Selective Functionalization of Two-Component Magnetic Nanowires

Gold, nickel, and two-segment nickel−gold nanowires have been synthesized by electrodeposition into alumina templates. The nanowires have ∼350-nm ...
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Langmuir 2003, 19, 7043-7048

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Selective Functionalization of Two-Component Magnetic Nanowires Laura Ann Bauer,† Daniel H. Reich,*,‡ and Gerald J. Meyer*,†,§ Departments of Chemistry, Physics and Astronomy, and Materials Science and Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218 Received April 9, 2003. In Final Form: May 9, 2003 Gold, nickel, and two-segment nickel-gold nanowires have been synthesized by electrodeposition into alumina templates. The nanowires have ∼350-nm diameters and were typically 12-22 µm in length. The nanowires were removed from the templates and were functionalized with organic molecules. Adsorption isotherms were constructed for the binding of 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23Hporphine-2,18-dipropionic acid to nickel nanowires in ethanol solution at 298 K. Adduct formation constants of 9 ( 5 × 106 M-1 and limiting surface coverages of 8 × 10-10 mol/cm2 were abstracted from the isotherms. Surface functionalization conditions were identified where thiols bind selectively to gold and carboxylic acids bind to nickel. Nanowires with free amino or thiol functional groups were reacted with activated dyes to yield amide, thiourea, and thioether covalent linkages that were quantified by fluorescence microscopy. These reactions with two-segment nickel-gold nanowires produced materials that emitted light only on one segment of the nanowire or emitted light of different colors on each segment.

Introduction The templated electrodeposition of wires with nanometer diameters was first demonstrated in the late 1960s.1 Since that time researchers have utilized this approach to fabricate a variety of novel nanostructured materials.2-4 An advantage of electrodeposition is that different materials can be sequentially deposited in the templates yielding nanowires comprised of segments of different materials. These multisegmented or multicomponent wires have found interesting applications in biology5 and magnetics6,7 and bring exciting new perspectives for surface functionalization.8 By taking advantage of known surface coordination chemistry, it is possible to selectively functionalize multicomponent nanowires with receptors of interest for applications in sensing, molecular recognition, drug delivery, separations, and imaging. We have recently initiated studies to assemble magnetic nanowires with the goal of creating materials with enhanced anisotropy.9-13 Fluorescent and nonfluorescent nickel nanowires have been magnetically oriented and * Authors to whom correspondence should be addressed. † Department of Chemistry, Johns Hopkins University. ‡ Department of Physics and Astronomy, Johns Hopkins University. § Department of Materials Science and Engineering, Johns Hopkins University. (1) Possin, G. E. Rev. Sci. Instrum. 1970, 41 (5), 772. (2) Martin, C. R.; Mitchell, D. T. Electroanal. Chem. 1999, 21, 1-74. (3) Kovtyukhova, N. I.; Martin, B. R.; Mbindyo, J. K. N.; Smith, P. A.; Razavi, B.; Mayer, T. S.; Mallouk, T. E. J. Phys. Chem. B 2001, 105 (37), 8762-8769. (4) Wade, N.; Gologan, B.; Vincze, A.; Cooks, R. G.; Sullivan, D. M.; Bruening, M. L. Langmuir 2002, 18 (12), 4799-4808. (5) Nicewarner-Pena, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pena, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294 (5540), 137-141. (6) Liu, K.; Nagodawithana, K.; Searson, P. C.; Chien, C. L. Phys. Rev. B 1995, 51 (11), 7381-7384. (7) Blondel, A.; Meier, J. P.; Doudin, B.; Ansermet, J. P. Appl. Phys. Lett. 1994, 65 (23), 3019-3021. (8) Martin, B. R.; Dermody, D. J.; Reiss, B. D.; Fang, M. M.; Lyon, L. A.; Natan, M. J.; Mallouk, T. E. Adv. Mater. 1999, 11 (12), 10211025. (9) Chien, C. L.; Sun, L.; Tanase, M.; Bauer, L. A.; Hultgren, A.; Silevitch, D. M.; Meyer, G. J.; Searson, P. C.; Reich, D. H. J. Magn. Magn. Mater. 2002, 249 (1-2), 146-155.

Chart 1

self-assembled into long chains and “trapped” on lithographically deposited electrodes for single wire measurements.9-12 In addition, we have used magnetic nanowires for cell separations.13 Here, we report the selective surface functionalization and subsequent coupling chemistry with nickel, gold, and two-component nickel-gold nanowires. The functionalization and coupling chemistry with magnetic beads is a well-developed science, and commercial materials are widely available.14,15 However, to date these magnetic carriers have a single chemical functionality per bead, as is shown in Chart 1a. The approach described herein is to utilize multisegmented nanowires with inherent multiple chemical specificity through the use of ligands that bind selectively to the different segments. Chart 1b shows the example under study, where a two-segment nanowire is functionalized with ligands whose headgroups selectively bind to a desired segment and whose tail groups (R and R’) will target two different materials, cells, or biomolecules. We demonstrate that this coupling chemistry can be quite (10) Tanase, M.; Silevitch, D. M.; Hultgren, A.; Bauer, L. A.; Searson, P. C.; Meyer, G. J.; Reich, D. H. J. Appl. Phys. 2002, 91 (10), 85498551. (11) Tanase, M.; Bauer, L. A.; Hultgren, A.; Silevitch, D. M.; Sun, L.; Reich, D. H.; Searson, P. C.; Meyer, G. J. Nano Lett. 2001, 1 (3), 155158. (12) Reich, D. H.; Bauer, L. A.; Tanase, M.; Hultgren, A.; Chen, C. S.; Meyer, G. J. J. Appl. Phys. 2003, 93, 7275-7280. (13) Hultgren, A.; Tanase, M.; Chen, C. S.; Meyer, G. J.; Reich, D. H. J. Appl. Phys. 2003, 93, 7554-7556. (14) Polysciences, Inc. Web Page. www.polysciences.com (accessed Apr 2003). (15) Dynal Biotech Web Page. www.dynalbiotech.com (accessed Apr 2003).

10.1021/la034613b CCC: $25.00 © 2003 American Chemical Society Published on Web 07/15/2003

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general and can be directed to a specific segment of the magnetic nanowire. Experimental Section Materials. Hematoporphyrin IX dihydrochloride (HemIX) [8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine2,18-dipropionic acid, Aldrich], tetraphenylporphyrin (5,10,15,20tetraphenylporphin, Aldrich), 1,9-nonanedithiol (Aldrich), nonylmercaptan (Aldrich), boric acid (Aldrich), palmitic acid (Aldrich), nickel(II) chloride (NiCl2‚H2O, Alfa Aesar), copper(II) sulfate (CuSO4‚5H2O, Alfa Aesar), nickel sulfamate [Ni(H2NSO3)2‚4H2O, Alfa Aesar], Alexa Fluor 488 carboxylic acid, succinimidyl ester (AF488, Molecular Probes, Eugene, OR), Alexa Fluor 546 maleimide (AF546, Molecular Probes, Eugene, OR), fluorescein5-isothiocyanate (Molecular Probes, Eugene, OR), 11-aminoundecanoic acid (Acros), PBS (phosphate-buffered saline solution, pH 7.4, BioSource International, Camarillo, CA), and all other chemicals were used as supplied without further purification. The gold electrodeposition solution, 2.5 pennyweight/qt, (Technic, Inc., Cranston, RI) was an aqueous potassium aurocyanide and potassium oxalate solution. Alumina templates (Anodisc 47) with a 0.2-µm nominal pore diameter were obtained from Whatman (Maidstone, England). Nanowire Synthesis. Electrodeposition of nanowires was done in a three-electrode arrangement. An ∼0.4-µm-thick copper film sputter-deposited on the alumina membrane served as the working electrode with a platinum counter electrode and a Ag/ AgCl reference electrode. Potential control was accomplished with a BAS model CV-50W potentiostat. Prior to deposition of the nanowire, a small amount of copper (∼3 C) was grown in the pores to reinforce the sputtered copper layer. Copper was deposited from a 1 M CuSO4‚5H2O aqueous solution at -1.0 V. Nickel was deposited from a 20 g L-1 NiCl2‚H2O, 15 g L-1 Ni(H2NSO3)2‚4H2O and 20 g L-1 H3BO3 aqueous solution at -1.0 V. Gold nanowires were deposited from a 2.5 DWT/Qt. gold electrodeposition solution (Technic, Inc.) at -0.7 V. Twocomponent nickel-gold nanowires were prepared by first depositing gold, changing the solution, and then depositing nickel. Template Removal. After electrodeposition of the nanowires, the copper layer was removed with an aqueous solution of 0.5 M CuCl2 in 30% HCl. The alumina membrane was then dissolved in 0.5 M KOH at 60 °C. The magnetic nanowires were then isolated using a rare-earth magnet (JobMaster Magnets, Randallstown, MD) and placed in clean water. Gold nanowire collection was done via centrifugation at 14 000 rpm. Washing Procedure. Nickel and nickel-gold nanowires were collected from solutions with a rare-earth magnet or by centrifugation. The supernatant was decanted and replaced with 1.5 mL of neat solvent. The nanowires were then sonicated for 1 min in a Cole Parmer Model 8890 ultrasonicator at 50 kHz. This collection/solvent replacement/sonication process was usually performed five times. In cases where fluorescent molecules were used, the procedure was repeated until the supernatant was nonfluorescent. Especially rigorous washing methods were employed for AF488 and AF546 because of their high fluorescence quantum yields and low solubilities. Spectroscopy. Steady-state fluorescence measurements were performed in polystyrene cuvettes with a SPEX fluorolog spectrometer operating in the front face mode. UV-visible absorption measurements were made with a HP 8451A diode array spectrometer. Scanning electron microscopy measurements were performed with an JEOL high-resolution scanning electron microscope. Optical Microscopy. To isolate single nanowires for microscopy, sonicated nanowire solutions were quickly handpipetted onto a glass coverslip (Fisher) rotating at 2000 rpm on a model WS-400A-6NPP-Lite single wafer spin processor (Laurel Technologies, North Wales, PA). The sample was spun for an additional 10 s after the solution addition was complete. Fluorescence and reflection images were taken with a Nikon (Japan) TE200 microscope equipped with an Orca 100 monochrome charge-coupled device (CCD) camera. The light source used was a 100-W mercury arc lamp. The objective used was a Plan Apo 60× Oil DIC. When reflection images were taken, the light source was tempered with a 32× ND filter. Fluorescence

Bauer et al. images were taken with a G-2E/C TRITC filter set, which gives excitation light in the wavelengths from 525 to 555 nm and detects emission from 590 to 650 nm, or a B-2E/C FITC filter set, which gives excitation light in the wavelengths from 465 to 495 nm and detects emission from 515 to 555 nm. The exposure times were 7 s for fluorescence images and 35 ms for reflection images. Images were recorded and analyzed with CamMedia software. Nanowire segments were outlined along the edges of the nanowire as seen on the reflectance image. This outline was then transferred to the fluorescence image. The fluorescence intensity was determined using the average counts per pixel from the CCD in the outlined area. Background counts were determined from the average counts per pixel in an area with no fluorescence. The fluorescence intensity was corrected for dark counts by subtracting the background counts per pixel from the fluorescence counts per pixel. The linearity of the counts on the CCD with the fluorescence intensity was verified by repeating this procedure with calibrated fluorescent beads with known intensities.16

Results Nickel, gold, and two-segment nickel-gold nanowires were electrochemically synthesized in alumina templates, as was previously described.2-4,17 The length of the nanowires was calculated from the deposition charge and was later verified by scanning electron microscopy. The nanowire length was typically between 12 and 22 µm, and the nominal diameter was 350 ( 20 nm. The alumina template was removed in a basic aqueous solution, and the nanowires were isolated and cleaned. During this process, the nanowires had a tendency to bend. For twosegment nickel-gold nanowires, the bending often occurred at the junction between the two metals. Magnetic collection of the nanowires generally gave less bending than did centrifugation, and shorter nanowires were less susceptible to bending than were longer nanowires. Porphyrin Functionalization of Nickel Nanowires. Clean nickel nanowires were dried in an oven at 60 °C. They were then weighed and suspended in neat ethanol. A series of HemIX ethanol solutions were made, with known concentrations between 0.24 and 18 µM. For each concentration, two cuvettes were filled with 2.5 mL of the solution. One solution was reacted with nickel nanowires (0.5 mg) in ethanol, and the other cuvette served as a blank. After 48 h, a magnet was placed at the bottom of the reaction cuvette, and the solution absorption was measured. The concentration of HemIX associated with the nanowires was then determined by the difference between the absorption of the reaction cuvette and the absorption of the blank. Concentrations were then determined using Beer’s Law, A ) cl, where c is the molar concentration and l is the path length. The extinction coefficient is  ) 150 000 M-1cm-1. Shown in Figure 1 is a plot of the fractional surface coverage, θ, versus the solution concentration. At high solution concentrations, the surface coverage reached a limiting value, θ ) 1. The inset shows a double reciprocal plot of these data from which an equilibrium binding constant of 9 × 106 M-1 was obtained. Ethylene glycol suspensions of the nickel nanowires were stable for tens of minutes and could be easily redispersed by ultrasonication. Steady-state fluorescence and excitation spectra of HemIX functionalized nickel nanowire suspensions were, within experimental error, the same as those measured for HemIX in neat ethylene glycol. Collection of the nanowires with a magnet outside the excitation beam resulted in a complete loss of fluorescence, indicating that the observed emission was (16) Molecular Probes Web Page. www.molecularprobes.com (accessed Apr 2003). (17) Hutchison, J. L.; Routkevitch, D.; Albu-Yaron, A.; Moskovitz, M.; Nayak, R. R. Inst. Phys. Conf. Ser. 1997, 157, 389-392.

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Figure 3. 14-µm nickel-gold nanowire that has been functionalized with 11-aminoundecanoic acid and nonylmercaptan and then reacted with AF488. Part a shows a reflection image of a nanowire, and part b shows a fluorescent image of the nickel segment. Figure 1. Adsorption isotherms for HemIX binding to nickel nanowires in an ethanol solution at 298 K, where θ is the fractional surface coverage. The inset gives the double reciprocal plots, the data from which an adduct formation constant, K ) 9 × 106 M -1, was abstracted. Table 1. Optical Data for HemIX in Solution and Bound to Nanowires fluorescence excitation absorption max (nm) max (nm) max (nm) HemIX in ethanol HemIX in ethylene glycol HemIX on nanowires in ethylene glycol

626 625 626

389 391 391

396 409

Figure 2. 22-µm nickel-gold nanowire that has been functionalized with HemIX and nonylmercaptan. Part a shows a reflection image of a nanowire, and part b shows a fluorescent image of the nickel segment.

from HemIX anchored to the nanowires. Table 1 shows the absorption and emission maxima for HemIX in a solution and on the nanowire surface. Optical images of individual nickel nanowires functionalized with HemIX showed uniform fluorescence on the nanowire surface. Nanowires that were not reacted with HemIX, or that were reacted with tetraphenylporphyrin that contains no carboxylic acid groups, were nonemissive. Two-component nickel-gold nanowires were reacted with HemIX in the manner described previously but with the inclusion of 1 mM nonylmercaptan. Figure 2 shows optical images of a nickel-gold nanowire with a nickelgold segment ratio of 2:3, functionalized with HemIX and nonylmercaptan. Figure 2a shows a reflection image of the nanowire, and Figure 2b shows a fluorescence image. When the nonylmercaptan was not present in the derivatizing solution, both the gold and nickel segments of the nanowire were found to be emissive. Amide Coupling. Nickel nanowires (0.5 mg) were placed in 3 mL of a 1 mM ethanol solution of 11aminoundecanoic acid for 24 h. Nickel-gold nanowires were treated in the same manner except that 1 mM nonylmercaptan was also present in the ethanol. After repeated collection and washing with neat ethanol, the nanowires were reacted with 0.05 mg of AF488 in 1 mL of dimethyl sulfoxide (DMSO) and pH 9 aqueous phosphate buffer (50:50 v/v). The solution was sonicated for 1 min

Figure 4. Pair of 12-µm nickel nanowires that have been functionalized with 11-aminoundecanoic acid and then reacted with fluoroscein isothiocyanate. Part a shows a reflection image, and part b shows a fluorescent image of the nanowires.

and then resonicated after 30 min and 1 h. The nanowires were then washed with DMSO, water, and ethanol. Figure 3 shows reflection and fluorescence images of a single 17-µm-long nickel-gold nanowire functionalized in this manner. Analysis of the image reveals fluorescence intensity from the nickel segment of the nanowire, which is six times the fluorescence intensity from the gold segment of the nanowire. A control experiment, performed by reacting a nickel nanowire with AF488 under the same conditions as those described previously, showed an identical reflection image, but no fluorescence was observed. In addition, no fluorescence was observed from nanowires where the base was omitted from the reaction mixture. Thiourea Coupling. Nickel nanowires (0.5 mg) were reacted with 1 mM 11-aminoundecanoic acid solution (3 mL) in ethanol for 24 h. The nanowires were collected and washed with ethanol. A 0.5-mL aliquot of 2.5 mM fluorescein-5-isothiocyanate in DMSO was then reacted with the nanowires. The solution was sonicated for 1 min, and then resonicated after 30 min and 1 h. The nanowires were then washed with ethanol. Figure 4 shows optical images of two 13-µm-long nanowires functionalized in this manner. Control experiments performed by reacting a bare nickel nanowire with fluorescein-5-isothiocyanate solution under the same conditions showed an identical reflection image, but no fluorescence was observed. Thioether Coupling. Gold nanowires (0.5 mg) were placed in a 1 mM 1,9-nonanedithiol solution (3 mL) for 24 h. They were then washed with ethanol and placed in DMSO-PBS (50:50 v/v; 1 mL). A solid sample of AF546 (0.05 mg) was immediately added to the solution that was then sonicated for 1 min, and repeatedly sonicated for 1 min every 30 min for 2 h. After 2 h, the nanowires were isolated via centrifugation and washed with DMSO, water, and ethanol. Microscopy showed uniform fluorescence intensity along the length of the wire. The control experiment performed in the absence of the PBS buffer, or the 1,9-nonanedithiol yielded nonfluorescent nanowires.

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Figure 5. 17-µm nickel-gold nanowire that has been functionalized with nonanedithiol and palmitic acid and then reacted with AF546. Part a shows a reflection image of a nanowire, and part b shows the fluorescent image of the gold segment.

Figure 6. 20-µm nickel-gold nanowire that has been functionalized with nonanedithiol and 11-aminoundecanoic acid and then reacted with AF488 and AF546. Part a shows a reflection image, part b shows a fluorescent image of the nanowire observed from 590 to 650 nm, and part c shows a fluorescent image of the nanowire observed from 515 to 555 nm.

The thiourea coupling of two-component nickel-gold nanowires was analogous to that of gold alone with the following exceptions. The nanowires were first reacted with 1.5 mM palmitic acid in ethanol solution (2 mL) for 24 h and subsequently placed in a 1 mM palmitic acid, 1 mM nonanedithiol ethanol solution (3 mL) for 24 h. The nanowires were then reacted with the AF546 for 30 min. Figure 5 shows typical reflectance and fluorescence images of a nickel-gold nanowire functionalized in this manner. Analysis of the images reveals fluorescence from the gold segment that had 12 times the intensity of that of the nickel segment of the nanowire. Two-Component Functionalization. Nickel-gold nanowires (0.5 mg) were placed in a 1.5 mM 11-aminoundecanoic acid solution (2 mL) for 24 h. An aliquot of a 3 mM 1,9-nonanedithiol solution (1 mL) was then added and allowed to react for 18 h. The nanowires were then washed with ethanol. The fluorophores, AF546 (0.05 mg) and AF488 (0.05 mg), were then reacted with the wires in DMSO-PBS (50:50 v/v; 1 mL). The solution was sonicated for 1 min initially and then for 1 min every 30 min for 2 h. After 2 h, the nanowires were washed with DMSO, water, and ethanol. Figure 6 shows typical reflectance and fluorescence images of a nickel-gold nanowire functionalized in this manner. Figure 6b shows a fluorescence image observed from 590 to 650 nm. Analysis of this and other images revealed that the gold segment was typically four times more fluorescent than the nickel segment of the nanowire. Figure 6c shows the fluorescence image observed from 515 to 555 nm, where it was found that the nickel segment had 2.5 times the fluorescence than the gold segment of the nanowire. Discussion The templated electrodeposition of nanowires yields a large number of nanowires with reasonably uniform lengths and diameters. The synthetic approach described affords approximately 500 million 350-nm-diameter, 1015-µm-long wires with magnetic and fluorescent segments.

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The pore diameters are irregular at the edges of the template,18 so the wires were electrodeposited in the template interior. We note that the preparation of templates with more uniform pore diameters has been described in the literature.19,20 The known surface coordination chemistry of planar surfaces was used to selectively functionalize the two segments of the nanowire with organic ligands. The end groups of the organic ligands were then covalently linked to dye molecules to yield nanowires that were fluorescent on that segment. These dual functionalized nanowires have potential applications as fluorescent probes and for magnetic separations in biology. In the following, we discuss details of the interfacial chemistry followed by the coupling chemistry. Surface Coordination Chemistry. It has been recognized for some time that hard-soft acid-base concepts originally developed in fluid solution are also applicable to surface coordination chemistry.21 Soft bases such as thiols coordinate to soft metals such as gold, whereas carboxylic acids bind to harder metal oxides such as NiO. However, the opposite reactions are also known, that is, thiols bind to nickel oxides22 and carboxylic acids bind to gold.23 The results described herein, and the results from published studies on planar surfaces, clearly show that the soft-soft and hard-hard interactions are preferred.21 High selectivity has been achieved by optimizing the reaction conditions and by simultaneously binding the two acids to the two-segment nanowires. Fluorescence microscopy was used as an assay to identify the physical location of the coordination chemistry. The relatively low surface area and the geometry of the nanowires preclude the use of many of the characterization techniques commonly applied to self-assembled monolayers (SAMS) on planar surfaces. Fluorescence quenching of fluorophores proximate to a metal surface is well documented in the literature.24,25 However, the surface fluorescence was not completely quenched on the nanowire and could easily be observed by microscopy. HemIX was chosen as a fluorescent probe to quantify directly the coordination of carboxylic acids to the native oxide on nickel.11 HemIX has two carboxylic acid groups linked to the porphyrin ring by a flexible ethane spacer. Adsorption isotherms were constructed for the binding of HemIX to nickel nanowires in an ethanol solution at 298 K by plotting the fractional surface coverage, θ, versus the solution concentration. The surface coverage was found to reach a limiting value at high solution concentrations, as was expected for the adsorption isotherm model proposed by Langmuir.26 Double reciprocal plots of θ-1 versus [HemIX]-1, shown in the inset of Figure 1, are linear and permit the adduct formation constant, K, to be abstracted from the slope. We find K to be 6 ( 5 × 106 M-1 for the nickel nanowires. Adduct formation constants of 105-106 M-1 are typical for the binding of carboxylic acid(18) Wilson, J. N.; Bancuyo, C. G.; Erdogen, B.; Myrick, M. L.; Bunz, U. H. F. Macromolecules 2003, 36, 1426-1428. (19) Sun, L.; Chien, C. L.; Searson, P. C. J. Mater. Sci. 2000, 35 (5), 1097-1103. (20) Choi, J.; Neilsch, M.; Reiche, M.; Wehrspoon, R. B.; Gosele, U. J. Vac. Sci. Technol., B 2003, 21 (2), 763-766. (21) Laibinis, P. E.; Hickman, J. J.; Wrighton, M. S.; Whitesides, G. M. Science 1989, 245 (4920), 845-847. (22) Mekhalif, Z.; Riga, J.; Pireaux, J. J.; Delhalle, J. Langmuir 1997, 13 (8), 2285-2290. (23) Zhang, Z.; Imae, T. Nano Lett. 2001, 1 (5), 241-243. (24) Chen, S. H.; Frank, C. W. Langmuir 1991, 7, 1719-1726. (25) Karpovich, D. S.; Blanchard, G. J. Langmuir 1997, 13 (15), 40314037. (26) Langmuir, I. J. Am. Chem. Soc. 1918, 40 (9), 1361-1403.

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containing molecules to metal oxide surfaces.27-31 The limiting concentration of HemIX bound to the nanowire surface was determined spectroscopically to be 8 × 10-10 mol/cm2. With a surface area of 6.4 cm2/mg and assuming that the porphyrin footprint on the surface is 100 Å2,32 this corresponds to about 1.8 monolayers. With the inherent uncertainty in the true surface area, the limiting surface coverage most probably corresponds to the monolayer coverage. Attempts to construct adsorption isotherms of HemIX on gold were unsuccessful, presumably due to the weak -CO2H|Au adducts formed and the high solubility of HemIX in ethanol.23 When two-segment nickel-gold nanowires were reacted with HemIX, the nickel segment showed uniform fluorescence with weak and nonuniform fluorescence from the gold segment. The gold segment could be made nonfluorescent by adding a long-chain thiol to the reaction mixture. The thiol likely displaces any weakly bound HemIX. We note that similar behavior has been observed in the reactions of long-chain carboxylic acids and thiols with patterned gold-aluminum surfaces.21 Coupling Chemistry. Three coupling reactions, based on amide, thiourea, and thioether linkages, were studied to bind fluorophores to functionalized segments of the nanowires, as is shown in Scheme 1. We utilize reactive fluorescent probes available commercially for labeling proteins and other biomolecules.16,33 Related coupling chemistry has also been reported for SAMS on planar (27) Meyer, T. J.; Meyer, G. J.; Pfennig, B. W.; Schoonover, J. R.; Timpson, C. J.; Wall, J. F.; Kobusch, C.; Chen, X. H.; Peek, B. M.; Wall, C. G.; Ou, W.; Erickson, B. W.; Bignozzi, C. A. Inorg. Chem. 1994, 33 (18), 3952-3964. (28) Argazzi, R.; Bignozzi, C. A.; Heimer, T. A.; Castellano, F. N.; Meyer, G. J. Inorg. Chem. 1994, 33 (25), 5741-5749. (29) Heimer, T. A.; Darcangelis, S. T.; Farzad, F.; Stipkala, J. M.; Meyer, G. J. Inorg. Chem. 1996, 35 (18), 5319-5324. (30) Pechy, P.; Rotzinger, F. P.; Nazeeruddin, M. K.; Kohle, O.; Zakeeruddin, S. M.; Humphrybaker, R.; Gratzel, M. J. Chem. Soc., Chem. Commun. 1995, No. 1, 65-66. (31) Nazeeruddin, M. K.; Liska, P.; Moser, J.; Vlachopoulos, N.; Gratzel, M. Helv. Chim. Acta 1990, 73 (6), 1788-1803. (32) Pilloud, D. L.; Moser, C. C.; Reddy, K. S.; Dutton, P. L. Langmuir 1998, 14 (17), 4809-4818. (33) Lundblad, R. L.; Noyes, C. M. Chemical Reagents for Protein Modification; CRC Press: Boca Raton, FL, 1984; Vol 1.

surfaces and is the subject of a recent review article.34 In all three coupling reactions studied, we found no evidence for the fluorophores binding directly to the unfunctionalized gold or nickel segments. The first two coupling reactions, Scheme 1a,b, rely on the reactivity of amine groups. Scheme 1a utilizes a succinimidyl ester of the AF488 dye that reacts readily with terminal amine groups on the nanowire surface to form amide linkages. The succinimidyl ester is activated, and the coupling reactions do not require the use of dehydrating agents such as dicyclohexylcarbodiimide. Scheme 1b uses fluoroscein isothiocyanate that reacts to form a thiourea linkage. In both cases, fluorescence microscopy clearly shows that the selective functionalization occurs to yield nanowires with a green fluorescent magnetic segment and a nonfluorescent gold segment. Both coupling reactions have been well studied in biology with fluoroscein labeling historically being the most common.33 However, fluoroscein has a relatively high rate of photobleaching, and the fluorescence intensity is significantly diminished below pH 7.16 The AF488 dye has a significantly higher stability and a higher fluorescence quantum yield that is independent of the pH from 4 to 10.16 While the stability of both dyes coupled to the nanowires appeared to be comparable on the laboratory time scale of a few weeks, the amide-linked AF488 dye is expected to be superior for long-term applications. The amide bonds formed with AF488 are expected to be as stable as peptide bonds in biology.33 Scheme 1c is the reaction of thiols with maleimide substituents of AF546. In this reaction, the thiol acts as a nucleophile, adding to the double bond in maleimide to yield a stable thioether linkage. Related reactions are well studied for fluorescence tagging in biology because the sulfhydryl group of cysteine is generally the most reactive functional group in a protein.16,33 Evidence for the formation of thioether links is apparent in the appearance of red fluorescence in the gold segment of two-segment nickel-gold nanowires. (34) Sullivan, T. P.; Huck, W. T. S. Eur. J. Org. Chem. 2003, No. 1, 17-29.

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Coupling two unique fluorescent probes to discrete segments of a two-segment nanowire was also examined (Figure 6). In this case, selectivity depends on (1) carboxylic acids binding to nickel and thiols binding to gold and (2) the reactions of the maleimide (AF546) with the thiol and the succinimidyl ester (AF488) with the amine. The gold segment shows an intense red fluorescence after reaction with AF546. A weak red fluorescence, ∼one-fourth the intensity, from the nickel segment must signal the presence of a small amount of dithiol on nickel because the maleimide is not know to react with amines. The nickel segment shows an intense green fluorescence after reaction with AF488, and the gold segment displays some green fluorescence. This is presumably a result of a side reaction between the thiol and the succinimidyl ester. Such side reactions have previously been observed in proteinlabeling studies and should be minimal at the pH used.16 The relatively weak green emission from the gold segment (∼40% that of nickel) implies that the coupling chemistry is much less efficient. This approach yields nanowires that display unique fluorescence spectra on each segment of the nanowire. Conclusion Selective surface functionalization and coupling chemistry with two-segment nickel-gold nanowires has been

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achieved. The selective surface coordination chemistry takes advantage of hard-soft acid-base chemistry and can yield magnetic nanowires that emit light on only one segment of the nanowire. Subsequent coupling reactions with free amine or thiol groups were also quantified and generated nanowires that emit different colored light on the two ends. Even though a limited number of coupling reactions (amide, thioether, and thiourea) and dyes were studied, the results and approach appear to be general and are not limited to nickel and gold or to fluorescent molecules.8 The fluorescent and magnetic asymmetry of the nanowires have potential advantages over spherical beads and may be exploited as orientational probes for biological samples. In addition, external magnetic fields can be used for the in situ assembly or isolation of the nanowires.11 Applications of these nanowires in biotechnology are currently underway in our laboratories. Acknowledgment. The authors acknowledge equipment support from the NSF MRSEC, Grant DMR0080031. The authors acknowledge support from DARPA/ AFOSR, Grant F49620-02-1-0307, and from The David and Lucille Packard Foundation, Grant 2001-17715. We thank Professor P. C. Searson for helpful discussions and assistance. LA034613B