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Figure 1. (A) C60 fullerene binding to monoclonal antibody with specificity for C60-fullerene. (B) [10,10]-tube binding to modified anti-C60 antibody |
The anti-fullerene antibody has been tested by biological techniques that determine its binding specificity for various fullerenes, fullerene derivatives, and carbon nanotubes. In accordance with expectations, the antibody binds to the fullerene sphere, rather than attached side chains. For example, a C60-dendrimer fullerene derivative synthesized by Hirsch and coworkers [12] has a significantly lower IC50 value than the C3 and D3 tris-malonate derivatives. Mono-fullerene adducts bind better than tris-fullerenes adducts, possibly because more of the carbon sphere is accessible to the antibody-binding site. Molecular modeling performed on the binding of C60 and several fullerene derivatives to the antibody agree with this experimental observation [13]. Furthermore, the antibody has been shown to bind strongly to carbon nanotubes, since they possess similar properties to fullerenes (unpublished observations).
Experimental binding studies of the anti-fullerene monoclonal antibody has previously only been performed using standard biological techniques. Extensive molecular modeling has resulted in theoretical corroboration of the experimental data. This work presents the first binding data of the antibody using real-time surface plasmon resonance methods. In addition, binding data for several molecules that have not been previously tested are presented herein. Surface plasmon resonance allows for testing and analysis of binding in a relatively easy, fast, and repeatable manner.
The synthesis of C3-trisamine-C60 (molecule 1) has previously been reported [14]. Modification of 1 was accomplished as depicted in Scheme 1 to produce the biotinylated fullerene, molecule 2. [15] This preparation will be reported elsewhere.
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Scheme 1. Attachment of biotin to a C3-symmetric trisamine C60-fullerene (1) to form a biotinylated fullerene derivative (2). |
Physical cleaning of the Spreeta gold sensor surface is required before assembly of the flow cell for binding studies. The gold surface was cleaned by removal of macroscopic dirt particles by applying pure deionized water, 100% ethanol, and 0.1 M NaOH in 1% Triton X-100 in H2O, in alternating solutions, gentle wiping with a soft Kim wipe (Kimberly-Clark, Roswell, GA) and drying with nitrogen gas. The flow cell was assembled according to the Spreeta User's Manual [16] using the standard Teflon block with a small black O-ring sealing the gold sensor channel. A syringe pump was connected to the flow cell through a short length of tubing, allowing solutions in 10-mL disposable plastic syringes (Becton Dickinson, Franklin Lakes, NJ) to be delivered across the sensor surface.
Buffers and detergent solutions must be used to clean the sensor surface before biomolecules can be adsorbed on the gold. Buffers used to clean the sensor and flow all solutions for the protein binding experiments included purified deionized water and phosphate-buffered saline (PBS), pH 7.0-7.2 (GibcoBRL, Gaithersburg, MD). The changes in index of refraction during a typical solution cleaning procedure for a newly assembled Spreeta sensor with the flow cell apparatus are depicted in Graph 1.
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Graph 1. Typical cleaning of gold sensor surface by washing with successive flows of H2O, (A) 0.1 M NaOH in 1% Triton X-100 in H2O, (B) H2O again with (C) recalibration to 1.333, and (D) final PBS baseline, followed by (E) adsorption of Neutravidin. |
The first 600 seconds in Graph 1 indicates the stable pre-clean baseline flow of deionized water at 7.2 µL s-1 arbitrarily set at 1.333. At point A, the flow was switched to a detergent solution of 0.1 M NaOH in 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in water for 10 minutes. At point B, water was flowed again, and the index of refraction was allowed to decrease to a stable value below the original pre-clean baseline. This stable index of refraction value is the real index of refraction of water for a clean gold surface, and was reset to 1.333 to reflect this fact at point C. At point D, the flow was changed to PBS for 10 minutes. A 100 µg mL-1 solution of purified streptavidin protein known as Neutravidin (Pierce, Rockford, IL) in PBS was flowed across the surface at a rate of 0.72 µL s-1 at point E. Adsorption of Neutravidin was allowed to progress until saturation at a stable index of refraction value was observed. Subsequent flow of PBS only did not decrease the index of refraction.
At a concentration of 180 µg mL-1, compound 2 dissolved in PBS was flowed across the sensor surface at 5 - 6 µL s-1. The solution (total volume of 10 mL) was recirculated through the flow cell for 40 minutes. A rinse for 10 minutes (10 - 12 µL s-1) with PBS followed. At this point, the sensor was primed to detect binding proteins to C60. The change in index of refraction by the binding of compound 2 to streptavidin on the gold biosensor surface versus time is contained in Graph 2. (The flow was changed during the time gap indicated on the graph.)
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Graph 2. Binding biotinylated fullerene compound 2 to streptavidin adsorbed to the gold sensor surface, (A) beginning and (B) ending with PBS flows. |
After the baseline was established by flowing PBS at 10 - 12 µL s-1 for 10 minutes, a solution of anti-C60 antibody [17] 4 µg mL-1 (20 µL of a 2 mg mL-1 solution, diluted in 10 mL of PBS) was recirculated through the system at 5 - 6 µL s-1 until the index of refraction measurements reached a plateau level. Extra time was allotted to compensate for flow impedance, mass transport, and the alignment of the binding site with fullerene ligand. Afterwards, PBS buffer was run through the flow cell system across the sensing surface for 10 minutes (10 - 12 µL s-1) to remove all traces of the bulk antibody solution and establish a final binding index of refraction baseline. Graph 3 shows index of refraction data gathered during typical anti-C60 antibody binding. The expected arrangement of biomolecules on the Spreeta sensor surface after the antibody is bound is depicted in Figure 2.
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Graph 3. Typical binding of anti-C60 antibody to fullerene 2 on biosensor surface. |
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Figure 2. Cartoon of molecules on biosensor surface. Figure not representative of scale of molecules, except where indicated. |
The data gathered from two typical competition binding experiments involving preformed solutions of anti-C60 antibody and a water-soluble fullerene are presented in Graph 4. In curve A, a solution of both compound 1 and anti-C60 antibody in PBS was flowed across the surface that has Neutravidin-bound compound 2 already on it. The final change in index of refraction was less intense than binding of anti-C60 antibody alone. Similarly, a solution of the antibody and the water-soluble C60-dendrimer produced a significantly smaller change in index of refraction, as curve B indicates.
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Graph 4. Typical solution phase competition binding experiments using preformed solutions of anti-C60 antibody and a water-soluble fullerene. Curve A shows using molecule 1 and curve B shows using the C60-dendrimer [12]. |
Before performing any data analysis, the SPR curve was smoothed by a least-squares method that incorporated one point on either side of a data point [18,19]. This reduced the noise when tracking refractive index versus time.
The preliminary binding experiments described herein involved procedures quite similar to those presented by Texas Instruments in the supplementary information supplied for the Spreeta device [20]. The first experiment involved the cleaning of the gold sensor surface, adsorption of streptavidin, and subsequent attachment of the biotinylated fullerene, molecule 2. This was successful and corresponded to data presented in the Spreeta preliminary information. The SPR data indicating binding of material to the biosensor was supplemented by comparison of UV-Vis absorption spectra of solutions before and after they were used, revealing consistent decreases in absorption intensity corresponding to the material deposited on the sensor surface (data not shown).
Small molecules are more difficult to detect because they contribute only small changes in mass to a solution, giving also only minimum refractive index changes [21]. For this reason, a fullerene was immobilized on the sensor rather than the fullerene binding protein so that larger SPR signals would result. The molecular weight of the anti-C60 antibody is about 150 000 (150 kDa), whereas a typical fullerene derivative has a molecule weight of 1000 to 3000 (1 to 3 kDa).
Control binding experiments were performed to check for possible non-specific binding interactions. Bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) was used as a substitute protein for all phases of the binding interaction tests. BSA was used to check for non-specific protein-protein interactions that might exist between streptavidin and the antibody. In addition, without fullerenes present the antibody had only small non-specific interactions with streptavidin on the biosensor surface. There was no significant binding for non-biotinylated fullerenes to streptavidin on the gold sensor surface. Data from all these experiments never contradicted the data gathered for successful binding of the antibody to fullerenes. Finally, flows of detergents after binding experiments confirmed that only specific binding interactions existed.
Binding anti-C60 antibody to the biotinylated-fullerene on the biosensor surface resulted in an average change in index of refraction of 1.4 x 10-3 RIU. The concentration of antibody used in the binding experiments was shown to be more than sufficient to reach saturation of binding, since consistent results were achieved upon repeated use of the same antibody solutions.
The data presented in Graph 4 indicate that C60-dendrimer binds to the anti-C60 antibody better than molecule 1. The smaller overall increase in index of refraction shows that solution phase binding is more competitive with C60-dendrimer [11] in solution than molecule 1.
Finally, preliminary experiments with carbon nanotubes indicate that they can be detected by SPR in experiments similar to those performed with fullerenes. The experiments showed that the anti-C60 antibody binds strongly to very dilute solutions of single-walled carbon nanotubes (SWNTs, from tubes@rice) in PBS. In fact, the binding of the antibody to SWNTs produced a larger change in index of refraction than for binding to fullerenes, (data not shown). While part of the reason for the strong SPR signal could be due to the high mass of SWNTs, there also may be more binding sites for the antibody and more "irreversible" binding interactions between the antibody and SWNTs. Further research in this area is currently in progress.
The data presented in this paper show that a biosensor surface can be prepared that detects the binding of fullerenes and carbon nanotubes. The qualitative analysis of these experiments demonstrates the extent of antibody-fullerene binding through surface and solution phase interactions. The data from these binding experiments will be rigorously analyzed to determine useful quantitative information, such as stoichiometry of interaction, association and dissociation binding constants, and equilibrium constants. Data and techniques presented in this paper, and future SPR experimentation, will improve our understanding of fullerene-protein interactions and provide new tools for nanotube analysis.
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[2] See information at http://plasmon.fiu.edu/spr
[3] Refractive index relative to air at 20°C with 589 nm light. 1972 American Institute of Physics handbook ed Gray D E (New York: McGraw-Hill)
[4] (a) Parsons I D, Persson B, Mekhalfia A, Blackburn G M and Stockley P G 1995 Nucleic Acids Res. 23 211-216 (b) Parsons I D and Stockley P G 1997 Anal. Biochem. 254 82-87 (c) Stockley P G, Baron A J, Wild C M, Parsons I D, Miller C M, Holtham C A M and Baumberg S 1998 Biosensors. Bioelect. 13 637-650
[5] (a) Elkind J L, Stimpson D I, Strong A A, Bartholomew D U and Melendez J L 1999 Sensors and Actuators B 54 182-190 (b) Kukanskias K, Elkind J, Melendez J, Murphy T, Miller G and Garner H 1999 Analytical Biochem 274 7-17
[6] Wilson S R 2000 Biological Aspects of Fullerenes (Fullerenes: Chemistry, Physics, and Technology), ed K M Kadish and R S Ruoff (New York: John Wiley & Sons) p 439
[7] Ku H H, Cleveland W L and Erlanger B F 1987 J. Immunol. 139 2376-2384
[8] Ownby D R, Ownby H E, McCullough J and Shafer A W 1996 J. Allergy Clin. Immunol. 97 1188-1192 Also, see article at http://www.cdc.gov/niosh/latexalt.html
[9] Moussa F, Trivin F, Céolin R, Hadchouel M, Sizaret P-Y, Greugny V, Fabre C, Rassat A and Szwarc H 1996 Full. Sci. Tech. 4 21-29
[10] Chen B-X, Wilson S R, Das M, Coughlin D J and Erlanger B F 1998 Proc. Natl. Acad. Sci. USA 95 10809-10813
[11] Braden B C, Goldbaum F A, Chen B-X, Kirschner A N, Wilson S R and Erlanger B F 2000 Proc. Natl. Acad. Sci. USA 97 12193-12197
[12] Brettreich M and Hirsch A 1998 Tetrahedron Lett. 27 2731
[13] Chen B-X, Braden B C, Erlanger B F, Kirschner A N and Wilson S R 2000 Proc. Electrochem. Soc. 9 233-239
[14] Richardson C F, Schuster D I and Wilson S R 2000 Org. Lett. 2 1011-1014
[15] Synthesized and supplied by C. F. Richardson (Department of Chemistry, New York University, NY)
[16] Spreeta User's Manual, Texas Instruments, Inc., August 1999.
[17] Generously donated by B F Erlanger and B-X Chen (Department of Microbiology, Columbia University, NY)
[18] Savitzky A C and Golay M J E 1964 Analytical Chem. 36 1627-1639
[19] Savitzky A C and Siggia S 1972 Analytical Chem. 44 1906-1909
[20] See TI Spreeta website at http://www.ti.com/spreeta
[21] Vangent J, Lambeck P V, Kreuwel H J M, Gerritsma G J, Sudehölter E J R, Reinhoudt D N and Popma Th J A 1989 Sens. Actuators 17 297-305
We would like to thank NASA Graduate Student Researchers Program for financial support. We are very appreciative of the helpful correspondence and technical assistance concerning TI Spreeta provided by Jerry Elkind, Anita Strong, Dwight Bartholomew, and other members of Texas Instruments, Inc.
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