; while the relative enzyme activity decreases. The rough surface can increase the steric hindrance of immobilized enzyme, thereby impeding the accessibility of the substrate with enzyme active center. However, the nano-surface roughness has little effect on the stability of immobilized enzyme.

Acknowledgements

The authors thank the financial support from the National Science Fund for Distinguished Young Scholars (21125627), the Program of Introducing Talents of Discipline to Universities (B06006) and the National Natural Science Funds of China (21406163).

Notes and references

1. J. Kim, J. W. Grate and P. Wang, Trends Biotechnol., 2008, 26, 639–646.
2. J. Ge, C. Yang, J. Zhu, D. Lu and Z. Liu, Top. Catal., 2012, 55, 1070–1080.
3. J. Kim, B. C. Kim, D. Lopez-Ferrer, K. Petritis and R. D. Smith, Proteomics, 2010, 10, 687–699.
4. K. Min and Y. J. Yoo, Biotechnol. Bioprocess Eng., 2014, 19, 553–567.
5. K.-P. Lee, S. Komathi, N. J. Nam and A. I. Gopalan, Microchem. J., 2010, 95, 74–79.
6. A. S. S. Ibrahim, A. A. Al-Salamah, A. M. El-Toni, M. A. El-Tayeb and Y. B. Elbadawi, Electron. J. Biotechnol., 2014, 17, 55–64.
7. Q. Li, F. Fan, Y. Wang, W. Feng and P. Ji, Ind. Eng. Chem. Res., 2013, 52, 6343–6348.
8. H. Lee, S. M. Dellatore, W. M. Miller and P. B. Messersmith, Science, 2007, 318, 426–430.
9. S. M. Kang, S. Park, D. Kim, S. Y. Park, R. S. Ruoff and H. Lee, Adv. Funct. Mater., 2011, 21, 108–112.
10. J. Sedó, J. Saiz-Poseu, F. Busqué and D. Ruiz-Molina, Adv. Mater., 2013, 25, 653–701.
11. M. E. Lynge, R. van der Westen, A. Postma and B. Städler, Nanoscale, 2011, 3, 4916–4928.
12. L. Guo, Q. Liu, G. Li, J. Shi, J. Liu, T. Wang and G. Jiang, Nanoscale, 2012, 4, 5864–5867.
13. M. Park, M. Shin, E. Kim, S. Lee, K. I. Park, H. Lee and J.-H. Jang, J. Nanomater., 2014, 793052.
14. S. M. Kang and H. Lee, Bull. Korean Chem. Soc., 2013, 3, 960–962.
15. K. Yang, J. S. Lee, J. Kim, Y. B. Lee, H. Shin, S. H. Um, J. B. Kim, K. I. Park, H. Lee and S. W. Cho, Biomaterials, 2012, 33, 6952–6964.
16. T. Kasuga, Thin Solid Films, 2006, 496, 141–145.
17. M. D. Hernandez-Alonso, S. Garcia-Rodriguez, B. Sanchez and J. M. Coronado, Nanoscale, 2011, 3, 2233–2240.
18. L. Zhao, J. Yu, J. Fan, P. Zhai and S. Wang, Electrochem. Commun., 2009, 11, 2052–2055.
19. L. Xiong, W. Sun, Y. Yang, C. Chen and J. Ni, J. Colloid Interface Sci., 2011, 356, 211–216.
20. A. Liu, M. D. Wei, I. Honma and H. Zhou, Adv. Funct. Mater., 2006, 16, 371–376.
21. F. Fenyvesi, Z. Kónya, Z. Rázga, M. Vecsernyés, P. Kása Jr, K. Pintye-Hódi and I. Bácskay, AAPS PharmSciTech, 2014, 15(4), 858–861.
22. Q. Ai, D. Yang, Y. Li, J. Shi, X. Wang and Z. Jiang, Biochem. Eng. J., 2014, 83, 8–15.
23. A. M. Amorim, M. D. Gasques, J. Andreaus and M. Scharf, An. Acad. Bras. Cienc., 2002, 74, 433–436.
24. N. Lončar and M. W. Fraaije, Appl. Microbiol. Biotechnol., 2015, 99, 3351–3357.
25. P. Montavon, K. R. Kukic and K. Bortlik, Anal. Biochem., 2007, 360, 207–215.
26. S. Hong, Y. S. Na, S. Choi, I. T. Song, W. Y. Kim and H. Lee, Adv. Funct. Mater., 2012, 22, 4711–4717.
27. H. Liu, C.-A. Tao, Z. Hu, S. Zhang, J. Wang and Y. Zhan, RSC Adv., 2014, 4, 43624–43629.
28. S. M. Kang, J. Rho, I. S. Choi, P. B. Messersmith and H. Lee, J. Am. Chem. Soc., 2009, 131, 13224–13225.
29. G. Liu, D. Yang, Y. Zhu, J. Ma, M. Nie and Z. Jiang, Chem. Eng. Sci., 2011, 66, 4221–4228.
30. M. Martín, P. Salazar, R. Villalonga, S. Campuzano, J. M. Pingarrón and J. L. González-Mora, J. Mater. Chem. B, 2014, 2, 739.
31. J.-H. Jiang, L.-P. Zhu, X.-L. Li, Y.-Y. Xu and B.-K. Zhu, J. Membr. Sci., 2010, 364, 194–202.
32. S. Hong, J. Kim, Y. S. Na, J. Park, S. Kim, K. Singha, G. I. Im, D. K. Han, W. J. Kim and H. Lee, Angew. Chem., 2013, 52, 9187–9191.
33. L. A. DeLouise and B. L. Miller, Anal. Chem., 2005, 77, 1950–1956.
34. B. Karimi, S. Emadi, A. A. Safari and M. Kermanian, RSC Adv., 2014, 4, 4387–4394.
35. E. Basak, T. Aydemir, A. Dincer and S. C. Becerik, Artif. Cells, Nanomed., Biotechnol., 2013, 41, 408–413.
36. R. Fernandez-Lafuente, Enzyme Microb. Technol., 2009, 45, 405–418.
37. N. Aissaoui, J. Landoulsi, L. Bergaoui, S. Boujday and J.-F. Lambert, Enzyme Microb. Technol., 2013, 52, 336–343.
38. N. Aissaoui, L. Bergaoui, S. Boujday, J.-F. Lambert, C. Méthivier and J. Landoulsi, Langmuir, 2014, 30, 4066–4077.

---