Attaching Proteins to Carbon Nanotubes via Diimide-Activated

NANO LETTERS

Attaching Proteins to Carbon Nanotubes via Diimide-Activated Amidation

2002 Vol. 2, No. 4 311-314

Weijie Huang,† Shelby Taylor,† Kefu Fu,† Yi Lin,† Donghui Zhang,‡ Timothy W. Hanks,‡ Apparao M. Rao,§ and Ya-Ping Sun*,† Department of Chemistry and Center for AdVanced Engineering Fibers and Films, Howard L. Hunter Chemistry Laboratory, Clemson UniVersity, Clemson, South Carolina 29634-0973, Department of Chemistry, Furman UniVersity, GreenVille, South Carolina 29613-0420, and Department of Physics and Astronomy, Kinard Laboratory, Clemson UniVersity, Clemson, South Carolina 29634-0978 Received December 13, 2001; Revised Manuscript Received February 15, 2002

ABSTRACT Carbon nanotubes are functionalized by bovine serum albumin (BSA) proteins via diimide-activated amidation under ambient conditions. The nanotube-BSA conjugates thus obtained are highly water-soluble, forming dark-colored aqueous solutions. Results from characterizations using atomic force microscopy (AFM), thermal gravimetric analysis, Raman, and gel electrophoresis show that the conjugate samples indeed contain both carbon nanotubes and BSA proteins and that the protein species are intimately associated with the nanotubes. Bioactivities of the nanotube-bound proteins are evaluated using the total protein micro-determination assay (the modified Lowry procedure). The results show that the overwhelming majority (∼90%) of the protein species in the nanotube-BSA conjugates remain bioactive.

Potential biological applications of both single-wall (SWNT) and multiple-wall (MWNT) carbon nanotubes have captured much imagination.1 There have been several recent investigations concerning the use of carbon nanotubes for biological purposes and the introduction of carbon nanotubes into biological systems.2-5 For example, Balavoine, et al. used MWNTs for helical crystallization of proteins.2 Mattson, et al. took advantage of the shape and exceptional rigidity of nanotubes for the growth of embryonic rat-brain neurons on MWNTs.3 Sadler and co-workers immobilized biological species on both SWNTs and MWNTs for potential biosensor and bioreactor systems.4 Their results suggest that carbon nanotubes are better substrates than amorphous glassy carbon in studying the immobilization or growth of biomolecules on materials of a high surface area, especially concerning the structure and conformation of anchored molecules on the surfaces.4 For many proposed biologically relevant applications, aqueous solubility of carbon nanotubes as a part of biocompatibility is a significant issue.6,7 The decoration of carbon nanotubes with biological species such as proteins represents an effective approach in this regard. Here, we report the functionalization of carbon nanotubes with bovine serum albumin (BSA) proteins for the preparation of nanotube-protein conjugates. The functionalization is based on † Department of Chemistry and Center for Advanced Engineering Fibers and Films, Howard L. Hunter Chemistry Laboratory, Clemson University. ‡ Department of Chemistry, Furman University. § Department of Physics and Astronomy, Kinard Laboratory, Clemson University.

10.1021/nl010095i CCC: $22.00 Published on Web 03/16/2002

© 2002 American Chemical Society

the diimide-activated amidation of nanotube-bound carboxylic acids under the experimental conditions designed to cause no denaturing of the protein species.8

SWNTs and MWNTs were produced via the arc discharge method and the chemical vapor deposition (CVD or sometime called catalytic pyrolysis) method, respectively.9-11 The carbon nanotube samples were purified by refluxing in an aqueous HNO3 solution (2.6 M, 48 h), washing repeatedly with water, and drying.7 The nanotube-bound carboxylic acids are associated with the defect sites and the terminal carbons in chemically shortened nanotubes as a result of the purification process.12 They have been used to attach aminopolymers such as poly(propionylethylenimine-co-ethylenimine) (PPEI-EI) to the nanotubes via amide linkages.6a,7 In a typical experiment for the amidation reaction with BSA proteins, a SWNT sample (35 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, 155 mg) were added to an aqueous KH2PO4 buffer solution (15 mL). After the mixture was sonicated for 2 h, BSA (350 mg) was added.

Figure 1. UV-vis absorption spectrum of the SWNT-BSA conjugate in aqueous solution. Shown in the inset is LambertBeer’s plot for the absorption at 500 nm.

The resulting mixture was stirred at room temperature for 24 h and then transferred to a membrane tubing (cutoff molecular weight 12 000) for dialysis against freshwater. Following the dialysis for 3 d, the mixture was vigorously centrifuged at a high speed (7 800 rpm) to separate the insoluble nanotubes, yielding a dark-colored homogeneous solution. Free BSA in the solution was removed via dialysis again in a membrane tubing of a larger pore size (cutoff molecular weight 100 000) for 3 days.13 Upon the evaporation of water, a solid-state SWNT-BSA conjugate sample was obtained. The same procedure and conditions were used in the functionalization of MWNTs with BSA proteins for both aqueous solution and solid-state samples of the MWNT-BSA conjugate. The BSA protein contains 60 amino moieties in lysine residues and 26 arginine moieties in guanidino side-chains,14 amenable to the coupling with carboxylic acids in the diimide-activated amidation reaction.15 The protein sample is colorless in an aqueous buffer solution and remains colorless upon being subjected to the reaction conditions in the absence of nanotubes. Thus, the dark-color in the samples obtained from reactions of BSA with SWNTs and MWNTs must be due to the functionalized nanotubes. The UV-vis absorption spectra of these samples in aqueous solutions are similar to those of the PPEI-EI polymer-functionalized SWNTs and MWNTs,6a,7 with the dependence of absorbance on the solution concentration obeying the Lambert-Beer’s law (Figure 1). The nanotube-BSA conjugate samples were deposited on mica substrate for atomic force microscopy (AFM) analyses. Both height and phase images were recorded at a scan rate of 4 Hz and over a scan area of 2-10 µm2. As shown in Figure 2 and Figure 3, the BSA proteins are intimately associated with the nanotubes in the conjugates.16 The results are consistent with the functionalization of nanotubes with 312

Figure 2. Height (left) and phase (right) images from the AFM analysis of the SWNT-BSA conjugate sample on mica substrate.

Figure 3. Height (left) and phase (right) images from the AFM analysis of the MWNT-BSA conjugate sample on mica substrate.

BSA proteins under the diimide-activated amidation reaction conditions. The AFM images also seem to suggest that the proteins are immobilized at various points of a nanotube, consistent with the notion that the functionalization is likely associated with the carboxylic acids at defect sites of the nanotube. For a comparison between SWNT-BSA and MWNT-BSA conjugates, the latter has a significantly higher BSA loading. This is again consistent with the same notion of functionalization at the defect sites. The CVD-produced MWNTs are known to contain more defects,10 so as to provide more sites for functionalization with BSA proteins. In the nanotube-BSA conjugate samples, the carbon nanotubes were well-dispersed. As a result, Raman spectra of the conjugate samples were overwhelmed by the strong luminescence from the functionalized nanotubes (Figure 4). To confirm that the samples did contain a substantial quantity of carbon nanotubes and that the luminescence interference was only associated with well-functionalized nanotubes, the conjugate samples were thermally defunctionalized in a thermal gravimetric analysis (TGA) experiment before Raman measurements. For example, the SWNT-BSA sample Nano Lett., Vol. 2, No. 4, 2002

Figure 4. Raman spectra (780 nm excitation) of the SWNT-BSA conjugate sample before (top) and after (bottom) thermal defunctionalization in a TGA scan.

was first treated in a TGA scan (to 550 °C at 10 °C/min in the presence of oxygen) and then submitted for Raman measurements. The Raman spectrum thus obtained exhibits the disorder-band peak at 1312 cm-1, tangential-mode peaks at 1568 cm-1 and 1591 cm-1, and the breathing-mode peak at ∼160 cm-1 (Figure 4).1,17 The nanotube-BSA conjugates were analyzed by electrophoresis using polyacrylamide gel (7.5%) and SDS-PAGE under reducing (2-mercaptoethanol) condition.18 The pristine BSA was used as a reference in the analysis. For SWNTBSA as an example, the gel pattern shows essentially two sections (Figure 5).19 The spots at the front that appear diffuse but generally parallel to those of the pristine BSA sample may be explained in terms of proteins in the conjugate sample that vary from being weakly bound to the nanotubes (thus, not being able to remain intact in the electrophoretic field) to some residual free BSA. The high molecular weight species are in the tail section, which are logically attributed to the proteins that are bound strongly to the nanotubes. These results suggest that the proteins are largely intact in the conjugate samples. The biocompatibility of the nanotube-BSA conjugates was examined in terms of bioactivities of the nanotube-bound BSA proteins. As reported in the literature, there are generally two somewhat different approaches for testing bioactivities of proteins: the immunochemical method and the total protein analysis.21-24 The former, represented by the wellknown ELISA (Enzyme-Linked Immunosorbent Assay), involves antigen-antibody interactions and thus offers excellent sensitivity.20,21 The latter, on the other hand, targets the amino acid building blocks in proteins. Among the most commonly used total protein analysis methods is the modified Lowry procedure, in which the tryptophan and tyrosine contents are measured to determine the protein concentration Nano Lett., Vol. 2, No. 4, 2002

Figure 5. Results from electrophoresis analyses of the SWNTBSA conjugate sample and the pristine BSA protein.

in the analyte.23,24 In the analysis of the nanotube-BSA conjugates reported here using the modified Lowry procedure,23 a dilute solution of SWNT-BSA conjugate in sodium chloride buffer was prepared. To a small aliquot of the solution (0.2 mL) was added Biuret reagent (2.2 mL). After the solution was allowed to stand for 10 min at room temperature, Folin-Ciocalteu’s phenol reagent (0.1 mL) was added. The resulting solution was allowed to stand at room temperature for another 30 min. The same experimental procedure was applied to pristine BSA to produce a solution for reference. Visible absorption spectra of the final solutions for SWNT-BSA conjugate and pristine BSA reference were measured and compared. The results show that the overwhelming majority of the BSA proteins (∼90% based on the difference in absorbance at 700 nm) in the conjugate remain bioactive. In summary, we have shown that BSA proteins can be covalently attached to carbon nanotubes via diimide-activated amidation under ambient conditions. The nanotube-BSA conjugates thus obtained are highly water-soluble, forming dark-colored aqueous solutions. Results from the characterization of the conjugates show that the proteins are intimately associated with the nanotubes. According to the total protein micro-determination assay, the overwhelming majority of the nanotube-bound BSA proteins remain bioactive. The same method may be used to introduce carbon nanotubes into other biologically and biomedically important systems. Acknowledgment. Y-P.S. acknowledges NSF (CHE9727506 and, in part, EPS-9977797), NASA (NCC1-01036, NGT1-52238, and NAT1-01036), the South Carolina Space Grant Consortium, and the Center for Advanced Engineering Fibers and Films (NSF-ERC at Clemson University) for financial support. A.M.R. acknowledges financial support from the NASA Ames Research Center (NCC2-5421). 313

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