NANO LETTERS

Location-Specific Biological Functionalization on Nanotubes: Attachment of Proteins at the Ends of Nanotubes Using Au Nanocrystal Masks
Ipsita A. Banerjee, Lingtao Yu, and Hiroshi Matsui*
Department of Chemistry and Biochemistry at Hunter College and the Graduate Center, The City UniVersity of New York, New York, New York 10021
Received January 20, 2003; Revised Manuscript Received February 6, 2003

2003 Vol. 3, No. 3 283-287

ABSTRACT
We developed a novel method to immobilize proteins at a specific location on nanotubes. In this report, avidin was immobilized only at the ends of peptide nanotubes using Au nanocrystals as protective masks on the sidewalls of nanotubes. While the Au nanocrystal-masked nanotubes adsorbed avidin on their entire surfaces, the chemical etching of the Au nanocrystal masks removed avidin molecules from the sidewalls, but avidin at the nanotube ends remained bound. The chemical etching process did not denature avidin and the nanotube ends could recognize and immobilize onto the complimentary biotin SAMs.

Over the past decade, there has been an immense interest in the fabrication of nanocomponents such as nanotubes, nanowires, and nanocrystals due to their potential to serve as building blocks for emerging nanometer-sized devices.1-12 While those nanocomponents have been assembled into electronic and sensor device configurations,13-18 the synthesis of smart nanotubes and nanocrystals that recognize specific complimentary molecules has become increasingly important to guide those nanometer-scaled building blocks to the right positions via molecular recognitions and self-assemblies. Recent trends involving the convergence of biotechnology and nanotechnology accelerate the development of hybrid nanomaterials incorporating the highly selective binding property of DNA.19-25 While DNA has been demonstrated as smart nanowires to be guided onto desired locations via their sequence-specific bindings,19 protein-functionalized nanowires may be more suitable for nanodevice building blocks due to their highly selective interactions toward their complimentary proteins.26-30 Recently, protein-functionalized nanotubes were positioned to interconnect patterned complimentary protein-SAM/Au surfaces via protein-protein interactions; however, the uniform coverage of proteins on template nanotubes also caused nanotube aggregations on the complimentary proteinSAM surfaces.29 One solution to produce nanotube-bridge configurations in higher yield could be selective protein immobilization at the ends of nanotubes, which prevents the sidewall-to-sidewall aggregation of nanotubes. Commonly used protein-patterning techniques are microcontact printing
10.1021/nl034038w CCC: $25.00 Published on Web 02/22/2003 2003 American Chemical Society

and AFM-based nanolithography;31-37 however, those techniques were applied to pattern proteins on flat surfaces, not on cylindrical interfaces such as nanotube surfaces. Recently, microrods encoded with sub-m stripes of metals and molecules were prepared by sequential electrochemical depositions inside porous templates.38 This method could be used for the end fabrication of nanotubes, but it requires the use of membrane templates and the membrane porous size also limits the diameter of nanotubes. One of the more straightforward methods to produce protein nanotubes is to immobilize proteins onto selfassembled peptide nanotubes as templates due to their high affinity to proteins.39 The peptide nanotubes are selfassembled from a peptide bolaamphiphile monomer, bis(NR-amido-glycyl-glycine)-1,7-heptane dicarboxylate, via intermolecular hydrogen bonds between amide and carboxylic acid groups.40-43 This peptide nanotube incorporates additional free amide and carboxylic groups on the surfaces as binding sites to anchor biological molecules and metal ions in order to produce various nanotubes such as metal nanotubes, porphyrin nanotubes, and protein nanotubes by simple incubation methods.39,44-46 Sequenced peptides were also incorporated onto this nanotube to control distribution and size of nanocrystals on the nanotube via biomineralization.47 Because these binding sites are distributed on the entire nanotube surface, proteins were immobilized uniformly on the surface of the nanotube.39 This was disadvantageous to immobilize the ends of protein nanotubes at the specific positions on the patterned complimentary protein surfaces

Scheme 1. Procedure to Immobilize Proteins at the Ends of Nanotubes Using Au Nanocrystals as Protective Masks

via biological interactions in order to fabricate fundamental device configurations such as a nanotube bridge. For example, avidin-coated nanotubes were interconnected between patterned biotin-SAMs, but the yield of the bridging nanotubes was less than 20% and most of the nanotubes were aggregated on the biotin-SAMs.29 In this study, we developed a method to immobilize proteins at specific locations, the ends, of the peptide nanotubes using Au nanocrystals as protective masks. The motivation of this report is that this type of protein nanotube can be assembled with higher accuracy into desired device configurations via biological recognitions of the nanotube ends. As a proof-of-concept, the end-functionalized nanotubes with avidin were demonstrated to recognize complimentary biotin SAMs on a patterned Au substrate, while the

population of aggregated nanotubes was reduced about 5 times compared to the nanotubes whose entire surfaces were coated by avidin.29 The outline for the proposed end-functionalization method is shown in Scheme 1. The approach involves thiolation of the nanotube sidewalls (step 1) and Au nanocrystal coating as a mask on the sidewall of the nanotube (step 2), followed by incubating sulforhodamine-labeled avidin in the nanotube solution (step 3). After avidin was immobilized at the ends (with no Au nanocrystals) and the sidewalls (with Au nanocrystals) of the nanotubes, the Au nanocrystals on the sidewalls were chemically etched and the proteins at the ends could remain attached without denaturing (step 4). In step 1, 2-mercaptoethylamine was covalently bound to COOH groups of the peptide nanotubes via NHS ester intermediates48,49 to introduce thiol groups to the sidewalls of nanotubes. Procedures to prepare the peptide nanotubes and the corresponding thiolated derivatives of the nanotubes are described in the Experimental Section. The thiolation of the nanotube surfaces was confirmed by FT-IR microscopic images, as shown in Figure 1 (Bruker IRScope II & Bruker Vector22 FT-IR spectrometer, Bruker Optics, Billerica, MA). Figure 1b is the IR intensity mapping of the CdO vibrational mode (1728 cm-1) on the bundled peptide nanotubes (Figure 1a) before the surface thiolation. In Figure 1b, brighter colors, corresponding higher IR intensity of the CdO mode, are distributed on the entire nanotube surface. This result is expected because the carboxylic acid groups are uniformly located on the entire nanotube surface.39 Figure 1c shows the IR intensity mapping of the CdO on the same nanotube bundles after thiolating the nanotube surfaces. As the sidewalls of nanotubes were thiolated, the 2-mercaptoethylamine was covalently bound to the carboxylic acid groups of peptide nanotubes as illustrated in Figure 1, and this thiolation

Figure 1. (a) Visible micrograph of the peptide nanotube bundles. (b) FT-IR microscopic image of the peptide nanotube bundles before the nanotube surface thiolation. Scale bar ) 2 m. (c) FT-IR microscopic image of the peptide nanotube bundles after the nanotube surface thiolation. In those images, the higher IR intensity of the CdO vibrational mode (1728 cm-1) was mapped with brighter colors.
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Figure 2. Transmission electron micrographs of (a) the Au nanocrystal-coated nanotube, scale bar ) 350 nm, (b) the nanotube after etching the Au nanocrystals from the nanotube surface, scale bar ) 300 nm.

induced the frequency shift of the CdO vibration. The absence of the bright color areas in Figure 1c suggests that the COOH group on the nanotube surfaces was substituted by the CONH(CH2)2SH group. Step 2 involved the deposition of Au nanocrystals onto the thiolated nanotubes. Au nanocrystals with a mean diameter of 40 nm were prepared using the seeded growth method.50 Procedures of the Au nanocrystal synthesis and their coatings on the sidewalls of the thiolated nanotubes are described in the Experimental Section. The Au nanocrystal immobilization on the nanotubes was confirmed by the transmission electron micrograph (TEM) in Figure 2a, showing that the aggregations of Au nanocrystals (dark spots in Figure 2a) coated the nanotube surface. The gradient of the Au nanocrystal distribution on the nanotubes shows that fewer Au nanocrystals were immobilized toward the tip of the nanotube. In step 3, sulforhodamine-labeled avidin was incubated into the Au nanocrystal-masked nanotube solution for 36 h. The fluorescence micrograph of the resulting nanotubes indicates that avidin was immobilized on the entire nanotube surface (Figure 3a; d in Scheme 1). This fluorescence image was dramatically changed after the Au nanocrystals were etched by iodine51 in the step 4, as shown in Figure 3b ((e) in Scheme 1). Procedures of the etching and solution cleaning processes are described in the Experimental Section. The removal of Au nanocrystals from the nanotube was observed in TEM micrograph of the Au nanocrystal-etched nanotube, which shows a transparent hollow nanotube structure without dark spots of the Au nanocrystals (Figure 2b). After etching the Au nanocrystals from the nanotube surface, fluorescing areas were limited only at the ends of the nanotube, and the fluorescence from the nanotube sidewall was diminished, as shown in Figure 3b. These fluorescence micrographs confirm that the protein remained bound to the peptide nanotube ends after the chemical Au nanocrystal etching. It should be noted that the concentration of 2-mercaptoethylamine must be controlled below 15 mM in 400 L solution in order to immobilize avidin at the ends of nanotubes. When the concentration of 2-mercaptoethylamine was 40 mM for the Au nanocrystal coating and underwent steps 3 and 4, no fluorescence was observed on the resulting nanotubes (Figure 3c). This observation suggests that 2-mercaptoethylamines bound not only the sidewall but also the ends of the nanotube when the 2-mercaptoethylamine concentration was too high in the nanotube solution. As the
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Figure 3. Fluorescence micrographs of (a) the Au nanocrystalmasked nanotube incubated with sulforhodamine-labeled avidin ((d) in Scheme 1), scale bar ) 2 m, (b) the nanotube with sulforhodamine-labeled avidin after etching the Au nanocrystals ((e) in Scheme 1), scale bar ) 4 m, (c) the nanotube, whose 2-mercaptoethylamine concentration on the nanotube surface was three times higher than (b), incubated with sulforhodamine-labeled avidin after etching the Au nanocrystals, scale bar ) 2 m, (d) the nanotube with avidin at the ends incubated with FITC-labeled biotin ((f) in Scheme 1), scale bar ) 1 m.

2-mercaptoethylamines bound the nanotube ends and induced the Au nanocrystal adsorption at the nanotube ends, those Au nanocrystals at the ends blocked the direct protein immobilization at the nanotube ends. If this process occurs, the Au nanocrystal etching removes all proteins from the entire nanotube surface, which is consistent with the no avidin observation on the nanotube with the higher 2-mercaptoethylamine concentration in Figure 3c. This hypothesis is also supported by a coarse calculation that the number of 2-mercaptoethylamine molecules in the concentration of 15 mM in 400 L solution, 3.6 1018, is comparable to the total number of the sidewall binding sites of the all-peptide nanotubes in the 200 L solution, 5.3 1018.52 One concern for this chemical Au nanocrystal etching process is that the etching treatment may denature proteins at the nanotube ends, which will prevent the use of this protein nanotube in the proposed device fabrication scheme due to the loss of the biological recognition function. To confirm whether the proteins at the nanotube ends can still recognize the complimentary molecules after the Au nanocrystal etching, FITC-labeled biotin was added to the avidinimmobilized nanotube solution after the Au nanocrystal removal ((f) in Scheme 1). After 36 h of the incubation, the biotin immobilization onto the avidin ends of the nanotubes was observed as green dots in the fluorescence micrograph in Figure 3d. Three fluorescing dots were observed since two nanotubes merged at the tips were imaged in this figure. Therefore, after the chemical etching of the Au nanocrystals, avidin at the nanotube ends is still active to recognize the complimentary biotin.
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Figure 4. Scanning electron micrograph of a patterned Au surface (brighter areas) with biotin SAMs and immobilized avidinfunctionalized nanotubes on their surfaces. Scale bar ) 10 m.

These end-functionalized nanotubes with avidin were deposited on a patterned Au substrate coated with biotin SAMs in solution to examine whether this system can reduce the nanotube aggregation while the protein nanotubes still recognize their complementary surfaces. After 1 day of the nanotube incubation, 85% of the nanotubes were immobilized on the biotin SAMs without aggregating, observed by scanning electron microscopy (SEM). Some of the nanotubes immobilized both ends on one SAM surface and some bridged two SAM surfaces, as shown in the SEM micrograph in Figure 4. When the nanotubes whose entire surfaces were coated by avidin were immobilized on the biotin SAMs, the ratio of aggregated nanotubes to whole nanotubes on the SAMs, evaluated in SEM images, was 83% on average.29 With this approach, the ratio of protein nanotube aggregation on the biotin SAMs could be reduced to 15%, which is about 5 times better than the previous method. In conclusion, avidin was immobilized only at the ends of peptide nanotubes using Au nanocrystals as protective masks. The chemical etching of the Au nanocrystal masks on the sidewalls of nanotubes removed avidin from the sidewalls, but avidin at the nanotube ends remained bound. The chemical etching process did not denature avidin, and the nanotube ends could recognize and immobilize on the complimentary biotin SAMs. This fabrication method is useful to guide each individual nanotube onto each corresponding location on complimentary protein-patterned surfaces because the nanotubes coated by proteins on the entire nanotube surfaces have tendency to yield the sidewall-tosidewall aggregations during the surface immobilization. Aggregation and branching of nanotubes will cause various problems in nanotube-based electronic device fabrications such as short-circuit and unnecessary nanotube-interconnection between electronic components after the nanotubes are metallized. Experimental Section. Materials. Bis(N-amido-glycylglycine)-1,7-heptane dicarboxylate was prepared as described elsewhere.40 Sulforhodamine-labeled avidin and FITC-labeled biotin were obtained from Sigma. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS) and hydrogen tetrachloroauric acid (HAuCl43H2O) were obtained from Aldrich. Tris buffer solution (pH 8) was obtained from Fisher Scientific. All chemicals were used as received. Preparation of Self-Assembled Peptide Nanotubes. To selfassemble the peptide nanotubes, the bis(N-amido-glycyl286

glycine)-1,7-heptane dicarboxylate monomer was dissolved in 30 mM NaOH to form the 10 mM solution. The solution was then acidified and the pH was adjusted to 4.36 with a 50 mM solution of citric acid. This solution was allowed to sit undisturbed at room temperature for two weeks to selfassemble into nanotubes. The formed nanotubes were then washed with Nanopure water (18 ) and centrifuged in a microcentrifuge (13,400 rpm) for 7 min. This process was repeated three times. To break up the nanotube aggregates, they were sonicated for an hour. Preparation of Thiolated Nanotubes. The peptide nanotubes (200 L) were immersed in an aqueous solution of 75 mM EDAC (200 L) and 15 mM N-hydroxy succinimide (200 L) for 30 min. This solution was mixed with 400 L of 15 mM 2-mercaptoethylamine and then added to the above reaction mixture in tris buffer (pH 8) for 24 h. The nanotubes were then washed exhaustively with the Nanopure water using a microcentrifuge. To introduce a higher concentration of thiol groups to the nanotube surfaces, 40 mM 2-mercaptoethylamine was added instead of 15 mM and the same procedure was used for thiolating the nanotube surfaces. Synthesis of Au Nanocrystals. Initially, Au nanocrystals with a mean diameter of 15 nm were prepared by reducing a boiling solution of 1wt % hydrogen tetrachloroauric acid (HAuCl43H2O) in deionized water with sodium citrate.53 Au nanocrystals with a mean diameter of 40 nm were subsequently prepared by the seed growth method.50 Briefly, 10 mL of the 15 nm gold colloid solution was diluted to 100 mL with water, and 0.5 mL of 0.2 M hydroxylamine hydrochloride (NH2OHHCl) was added to this solution with vigorous stirring. After 5 min, 1 mL of 1% HAuCl4 was added slowly and the reaction mixture was stirred for 15 min. In a separate beaker, 25 mL of this Au colloid solution was diluted to 100 mL with Nanopure water followed by addition of 0.5 mL of NH2OHHCl under stirring. After 5 min, 1 mL of 1% HAuCl4 was added slowly and the reaction mixture was stirred for 15 min to yield Au nanocrystals with 40 nm in diameter. The nanocrystals were then dialyzed against water for 24 h and used immediately. Deposition of Au Nanocrystals on Thiolated Nanotubes. The Au nanocrystal solution, 60 L, was mixed with 1 mL of the thiolated nanotube solution. This mixture was slowly stirred overnight, and the Au-coated nanotubes were then centrifuged and washed three times with the Nanopure water. Attachment of Sulforhodamine-Labeled AVidin onto AuCoated Nanotubes. The resulting Au-coated nanotube solution was mixed with 200 L of sulforhodamine-labeled avidin (0.5 mg/mL). This solution was wrapped with aluminum foil to prevent bleaching of the dyes and allowed to sit at 4 C for 36 h. The avidin-coated nanotubes were then washed twice with tris buffer using a microcentrifuge. Etching of Au Nanocrystals. Potassium iodide, 1 g, and iodine, 0.3 g, were dissolved in 1 mL Nanopure water. The nanotube solution was then mixed with 200 L of the iodine solution for 15 min and vortexed. The resulting nanotubes were then rinsed with methanol, and then acetone, followed by washing twice with the Nanopure water and centrifuging slowly (5000 rpm) for 3 min.
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Attachment of FITC-Labeled Biotin onto the AVidin Ends of Nanotubes. The nanotube solution was incubated with 100 L of FITC-labeled biotin (0.5 mg/mL). The vial of this mixture was wrapped in aluminum foil and allowed to sit at 4 C for 36 h. The resulting nanotubes were then washed twice with the tris buffer using a microcentrifuge. Immobilization of the End-Functionalized AVidin Nanotubes on Biotin SAMs. The detailed biotin-SAM deposition process on Au surfaces is described elsewhere.29,54 The biotin-SAM/Au substrate was placed in a citric acid solution (pH 5) along with 250 L of the end-coated avidin nanotubes prepared earlier. The end-coated avidin nanotubes were allowed to immobilize on the biotin SAMs for 24 h in the dark at 4 C. The SAMS were then rinsed with ethanolwater (1:1) before further analysis. The substrates were then analyzed by SEM. Acknowledgment. This work was supported by the National Science Foundation-CARRER (EIA-0133493), the National Science Foundation-NER (ECS-0103430), and the U.S. Department of Energy (DE-FG-02-01ER45935). I.B. and L.Y. acknowledge Mr. C. Matthaeus and Dr. M. Diem at the CUNY, College of Staten Island (Biology Department) for the assistance of FTIR microscopy, Dr. K. Fath for the use of the TEM facility at the CUNY, Queens College (Biology Department), and Dr. W. LAmoreaux for the use of the SEM facility at the CUNY, College of Staten Island (Biology Department). H.M. thanks Dr. S. Hong (Florida State University, Physics Department) for the donation of Au substrates. References
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