Peptide-Induced Affinity Binding of Carbonic Anhydrase to Carbon

Article pubs.acs.org/Langmuir

Peptide-Induced Affinity Binding of Carbonic Anhydrase to Carbon Nanotubes Xiaoxing Chen,† Yibing Wang,† and Ping Wang* State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China S Supporting Information *

ABSTRACT: Although affinity binding between short chain peptides and carbon nanotube (CNT) has been reported, little is known for the study of proteins with CNT recognition and specific binding capabilities. Herein, carbonic anhydrase (CA) was functionalized via protein fusion with a single-walled carbon nanotube (SWNTs)-binding peptide, thereby forming a bioactive protein with high affinity binding capability. TEM and AFM analyses showed that the fusion CA could firmly coat to SWNTs with a surface coverage over 51%, while the enzyme maintained its catalytic activity. Structural analysis revealed that slight conformation changes were induced as a result of the fusion; however, the affinity binding of CA to the hydrophobic surface of SWNTs restored the native structure of the protein, with the conformation of the SWNT-bound CA largely resembling that of the native parent enzyme. Interfacial interactions between the fusion CA and SWNT were further investigated with Raman spectrometry and microscopic analysis. The results suggested that such peptide-induced CNT-protein binding allows the development of bioactive hybrid materials with the native structures of the protein moieties largely undisrupted.

1. INTRODUCTION Single-walled carbon nanotubes (SWNTs) have been greatly attractive to many areas owing to their unique 1-D structural and physical properties in terms of mechanical strength, electrical thermal conductivity, and optical properties.1,2 The pure carbon structure affords SWNTs great chemical stability and at the same time makes their chemical manipulation for wet-chemistry applications generally challenging. Nevertheless, SWNTs have been explored for a wide range of applications including new functional nanocomposites, solid-state nanoelectronics, sensing, and novel nanodevices.3,4 More recently, studies have shown that biomolecules such as DNA,5,6 polysaccharides,7 polypeptides,8−10 and proteins11,12 could also benefit from the excellent physical properties of SWNTs for various biotechnological applications. Conjugation of proteins with SWNTs, in particular, promises new functional nanocomposites,13 biomedical devices,14 biosensor,15 biofuel cells,16 and drug delivery systems.17 Hitherto, several approaches have been explored for the attachment of proteins onto SWNTs. In general, previous conjugation strategies included covalent binding, nonspecific physical interactions, or more typically a combination of both. It has been reported that various proteins such as ferritin,18 cytochrome,18 glucose oxidase,19 lysozyme,20,21 α-chymotrypsin,22 and soybean peroxidase22 could be directly binding to SWNTs through hydrophobic, π−π stacking, or electrostatic interactions. Those were nonselective binding forces, and the resulting binding capacities were generally low, while many © 2014 American Chemical Society

water-soluble proteins with regular surface properties could not bind directly to SWNTs.23 In addition, the interactions between SWNTs and proteins often led to conformational changes and impaired protein activities. Covalent binding often employed carboxylation modification of nanotubes introduced by oxidation treatment. By using this approach, various proteins,24,25 including enzymes,26 have been conjugated with SWNTs. Covalent binding gives more stable conjugation yet generally requires several steps of harsh chemical treatments that may induce protein denaturation. Alternatively, protein− CNT conjugation was also achieved through coadsorption with other biomolecules with binding affinities (such as biotin/ pyrene,27 biotin/β-cyclodextrin,28 and β-cyclodextrin/pyreneadamantane29), small chemical molecules or surfactants,30 and synthetic polymers.31 While these noncovalent conjugation also showed effective binding, it generally requires multistep treatments, similar to that of covalent binding, and the modification chemicals may present impurities that are hard to remove from the final products. In addition, such chemical or physical manipulations generally lack control of protein orientation. Over the past decades, various material-binding peptides (MBPs)32,33 including carbon-nanotube-binding peptides (CBPs)8−10,34,35 have been identified through phage display Received: November 3, 2014 Revised: December 17, 2014 Published: December 18, 2014 397

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2.3. Conjugation of the CA on to SWNTs. Before attachment of enzymes to SWNTs, the SWNTs as-received were treated and purified with KMnO4 oxidation following a reported protocol.51 The CA and fusion CA were conjugated onto SWNTs following a procedure similar to that previously reported.52 In brief, a certain amount of purified SWNTs in DI water was bath-sonicated for 20 min to form homogeneous dispersions at a final concentration of ∼0.1 mg/mL. Then, an adequate amount of CA solution was added to the SWNTs solutions, followed by stirring at 200 rpm for 2 h. At last, the mixture was washed twice through TBS buffer. The precipitates were collected and resuspended in TBS buffer to form dispersed enzyme−SWNT conjugate solutions. 2.4. Characterization of the CA-SWNT Conjugates and Fusion CA-SWNT Conjugates. The morphology of SWNTs and CA-SWNTs conjugates was characterized by using TEM and AFM, while the CD spectroscopy and Raman spectrum were used for analysis of the interaction between CA and SWNTs. TEM analysis was conducted using a JEM-1400 transmission electron microscope (JEOL, Japan) at an accelerating voltage of 120 kV. For sample preparation, a drop of SWNTs or CA-SWNTs conjugate aqueous solution was placed onto a carbon support grid; then, the specimen was dried at room temperature completely before TEM imaging. AFM images were carried out on a Nanoscope IIIa microscope system (Bruker, USA) in a tapping model. Samples of SWNTs or CASWNT conjugate dilute solutions, typically 15 μL, were dropped onto fresh clean mica substrates and dried overnight under ambient conditions. Raman spectra were obtained using a Renishaw Iuvia Reflex Raman Spectrometer (Renishaw Apply Innovation, England) equipped with CCD. The excitation at 514.5 nm was achieved using an argon ion laser. Sample preparation for Raman analysis of SWNTs and CASWNT conjugate solutions (∼10 μL) was similar to the previously mentioned process. Before the spectra were recorded, wavenumber calibration was performed using the 521 cm−1 line of the silicon wafer. The spectra of both SWNTs and CA-SWNTs conjugate samples were recorded by scanning five times in the region of 1000−1800 cm−1. CD spectroscopy was used to analyze the secondary structures of the CA and CA-SWNTs conjugates with a Chirascan-plus system (Applied Photophysics, England). In this measurement, the CA was dissolved in potassium phosphate buffer (20 mM, pH 7.8, containing 150 mM sodium fluoride) at a concentration of 90 μg/mL. For protein−SWNT conjugation analysis, a certain amount of SWNTs dilute solution was added to interact with protein. The UV CD spectra were recorded over the wavelength between 185 and 260 nm with a bandwidth of 1.0 nm and a scan speed of 30 nm/min. For each test, three spectra were recorded, followed by automated averaging. The structure compositions of proteins were calculated by using the CDPro software package (http://lamar.colostate.edu/~sreeram/CDPro/). 2.5. Enzyme Activity Assay. The activities of free and immobilized CA were assayed through a spectrophotometric method based on its esterase activity according to previous report with a minor modification.53 The reaction mixture was prepared by mixing 1.9 mL of Tris buffer solution (20 mM, pH 8.0), 1.0 mL of substrate solution (p-NPA dissolved in acetonitrile), and 0.1 mL of CA solution. This reaction solution was mixed at room temperature for 5 min; then, the concentration of the p-nitrophenol (p-NP) product was measured (A348) with a UV−vis spectrophotometer (Hitachi U-3900, Japan). One unit of enzyme activity is defined as the quantity of enzyme required to produce 1 μmol of p-nitrophenol per minute under this assay condition. Control experiments were also conducted to eliminate the self-dissociation of p-NPA in each assay system.

peptide library technology as well as other combinatorial peptide biotechnologies. Studies indicated that these binding peptides with specific conformations may recognize functional groups or orderly arranged atoms on surface of solid materials.36−39 For CBPs, studies revealed that their sequences usually were rich in aromatic amino acids, such as histidine (H) or tryptophan (W), at specific locations and showed strong affinity for SWNTs.8−10,34,35,40,41 Experimental and molecular dynamics (MD) simulation studies have revealed that such peptide sequences could afford enhanced π−π stacking and hydrophobic interactions for peptide−SWNT bindings.8−10,42,43 Some of these MBPs have been already applied as binding linkers for attachment of proteins,44−46 other biomolecules,47 and nanoparticles48,49 onto specific substrate for constructing sensor devices or well-ordered hybrid structures in nanotechnology. However, knowledge of CBPfunctionalized fusion proteins on surface of SWNTs is still limited. As far as retention of protein structure is concerned, fusion proteins with terminal-linked peptides may afford orientation-controlled conjugation of the protein, as has been observed for protein binding on Ag nanoparticles.50 We may further assume that fusion CBPs can provide a spacer between the carbon surface and the conjugating protein moiety, thereby minimizing the interruption of the protein structure from interfacial interactions. Toward that, in this work, we demonstrate specific affinity conjugation of a model protein carbonic anhydrase (CA) to the surface of SWNTs using CBP. CA is the enzyme that catalyzes the dissolution of carbon dioxide into water and has been explored extensively in recent years for carbon capture technologies. The enzyme was cloned from Bacillus subtilis str. 168 into Escherichia coli BL21(DE3) with a terminal CBP sequence. The interactions between CBPCA and SWNTs were examined in terms of protein structure stability and enzyme activity.

2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. SWNTs (purity>90%, an average diameter of 2 nm, 1 μm in length) were purchased from XFNANO (Nanjing, China). Escherichia coli BL21(DE3) was sample-preserved in our laboratory (original from Novagen). Bacillus subtilis str. 168 was provided by Institute of Microbiology, Chinese Academy of Sciences (Beijing, China). Restriction endonuclease, Premix Taq DNA polymerase, and T4 ligase were purchased from TaKaRa Biotechnology (Dalian, China). pET24a(+) Vector was purchased from Novagen (Shanghai, China). Other chemical reagents were purchased from Sinopharm Chemical Reagent (Shanghai, China) and Sigma-Aldrich Chemical (Shanghai, China). 2.2. Cloning, Expression, and Purification of CA and CAAffinity Peptide Fusion Proteins. The gene yvdA (GenBank ID: NC_000964.3) encoding the carbonic anhydrase was cloned from the genomic DNA of Bacillus subtilis str. 168. The CBP was expressed at the C terminal of CA. The primers used in PCR cloning are listed in Table S1 in the Supporting Information. The target gene fragments (CA and fusion CA) were amplified via PCR. Finally, target gene fragments were inserted into pET24a(+). These recombinant plasmids were transformed into E. coli DH5α for further subcloning analysis. The recombinant plasmids were verified by gene sequencing. After that, the recombinant plasmids were transformed into E. coli BL21(DE3) for protein expression. The expressed proteins were purified through Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography and were then analyzed through SDS-PAGE. Bicinchoninic acid (BCA) protein assay kit (Sangong, Shanghai) was used to measure the concentration of purified proteins. The products were stored at 4 °C before any further using. Detailed processes of cloning, expression, and purification are given in the Supporting Information.

3. RESULTS AND DISCUSSION 3.1. Morphology Characterization of CA-SWNTs Conjugates. SDS-PAGE analysis (Figure S1 in the Supporting Information) showed that CA and fusion CA were expressed and purified successfully with good purity. To examine the efficiency of peptide-induced enzyme binding to SWNTs, we 398

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This strong binding of CBP-fusion CA on the SWNTs surface was also confirmed with TEM imaging analysis. Figure 1c shows the morphology of fusion CA-SWNTs conjugate. As observed, the proteins appeared to be dense on SWNTs as indicated by the amorphous structures on surface of CNTs and increased diameter of CNT bundles. As a result of the protein binding, some of individual SWNTs also can be identified clearly (Figure 1c pointing with a red arrow), which could not be seen clearly otherwise under the same resolution. In contrast, TEM image of the SWNTs conjugated with native CA did not show apparent changes on surface of the CNT sample (Figure 1d). To further examine the protein distribution on the surface of SWNTs, we conducted AFM that offers a higher resolution than TEM. Figure 2 shows the AFM image of fusion CASWNT conjugate. From the image, a layer of matter coated on SWNTs was evident. The height measurement in the end of SWNT, which has no enzyme adsorbed, showed that the diameter was ∼2.2 nm, which is approximately the diameter of a single SWNT. However, the diameter is ∼6.6 nm in the middle of SWNT, which indicates conjugation with proteins because the difference in the height measurement matches well the size of CA, which is ∼4.5 nm in diameter (Figure S3 in the Supporting Information). Previous studies also found that the height of a single molecule adsorbed on SWNTs was in the range of 1.0 to 3.0 nm for peptide54 and 2.5 to 6.0 nm for proteins.22,55 The SWNT−protein could be verified further with the phase images (Figure S4 in the Supporting Information), where single protein particles could also be distinguished one by one. These phase images reflect differences in hardness between protein and SWNTs, with bright dots in phage images showing protein structures. In contrast, AFM analysis of samples contacted with native CA could only barely detect the conjugation of the protein to the nanotubes (Figure S5 in the Supporting Information). 3.2. Raman Spectroscopy Analysis of the Interaction between Enzymes and SWNTs. Investigation of the interaction between protein and SWNTs was performed by using Raman spectroscopy. Figure 3 shows the Raman spectra of fusion CA-SWNTs conjugate, native CA-SWNTs conjugate, and pristine SWNTs. The spectra have two major characteristic bands, a D band at 1350.0 cm−1 and a G band at 1591.0 cm−1. The D band corresponds to sp3-hybridized carbons, and its intensity reflects the disorder and defects in the carbon materials. G band is related to the intrinsic vibration mode of graphite structure on the surface of nanotubes and is a characteristic of the sp2-hybridized carbon networks. The

conducted quantitative measurement of the protein adsorption and morphology characterization of SWNTs. TEM images showed that SWNTs samples as-received contained amorphous carbon structures (Figure 1a) but can be removed with

Figure 1. TEM images of SWNTs with conjugated proteins. (a) Pristine SWNTs; (b) purified SWNTs; (c) fusion CA-SWNT conjugate (an individual SWNT is pointed by red arrow); and (d) SWNTs conjugation with native CA.

oxidation and purification treatments (Figure 1b). The purified SWNTs were then contacted with protein solutions, with the protein concentrations in the original conjugation solution and subsequent washing supernatant measured using BCA protein assay. As a result, the protein loading was determined to be as high as 952 μg-protein/mg-CNT. However, CA without CBP could only bind at a level of ∼50 μg-protein/mg-CNT, indicating an enhancement factor of 20 as a result of peptide affinity binding. The CBP-induced protein loading observed in this work is comparable to the highest protein loading achieved via covalent binding (ranged from 80 to 1300 μg-protein/mgCNT26,52). By accounting the total surface area available for binding (assuming 100% dispersion of SWNTs in solution), the theoretical loading of CA on SWNTs is 1860 μg-protein/mgCNT. The details of calculation are given in Figure S2 in the Supporting Information. Apparently, the surface coverage of fusion CA is >51%. The actual surface coverage should be much higher, as the SWNTs may not disperse perfectly in solution and often appear in form of bundles with diameters much greater than that expected for individual SWNT.

Figure 2. AFM image of fusion CA-SWNTs conjugate (Left: AFM image of size 1.5 × 1.5 μm2; Right: cross-sectional topological profile taken along the red and green lines drawn in the left image). 399

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surface of SWNTs caused by the attachment of enzymes on SWNTs. This may serve as an indication of the noncovalent interactions between fusion CA and SWNTs, such as π−π stacking, hydrophobic, and charge-transfer interactions. Previously reported research has confirmed that the charge-transfer interaction between peptide acceptor and MWNT donor caused the G-band upshifting.57 We tend to believe that the slight downshift of the G band observed in the current work may indicate the existence of weak charge transfer with the fusion CA as donor and the SWNT as acceptor. Similar assumptions for charge transfer were also suggested previously for affinity binding of small peptides on SWNTs.10 Taking consideration of these results together, we may infer that the affinity binding peptide CBP at terminal of CA enabled the specific noncovalent interaction with the SWNT, and that aroused the enhancement of G band and downshift wavelength in the current case. All of these observations strongly supported the fact that the strong conjugation between fusion CA and SWNTs was indeed induced by the fused affinity peptide. 3.3. Circular Dichroism (CD) Spectroscopy Analysis of Conjugated Enzyme. We further conducted protein structural analysis to examine the impact of interfacial affinity binding on the native structure of the protein moiety. It is known that SWNT, with high hydrophobicity and high surface curvature, could influence the structure and bioactivity of enzymes.52 CD spectroscopy was used to analyze structures of CA. As shown in Figure 4a, the CD spectra of CA and fusion CA show obvious distinctions, indicating that the affinity binding peptide at terminal of CA may affect the secondary structure of CA. Table 1 lists the structure compositions of different forms of the enzymes, which are estimated from the average results of CDPro software programs (for original data see Tables S2−S4 in the Supporting Information). As shown in Table 1, the fraction estimates of total α-helix and β-sheet for the CA were 0.3154 and 0.1896, respectively, while the fusion CA exhibits increased total α-helix content (0.4703) and dramatically decreased β-sheet content (0.0647). The CD spectra of fusion CA, fusion CA-SWNTs conjugate, and bare SWNTs are shown in Figure 4b. The bare SWNTs show very weak CD signals, indicating that the influence of SWNTs on the structure analysis of enzyme−SWNTs conjugate is negligible. Compared with the free fusion CA, the amount of α-helix content in fusion CA-SWNTs conjugate was decreased, whereas the β-sheet conformation was significantly increased

Figure 3. Raman spectra of SWNTs with conjugated proteins. Black line: pristine SWNTs; blue line: fusion CA-SWNTs conjugate; red line: native CA-SWNTs conjugate.

intensity ratio (ID/IG) indicates the disordering and defect density of surface structures of carbon materials and can be used to characterize the functionalization of carbon nanotube.56 It is noteworthy that independent analysis of the Raman spectra of SWNTs containing native CA showed a weak D band at 1350.2 cm−1. The results in Figure 3 show that the G-band intensity of SWNTs enhanced significantly after conjugation with the fusion CA (blue line), while the intensity of D band decreased slightly. The ID/IG for pristine SWNTs was 0.238, but that for fusion CA-SWNTs conjugate was decreased to 0.059. The significant enhancement of G band and the decreased ID/IG ratio support the strong noncovalent interactions between SWNTs and fusion CA, that is, the affinity binding of the peptide.57 The ID/IG for SWNTs containing native CA was 0.254, which is very close to that of pristine SWNTs, indicating the lack of the specific interactions observed for the CBP-fusion protein. Furthermore, changes in peak position of the G band have been used to investigate doping effects of nanotube.58 In the current work, a band shift appeared once the SWNT is conjugated with fusion CA. The peak position of G band for pristine SWNTs (1591.4 cm−1) was shifted to 1589.6 cm−1 after fusion CA binding (Figure 3). A slightly blue shift of the G band suggests the existence of the stress experienced in the

Figure 4. CD spectra of the free CA, fusion CA, and fusion CA-SWNTs conjugate. (a) Free CA and fusion CA. (b) Fusion CA-SWNTs conjugate, free fusion CA, and bare SWNTs. 400

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Langmuir Table 1. Secondary Structure Fractions of the Different Forms of Enzymes Calculated from CD Spectraa

a

sample

H(r)

H(d)

S(r)

S(d)

Trn

Unrd

CA CBP fusion CA fusion CA-SWNTs conjugate

0.1837 0.2830 0.1678

0.1317 0.1873 0.1310

0.1113 0.0217 0.1163

0.0783 0.0430 0.0813

0.2110 0.1990 0.2117

0.2743 0.2693 0.2790

H(r): regular α-helix, H(d): distorted α-helix, S(r): regular β-sheet, S(d): distorted β-sheet, Trn: turn, and Unrd: unordered.

Figure 5. Relative activity of the native CA and CA-SWNTs conjugate. (a) Effect of pH. (b) Relative activities of different preparations at pH 8.5. The relative activity is defined by taking the specific activity of free fusion CA at pH 8.5 as 100%.

native CA-SWNTs conjugate was too low to measure. Figure 5a shows the dependence of enzyme activity on pH for native CA, fusion CA, and fusion CA-SWNTs conjugate. The same optimum pH 8.5 was found among these enzymes, indicating that the surface properties of the nanotube did not impose any sensitive microenvironment changes for the enzymatic reaction. However, the fusion CA-SWNTs demonstrated a slower decrease in activity as the pH increased from 7.0 to 9.0, indicating an enhanced pH tolerance as a result of the conjugation. The increased pH tolerance was also found in previous studies.59 Figure 5b shows the relative activities of the different enzymes. It appears that the activity of native CA was slightly lower than but close to that of fusion CA, indicating that the secondary structure change of CA after fusion with CBP had no significantly impact on its enzyme activity. After binding at SWNTs, the freedom of enzymes should have been restricted. Therefore, bioactivity of CA would be affected. From Figure 5b, the decreased activity of fusion CA-SWNTs was observed. The specific activity of SWNT-conjugated fusion CA is 67 ± 5% that of free fusion CA. The retention of enzyme activity is indeed pretty high compared with traditional immobilized enzymes with macroscopic supports, which generally induced over 90% reduction in apparent enzyme activities. As far as biocatalysis concerned, conjugation with CNTs could allow repeated catalyst recovery and reuse, thus improving overall processing efficiency ultimately.

after attachment on SWNTs, as shown in Table1. Here fusion CA conjugated to SWNTs retained approximately 63.53% of its α-helix content, while regular β-sheet content distinctly increased from 0.0217 to 0.1163, suggesting that the fusion CA binding onto SWNTs possibly associated with the formation of the specific β-sheet conformation of the affinity binding peptides interacting with SWNTs. This observation is consistent with what previously reported, which revealed that the same affinity binding peptide formed a β-turn conformation when bound to SWNTs at its free form.9 A similar conformational change happened in fusion protein when interacting with SWNTs. When this peptide fused to a carrier protein MFH, the peptide segment was induced to a folded structure upon the MFH-fusion protein interacted with SWNTs.9 Comparing secondary structure data (Table 1) of SWNTs conjugate with native and fusion CA, it seems that fusion CA-SWNTs conjugate is closer to native CA. All of these observation indicated that, interestingly, the affinity binding of the fusion CA to nanotubes actually restored the native structure of the protein, which was interrupted by the fusion of peptide. That agrees with our assumption that the spacing effect of the terminal peptides helps to screen off the impacts of interfacial interactions on the conjugated proteins. Spectra of native CA conjugated with SWNTs are shown in Figure S6 in the Supporting Information. The spectra of CA (pink line) and CA in diluted dispersions of SWNTs are more or less the same. This result shows that the secondary structures of the CA after contact with SWNTs was almost the same as that of native CA, an indication of weak interfacial interactions. 3.4. Enzyme Activity of Free CA and Immobilized CA. We further probed the correlation between protein conformational changes and catalytic activity of the nanotube conjugated enzyme. Bioactivities of native CA, fusion CA, and fusion CASWNTs conjugate were investigated (Figure 5). Because the loading of native CA on SWNTs was low, enzyme activity of

4. CONCLUSIONS In conclusion, a new fusion protein CA was functionalized via affinity binding peptide CBP, forming a bioactive protein with highly SWNTs affinity binding capabilities. Fusion CA-SWNT conjugate could improve the binding efficiency by a factor of 20 times, reaching >51% total surface coverage of nanotubes. Peptide fusion causes changes in secondary structures of the protein, yet the native protein structure appeared to be restored, presumably due to the spacing effect of the peptide. 401

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(10) Su, Z.; Mui, K.; Daub, E.; Leung, T.; Honek, J. F. Single-Walled Carbon Nanotube Binding Peptides: Probing Tryptophan’s Importance by Unnatural Amino Acid Substitution. J. Phys. Chem. B 2007, 111, 14411−14417. (11) Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen, X.; Dai, H. In Vivo Biodistribution and Highly Efficient Tumour Targeting of Carbon Nanotubes in Mice. Nat. Nanotechnol. 2006, 2, 47−52. (12) Messersmith, P. B.; Textor, M. Enzymes on Nanotubes Thwart Fouling. Nat. Nanotechnol. 2007, 2, 138−139. (13) Asuri, P.; Karajanagi, S. S.; Kane, R. S.; Dordick, J. S. PolymerNanotube-Enzyme Composites as Active Antifouling Films. Small 2007, 3, 50−53. (14) Liu, Z.; Yang, K.; Lee, S. T. Single-Walled Carbon Nanotubes in Biomedical Imaging. J. Mater. Chem. 2011, 21, 586−598. (15) McDonald, T. J.; Svedruzic, D.; Kim, Y. H.; Blackburn, J. L.; Zhang, S. B.; King, P. W.; Heben, M. J. Wiring-up Hydrogenase with Single-walled Carbon Nanotubes. Nano Lett. 2007, 7, 3528−3534. (16) Little, S. J.; Ralph, S. F.; Mano, N.; Chen, J.; Wallace, G. G. A Novel Enzymatic Bioelectrode System Combining a Redox Hydrogel with a Carbon NanoWeb. Chem. Commun. 2011, 47, 8886−8888. (17) Kam, N. W. S.; Dai, H. Carbon Nanotubes as Intracellular Protein Transporters: Generality and Biological Functionality. J. Am. Chem. Soc. 2005, 127, 6021−6026. (18) Azamian, B. R.; Davis, J. J.; Coleman, K. S.; Bagshaw, C. B.; Green, M. L. H. Bioelectrochemical Single-Walled Carbon Nanotubes. J. Am. Chem. Soc. 2002, 124, 12664−12665. (19) Tsai, T. W.; Heckert, G.; Neves, L. F.; Tan, Y. Q.; Kao, D. Y.; Harrison, R. G.; Resasco, D. E.; Schmidtke, D. W. Adsorption of Glucose Oxidase onto Single-Walled Carbon Nanotubes and Its Application in Layer-By-Layer Biosensors. Anal. Chem. 2009, 81, 7917−7925. (20) Horn, D. W.; Tracy, K.; Easley, C. J.; Davis, V. A. Lysozyme Dispersed Single-Walled Carbon Nanotubes: Interaction and Activity. J. Phys. Chem. C 2012, 116, 10341−10348. (21) Nepal, D.; Geckeler, K. E. pH-Sensitive Dispersion and Debundling of Single-Walled Carbon Nanotubes: Lysozyme as a Tool. Small 2006, 2, 406−412. (22) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 2004, 20, 11594−11599. (23) Matsuura, K.; Saito, T.; Okazaki, T.; Ohshima, S.; Yumura, M.; Iijima, S. Selectivity of Water-soluble Proteins in Single-walled Carbon Nanotube Dispersions. Chem. Phys. Lett. 2006, 429, 497−502. (24) Gao, Y.; Kyratzis, I. Covalent Immobilization of Proteins on Carbon Nanotubes Using the Cross-Linker 1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide−A Critical Assessment. Bioconjugate Chem. 2008, 19, 1945−1950. (25) Bhirde, A. A.; Patel, V.; Gavard, J.; Zhang, G. F.; Sousa, A. A.; Masedunskas, A.; Leapman, R. D.; Weigert, R.; Gutkind, J. S.; Rusling, J. F. Targeted Killing of Cancer Cells in Vivo and in Vitro with EGFDirected Carbon Nanotube-Based Drug Delivery. ACS Nano 2009, 3, 307−316. (26) Asuri, P.; Bale, S. S.; Pangule, R. C.; Shah, D. A.; Kane, R. S.; Dordick, J. S. Structure, Function, and Stability of Enzymes Covalently Attached to Single-Walled Carbon Nanotubes. Langmuir 2007, 23, 12318−12321. (27) Holzinger, M.; Haddad, R.; Maaref, A.; Cosnier, S. Amperometric Biosensors Based on Biotinylated Single-Walled Carbon Nanotubes. J. Nanosci. Nanotechnol. 2009, 9, 6042−6046. (28) Holzinger, M.; Singh, M.; Cosnier, S. Biotin/β-Cyclodextrin: A New Host−Guest System for the Immobilization of Biomolecules. Langmuir 2012, 28, 12569−12574. (29) Holzinger, M.; Bouffier, L.; Villalonga, R.; Cosnier, S. Adamantane/β-cyclodextrin Affinity Biosensors Based on Singlewalled Carbon Nanotubes. Biosens. Bioelectron. 2009, 24, 1128−1134. (30) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. Noncovalent Sidewall Functionalization of Single-Walled Carbon Nanotubes for Protein Immobilization. J. Am. Chem. Soc. 2001, 123, 3838−3839.

Raman spectrometry and microscopic analysis confirmed that the interfacial interactions between fusion CA and SWNTs were realized through specific noncovalent interactions between the fused CBPs and SWNTs. The results suggested that such CNT−protein affinity binding promises bioactive hybrid materials with protein structures largely retain undisrupted, while the protein functionalities can be best retained. That facilitates preparation of high-performance biomaterials, depending on the functionalities of the induced proteins, for a wide range of applications such as biosensors, smart materials, and biomedical devices.

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ASSOCIATED CONTENT

S Supporting Information *

Details of cloning, expression, and purification of CA and fusion CA; calculations of secondary structures, protein loadings, and structural data; and AFM images of fusion CA-SWNTs conjugates and related CD spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

X.C. and Y.W. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21303050 and 31471659), the China Postdoctoral Science Foundation (2013M540334), and the Engagement Fund of Interdisciplinary and Major Project of the Ministry of National Education (wk0913002).

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REFERENCES

(1) Ajayan, P. M. Nanotubes from Carbon. Chem. Rev. 1999, 99, 1787−1800. (2) Dresselhaus, M. S.; Dresselhaus, G.; Saito, R. Physics of Carbon Nanotubes. Carbon 1995, 33, 883−891. (3) Baughman, R. H.; Zakhidov, A. A.; de Heer, W. A. Carbon Nanotubes-the Route Toward Applications. Science 2002, 297, 787− 792. (4) Ouyang, M.; Huang, J. L.; Lieber, C. M. Fundamental Electronic Properties and Applications of Single-walled Carbon Nanotubes. Acc. Chem. Res. 2002, 35, 1018−1025. (5) Tu, X.; Manohar, S.; Jagota, A.; Zheng, M. DNA Sequence Motifs for Structure-specific Recognition and Separation of Carbon Nanotubes. Nature 2009, 460, 250−253. (6) Mantha, S.; Pedrosa, V. A.; Olsen, E. V.; Davis, V. A.; Simonian, A. L. Renewable Nanocomposite Layer-by-Layer Assembled Catalytic Interfaces for Biosensing Applications. Langmuir 2010, 26, 19114− 19119. (7) Numata, M.; Asai, M.; Kaneko, K.; Bae, A. H.; Hasegawa, T.; Sakurai, K.; Shinkai, S. Inclusion of Cut and As-Grown Single-Walled Carbon Nanotubes in the Helical Superstructure of Schizophyllan and Curdlan (β-1,3-glucans). J. Am. Chem. Soc. 2005, 127, 5875−5884. (8) Wang, S.; Humphreys, E. S.; Chung, S. Y.; Delduco, D. F.; Lustig, S. R.; Wang, H.; Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y. M.; Jagota, A. Peptides with Selective Affinity for Carbon Nanotubes. Nat. Mater. 2003, 2, 196−200. (9) Su, Z.; Leung, T.; Honek, J. F. Conformational Selectivity of Peptides for Single-Walled Carbon Nanotubes. J. Phys. Chem. B 2006, 110, 23623−23627. 402

DOI: 10.1021/la504321q Langmuir 2015, 31, 397−403

Article

Langmuir

Catalytically Produced Multi-wall nanotubes. J. Chem. Soc., Faraday Trans. 1998, 94, 3753−3758. (52) Campbell, A. S.; Dong, C.; Meng, F.; Hardinger, J.; Perhinschi, G.; Wu, N.; Dinu, C. Z. Enzyme Catalytic Efficiency: A Function of Bio−Nano Interface Reactions. ACS. Appl. Mater. Interfaces. 2014, 6, 5393−5403. (53) Vinoba, M.; Lim, K. S.; Lee, S. H.; Jeong, S. K.; Alagar, M. Immobilization of Human Carbonic Anhydrase on Gold Nanoparticles Assembled onto Amine/Thiol-Functionalized Mesoporous SBA-15 for Biomimetic Sequestration of CO2. Langmuir 2011, 27, 6227−6234. (54) Zorbas, V.; Smith, A. L.; Xie, H.; Ortiz-Acevedo, A.; Dalton, A. B.; Dieckmann, G. R.; Draper, R. K.; Baughman, R. H.; Musselman, I. H. Importance of Aromatic Content for Peptide/Single-Walled Carbon Nanotube Interactions. J. Am. Chem. Soc. 2005, 127, 12323−12328. (55) Ge, C. C.; Du, J. F.; Zhao, L. N.; Wang, L. M.; Liu, Y.; Li, D. H.; Yang, Y. L.; Zhou, R. H.; Zhao, Y. L.; Chai, Z. F.; Chen, C. Y. Binding of Blood Proteins to Carbon Nanotubes Reduces Cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16968−16973. (56) Ferrari, M. S.; Robertson, J. Interpretation of Raman Spectra of Disordered and Amorphous Carbon. Phys. Rev. B 2000, 61, 14095− 14107. (57) Deshpande, M. S.; Mazumdar, S. Sequence Specific Association of Tryptic Peptides with Multiwalled Carbon Nanotubes: Effect of Localization of Hydrophobic Residues. Biomacromolecules 2012, 13, 1410−1419. (58) Fagan, S. B.; Souza Filho, A. G.; Mendes Filho, J.; Corio, P.; Dresselhaus, M. S. Electronic Properties of Ag- and CrO3-filled Singlewall Carbon Nanotubes. Chem. Phys. Lett. 2005, 406, 54−59. (59) Verma, M. L.; Naebe, M.; Barrow, C. J.; Puri, M. Enzyme Immobilisation on Amino-Functionalised MultiWalled Carbon Nanotubes: Structural and Biocatalytic Characterisation. PLoS One 2013, 8, e73642.

(31) Shim, M.; Shi Kam, N. W.; Chen, R. J.; Li, Y.; Dai, H. Functionalization of Carbon Nanotubes for Biocompatibility and Biomolecular Recognition. Nano Lett. 2002, 2, 285−288. (32) Patwardhan, S. V.; Patwardhan, G.; Perry, C. C. Interactions of Biomolecules with Inorganic Materials: Principles, Applications and Future Prospects. J. Mater. Chem. 2007, 17, 2875−2884. (33) Seker, U. O.; Demir, H. V. Material Binding Peptides for Nanotechnology. Molecules 2011, 16, 1426−1451. (34) Brown, S.; Jespersen, T. S.; Nygard, J. A Genetic Analysis of Carbon-Nanotube-Binding Proteins. Small 2008, 4, 416−420. (35) Dieckmann, G. R.; Dalton, A. B.; Johnson, P. A.; Razal, J.; Chen, J.; Giordano, G. M.; Munoz, E.; Musselman, I. H.; Baughman, R.; Draper, R. K. Controlled Assembly of Carbon Nanotubes by Designed Amphiphilic Peptide Helices. J. Am. Chem. Soc. 2003, 125, 1770−1777. (36) Sano, K. I.; Shiba, K. A Hexapeptide Motif that Electrostatically Binds to the Surface of Titanium. J. Am. Chem. Soc. 2003, 125, 14234− 14235. (37) Serizawa, T.; Sawada, T.; Kitayama, T. Peptide Motifs That Recognize Differences in Polymer-Film Surfaces. Angew. Chem., Int. Ed. 2007, 46, 723−726. (38) Sawada, T.; Takahashi, T.; Mihara, H. Affinity-Based Screening of Peptides Recognizing Assembly States of Self-assembling Peptide Nanomaterials. J. Am. Chem. Soc. 2009, 131, 14434−14441. (39) Serizawa, T.; Sawada, T.; Matsuno, H.; Matsubara, T.; Sato, T. A peptide Motif Recognizing a Polymer Stereoregularity. J. Am. Chem. Soc. 2005, 127, 13780−13781. (40) Yu, T.; Gong, Y.; Lu, T.; Wei, L.; Li, Y.; Mu, Y.; Chen, Y.; Liao, K. Recognition of Carbon Nanotube Chirality by Phage Display. RSC Adv. 2012, 2, 1466−1476. (41) Pender, M. J.; Sowards, L. A.; Hartgerink, J. D.; Stone, M. O.; Naik, R. R. Peptide-Mediated Formation of Single-Wall Carbon Nanotube Composites. Nano Lett. 2006, 6, 40−44. (42) Tomásio, S. M.; Walsh, T. R. Modeling the Binding Affinity of Peptides for Graphitic Surfaces. Influences of Aromatic Content and Interfacial Shape. J. Phys. Chem. C 2009, 113, 8778−8785. (43) Sano, K.; Ajima, K.; Iwahori, K.; Yudasaka, M.; Iijima, S.; Yamashita, I.; Shiba, K. Endowing a Ferritin-Like Cage Protein with High Affinity and Selectivity for Certain Inorganic Materials. Small 2005, 1, 826−832. (44) Park, T. J.; Lee, S. Y.; Lee, S. J.; Park, J. P.; Yang, K. S.; Lee, K. B.; Ko, S.; Park, J. B.; Kim, T.; Kim, S. K.; Shin, Y. B.; Chung, B. H.; Ku, S. J.; Kim, D. H.; Choi, I. S. Protein Nanopatterns and Biosensors Using Gold Binding Polypeptide as a Fusion Partner. Anal. Chem. 2006, 78, 7197−7205. (45) Matsui, T.; Matsukawa, N.; Iwahori, K.; Sano, K. I.; Shiba, K.; Yamashita, I. Realizing a Two-Dimensional Ordered Array of Ferritin Molecules Directly on a Solid Surface Utilizing Carbonaceous Material Affinity Peptides. Langmuir 2007, 23, 1615−1618. (46) Kashiwagi, K.; Tsuji, T.; Shiba, K. Directional BMP-2 for Functionalization of Titanium Surfaces. Biomaterials 2009, 30, 1166− 1175. (47) Serizawa, T.; Hirai, Y.; Aizawa, M. Novel Synthetic Route to Peptide-Capped Gold Nanoparticles. Langmuir 2009, 25, 12229− 12234. (48) Kacar, T.; Ray, J.; Gungormus, M.; Oren, E. E.; Tamerler, C.; Sarikaya, M. Quartz Binding Peptides as Molecular Linkers towards Fabricating Multifunctional Micropatterned Substrates. Adv. Mater. 2009, 21, 295−299. (49) Grigoryan, G.; Kim, Y. H.; Acharya, R.; Axelrod, K.; Jain, R. M.; Willis, L.; Drndic, M.; Kikkawa, J. M.; DeGrado, W. F. Computational Design of Virus-Like Protein Assemblies on Carbon Nanotube Surfaces. Science 2011, 332, 1071−1076. (50) Sengupta, A.; Thai, C. K.; Sastry, M. S. R.; Matthaei, J. F.; Schwartz, D. T.; Davis, E. J.; Baneyx, F. A Genetic Approach for Controlling the Binding and Orientation of Proteins on Nanoparticles. Langmuir 2008, 24, 2000−2008. (51) Colomer, J. F.; Piedigrosso, P.; Willems, I.; Journet, C.; Bernier, P.; Tendeloo, G. V.; Fonseca, A.; Nagy, J. B. Purification of 403

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