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Langmuir 2008, 24, 2206-2211
Maleimide-Activated Aryl Diazonium Salts for Electrode Surface Functionalization with Biological and Redox-Active Molecules Jason C. Harper, Ronen Polsky, David R. Wheeler, and Susan M. Brozik* Biosensors and Nanomaterials, Sandia National Laboratories, P.O. Box 5800, MS-0892, Albuquerque, New Mexico 87185 ReceiVed August 23, 2007. In Final Form: NoVember 12, 2007 A versatile and simple method is introduced for formation of maleimide-functionalized surfaces using maleimideactivated aryl diazonium salts. We show for the first time electrodeposition of N-(4-diazophenyl)maleimide tetrafluoroborate on gold and carbon electrodes which was characterized via voltammetry, grazing angle FTIR, and ellipsometry. Electrodeposition conditions were used to control film thickness and yielded submonolayer-to-multilayer grafting. The resulting phenylmaleimide surfaces served as effective coupling agents for electrode functionalization with ferrocene and the redox-active protein cytochrome c. The utility of phenylmaleimide diazonium toward formation of a diazonium-activated conjugate, followed by direct electrodeposition of the diazonium-modified DNA onto the electrode surface, was also demonstrated. Effective electron transfer was obtained between immobilized molecules and the electrodes. This novel application of N-phenylmaleimide diazonium may facilitate the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays.
Introduction Control over functionalization of conducting or semiconducting microelectrode array surfaces with biological, redox-active, and photo/chemical sensitive molecules is a critical component in the development of bioelectronics, proteomic research, tissue engineering, clinical diagnostics, and chemical and biological sensing.1 Consequently, this topic is receiving a great deal of attention. A significant disadvantage in current array technology is the requirement for separate platforms for detection of different chemicals and/or biomolecules.2 Valuable information impacting research areas including cell signaling, genomic, and proteomic expression assays, and the proof-positive identification of biological organisms can be obtained via detection of several different biomarkers over the course of an experiment. The detection of virus or large bacterial-like particles, such as anthrax for instance, might require the identification of not just genomic signatures, but also several protein markers, greatly improving the confidence in the sensor’s results. Recently we demonstrated for the first time the simultaneous electrochemical detection of DNA and protein on the same electrode array using an electrically addressable deposition procedure to selectively immobilize the different biomolecule probes.3 This ability could possibly obviate the need for multiple orthogonal detection platforms for definitive identification of the target analyte(s). An important advancement that would facilitate development of future integrated microarray systems is the realization of a “universal toolset”4 that would enable the discrete immobilization of a vast number of chemicals and/or biomoleules onto individual electrodes in the same array. The use of aryl diazonium salts for * To whom correspondence should be addressed. E-mail: smbrozi@ sandia.gov. Phone: (505) 844-5105. Fax: (505) 845-8161. (1) (a) Willner, I.; Baron, R.; Willner, B. Biosens. Bioelectron. 2007, 22, 1841-1852. (b) Byrne, R.; Diamond, D. Nat. Mat. 2006, 5, 421-424. (c) Gooding, J. J. Anal. Chim. Acta 2006, 559, 137-151. (2) (a) Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol. Cell. Biol. 1999, 19, 1720-1730. (b) Ideker, T.; Thorsson, V.; Ranish, J. A.; Christmas, R.; Buhler, J.; Eng, J. K.; Bumgarner, R.; Goodlett, D. R.; Aebersold, R.; Hood, L. Science 2001, 292, 929-934. (3) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Dirk, S. M.; Brozik, S. M. Langmuir 2007, 23, 8285-8287. (4) Medintz, I. Nat. Mater. 2006, 5, 842.
the modification of electrode platforms shows great promise in addressing this need for a versatile and tunable surface chemistry for the selective immobilization of biological and chemical molecules. Introduced by Delamar et al.,5 aryl diazonium salts may be assembled onto conducting and semiconducting substrates forming a robust covalent bond with the surface.6 The electrochemical protocol used for bias-assisted assembly also provides control over functionalization density and electron-transfer kinetics through the film.7 Diazonium chemistry has been used to immobilize a wide variety of chemically sensitive groups8 and diverse biomolecules including biotin,9 DNA,10 proteins,11 and peptides.12 The direct electrically addressable immobilization of diazonium-modified proteins onto electrodes was used to obtain direct electron transfer to horseradish peroxidase13 and to construct a reagentless14 and a multianalyte electrochemical immunosensor.15 The maleimide functional group has been widely used for crosslinking and surface immobilization of biomolecules due to (5) Delamar, M.; Hitmi, R.; Pinson, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1992, 114, 5883-5884. (6) Pinson, J.; Podvorica, F. Chem. Soc. ReV. 2005, 34, 429-439. (7) Harper, J. C.; Polsky, R.; Dirk, S. M.; Wheeler, D. R.; Brozik, S. M. Electroanalysis 2007, 19, 1268-1274. (8) (a) Tammeveski, K.; Kontturi, K.; Nichols, R. J.; Potter, R. J.; Schiffrin, D. J. J. Electroanal. Chem. 2001, 515, 101-112. (b) Vaik, K.; Sarapuu, A.; Tammeveski, K.; Mirkhalaf, F.; Schiffrin, D. J. J. Electroanal. Chem. 2004, 564, 159-166. (c) Liu, S.; Shi, Z.; Dong, S. Electroanalysis 1998, 10, 891-896. (d) Downard, A. J.; Roddick, A. D.; Bond, A. M. Anal. Chim. Acta 1995, 317, 303-310. (9) Dequaire, M.; Degrand, C.; Limoges, B. J. Am. Chem. Soc. 1999, 121, 6946-6947. (10) (a) Shabani, A.; Mak, A. W. H.; Gerges, I.; Cuccia, L. A.; Lawrence, M. F. Talanta 2006, 70, 615-623. (b) Lee, C.-S.; Baker, S. E.; Marcus, M. S.; Yang, W.; Eriksson, M. A.; Hamers, R. J. Nano Lett. 2004, 4, 1713-1716. (11) (a) Liu, G.; Gooding, J. J. Langmuir 2006, 22, 7421-7430. (b) Wang, J.; Carlisle, J. A. Diam. Relat. Mater. 2006, 15, 279-284. (c) Corgier, B. P.; Marquette, C. A.; Blum, L. J. J. Am. Chem. Soc. 2005, 127, 18328-18332. (d) Bourdillon, C.; Delamar, M.; Demaille, C.; Hitmi, R.; Moiroux, J.; Pinson, J.J. Electronal. Chem. 1992, 336, 113-123. (12) Liu, G.; Bo¨cking, T.; Gooding, J. J. J. Electroanal. Chem. 2007, 600, 335-344. (13) Polsky, R.; Harper, J. C.; Dirk, S. M.; Arango, D. C.; Wheeler, D. R.; Brozik, S. M. Langmuir 2007, 23, 364-366. (14) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Rawlings, J. A.; Brozik, S. M. Chem. Commun. 2007, 2741-2743. (15) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Arango, D. C.; Brozik, S. M. Biosens. Bioelectron. 2008, 23, 757-764.
10.1021/la702613e CCC: $40.75 © 2008 American Chemical Society Published on Web 01/17/2008
Maleimide-ActiVated Aryl Diazonium Salts
its selectivity toward sulfhydryl groups under aqueous conditions.16 Conventional methods for producing maleimide-functionalized surfaces typically require an initial treatment to produce an amine-terminated surface followed by reaction with a heterobifunctional crosslinker that contains an amine reactive group and a maleimide group separated by a short alkane chain.10b,17 It would therefore be efficacious to produce aryl diazonium salts that would allow a one step preparation of a maleimide-active surface with control over film thickness and electron-transfer properties. A maleimide-functionalized aryl diazonium would allow for simple formation of maleimide surfaces, provide the ability to selectively modify closely spaced microelectrodes, and provide a degree of control over the deposited film thickness and electron-transfer properties of the film for improved electrochemical sensing applications.7 In this work we demonstrate for the first time the utility of phenylmaleimide diazonium as a tool for functionalizing electrode surfaces. Grazing angle FTIR, ellipsometry, and voltammetry are used to characterize electrodeposited phenylmaleimide thin films. Functionalization of electrodes with redox-active molecules, diverse biomolecules, and phenylmaleimide-thiol tagged biomolecule conjugates is demonstrated. This versatile and simple method for formation of maleimide-functionalized surfaces using electrodeposited maleimide-activated aryl diazonium salts provides a powerful new tool for the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays. Experimental Section Materials. Oligonucleotides were obtained from Synthegen (Houston, TX). The thiolated probe sequence, 5′-NH2-(CH2)6GGTTGGTGTGGTTGGCACC-(CH2)6-SH-3′, was conjugated to ferrocene succinimide ester at the 5′ amine of the oligo. Aqueous solutions were prepared with 18 MΩ water using a Barnstead Nanopure water purifier (Boston, MA). N-(4-Aminophenyl)maleimide was obtained from TCI (Boston, MA). Cytochrome c (horse heart, 90%), 2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid (HEPES), ferrocene, dithiothreitol (DTT), sodium perchlorate (NaClO4), sodium chloride, magnesium chloride and anhydrous acetonitrile (ACN), were purchased from Acros Organics (Beel, Belgium). Sodium phosphate monobasic, sodium phosphate dibasic, calcium chloride, and 30% H2O2 were purchased from Sigma (St. Louis, MO). Nitrosonium tetrafluoroborate, diethyl ether, and tetrabutylammonium tetrafluoroborate (Bu4NBF4) were obtained from Aldrich. Sulfuric acid, ethyl alcohol (95% denatured), and potassium chloride were purchased from Fischer Scientific (Pittsburgh, PA). Tris-HCl was purchased from Fluka (Buchs, Switzerland). 6-Ferrocenyl-1-hexanethiol was obtained from Dojindo Molecular Technologies (Gaithersburg, MD). All reagents were used as received unless otherwise noted. Synthesis of N-(4-Diazophenyl)maleimide tetrafluoroborate. In a drybox, a Schlenk flask with septum and stir bar was charged with 0.0975 g of nitrosonium tetrafluoroborate (0.854 mmol, 1.5 equiv). To the flask was added 5 mL of dry acetonitrile. After dissolution, the flask was cooled to -40 °C. A separate Schlenk flask, fitted with a stir bar and septum, was charged with 0.106 g of N-(4-aminophenyl)maleimide (0.563 mmol). After the flask was evacuated and backfilled with dry argon, 5 mL of dry acetonitrile was added. Upon dissolution, the solution of the N-(4-aminophenyl)maleimide was slowly cannulated into the cold nitrosonium tetrafluoroborate and the resulting solution was stirred for 1 h. The reaction mixture was allowed to warm to 0 °C over the course of (16) (a) Shen. G.; Anand, M. F. G.; Levicky, R. Nucleic Acids Res. 2004, 32, 5973-5980. (b) Lee, C.-Y.; Nguyen, P.-C. T.; Grainger, D. W.; Gamble, L. J.; Castner, D. G. Anal. Chem. 2007, 79, 4390-4400. (17) (a) Patolsky, F.; Weizmann, Y.; Katz, E.; Willner, I. Angew. Chem., Int. Ed., 2003, 42, 2372-2376. (b) Jin, L.; Horgan, A.; Levicky, R. Langmuir 2003, 19, 6968-6975.
Langmuir, Vol. 24, No. 5, 2008 2207 2 h. The solution was then cannulated into 300 mL of 0 °C rapidly stirred diethyl ether. After a few minutes, a white precipitate was observed. The solid was collected by filtration and dried under vacuum to afford 0.109 g of product, (0.380 mmol, 67%). 1HNMR (CD3CN); δ 8.55 pseudo dblt, 2H; 8.12 pseudo dblt, 2H; 7.07 singlet, 2H. Electrochemical Instrumentation. All electrochemical measurements were performed on a PGZ100 Voltalab potentiostat (Radiometer Analytical, Lyon, France) and were measured versus an Ag/ AgCl reference (3 M NaCl, aqueous solutions) or a Ag/AgNO3 reference (10 mM, nonaqueous solutions, -102 mV vs ferrocene couple) and a Pt counter electrode from Bioanalytical Systems (West Lafayette, IN). All potentials reported herein are with respect to the relevant reference electrode. Glassy carbon (GCE, 3 mm diameter) and 1.4 mm gold working electrodes were obtained from Bioanalytical Systems. Gold disk electrodes (5 mm diameter) were prepared via thermal evaporation of a 200 Å Ti adhesion layer followed by 2000 Å of Au onto a Pyrex wafer. Au electrodes were cleaned immediately before use with freshly prepared piranha (5:3 concd sulfuric acid/ 30% H2O2) for 5 min, washed with nanopure water, and dried under a stream of nitrogen. Glassy carbon electrodes were polished successively by 1, 0.3, and 0.05 µm alumina slurry on a cloth polishing pad (Buehler, Lake Bluff, IL) with sonication in ethanol between steps and a final sonication in nanopure water followed by drying under a stream of nitrogen. Grazing Angle FTIR and Ellipsometry Measurements. Lithographically defined gold disk electrodes, 5 mm diameter, were used for grazing angle FTIR and ellipsometry measurements. FTIR measurements were obtained with a Nicolet 6700 Fourier-transformed infrared spectrometer with a liquid-nitrogen-cooled mercurycadmium-telluride (MCT) detector. An external specular reflectance attachment (SMART SAGA) was used to obtain an incidence angle of 80° with unpolarized light. Scans (1024) were collected for each spectrum with a spectral resolution of 4 cm-1 using Happ-Genzel apodization. Background reference spectra were obtained immediately before collecting sample spectrum. All spectra are reported as log(R/R0) where R is the reflectivity of the modified gold and R0 is the reflectivity of the unmodified gold. Ellipsometry measurements were performed using a Gaertner Scientific Corporation L166 S Stokes Ellipsometer with a 2 mW HeNe (λ ) 632.8 nm) laser, an incidence angle of 70°, and film refractive index, nf ) 1.5. Initial substrate measurements were performed on each electrode following piranha cleaning. Electrode Functionalization. Phenylmaleimide thin films were assembled onto clean gold electrodes using chronoamperometry, linear sweep, or cyclic sweep methods in a solution of 1 mM N-phenylmaleimide diazonium and 0.1 M Bu4NBF4 in ACN. After electrodeposition, the electrodes were briefly rinsed with ACN, followed by a rinse with ethanol and a 15 s sonication in ethanol to remove any adsorbed phenylmaleimide diazonium. After sonication the electrodes were again rinsed in ethanol and dried under a stream of nitrogen. Functionalization of GCEs with ferrocene occurred via a 2 CV electrodeposition (0 to -1 to 0 V) of phenylmaleimide diazonium with rinsing and sonication steps as described above. The electrode was then treated with 500 µM 6-ferrocenyl-1-hexanethiol in ACN for 2 h. This was followed by rinsing with ACN, ethanol, an additional 15 s sonication in ethanol, and finally dried under a stream of nitrogen. Functionalization of GCEs with the protein, cytochrome c, occurred via a 30 s chronoamperometric electrodeposition (potential step to -1 V) of phenylmaleimide diazonium followed again by rinsing and sonication steps as described above. The electrode was then treated for 2 h with 500 µM cytochrome c in 100 mM HEPES buffer, pH 7.5. The electrode was then thoroughly rinsed with 0.1 M phosphate buffer, pH 7.4. Functionalization of GCEs with DNA probes occurred via a 10 CV electrodeposition (0 to -1 to 0 V) of 1 µM DNA-phenylmaleimide diazonium conjugate in 10 mM HCl. DNA-phenylmaleimide diazonium conjugate was produced by first reducing mercapto-modified DNA with DTT followed by filtering through a G-25 sephadex column. Reduced DNA was then reacted
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Figure 1. Cyclic voltammograms of N-phenylmaleimide diazonium (1 mM) at a gold electrode (red) and a glassy carbon electrode (blue) in 0.1 M Bu4NBF4 in ACN, V ) 100 mV s-1. Initial and second CV scans are shown for each electrode. Scheme 1. Synthesis of N-(4-Diazophenyl)maleimide Tetrafluoroborate
with 1 mM N-phenylmaleimide diazonium in 50 mM HEPES, pH 6.8, for 2 h. The solution was then filtered through a G-25 sephadex column.
Results and Discussion The synthesis of phenylmaleimide diazonium, as shown in Scheme 1, occurred via diazotization of the phenylmaleimide aniline derivative to form the aryl diazonium tetrafluoroborate (see Experimental Section). Cyclic voltammograms of gold and glassy carbon electrodes in acetonitrile solutions containing 1 mM phenylmaleimide diazonium and 0.1 M Bu4NBF4 are presented in Figure 1. A sharp irreversible peak was observed for the gold electrode, Ep,c ) -146 mV vs Ag/AgNO3, which is attributed to the reduction of the diazonium salt. A similar reduction peak was observed for the glassy carbon electrode at a more negative potential, Ep,c ) -262 mV. Also observed on both electrodes were a small pre and post wave, characteristic of strong adsorption of both the oxidized and reduced form of the electroactive species.18 The observed pre and post waves may therefore be attributed to adsorption of the phenylmaleimide diazonium to the electrode surface. The larger post wave for the GCE electrode suggests that phenylmaleimide diazonium adsorbs more strongly to GC than to Au. Following cyclic voltammetry deposition cycles show no further reduction peaks and a reduction in overall current response. It is worthwhile to note that this observed electrode passivation after the first potential sweep, common in bias-assisted diazonium assemblies, does not necessarily indicate an absence of diazonium grafting upon subsequent sweeps. Be´langer and co-workers used an electrochemical quartz crystal microbalance to show that diazonium film thicknesses increase with repeated potential scans despite the lack of an observable reduction wave.19 They attribute (18) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley and Sons, Inc.: New York, 2001.
Harper et al.
Figure 2. Grazing angle FTIR spectrum of N-phenylmaleimide20 and of a gold electrode prepared from a 10 CV electrodeposition of N-phenylmaleimide diazonium tetrafluoroborate.
this continued growth to diazonium cation reduction via electron transfer through the deposited film, although grafting at a decreased rate was observed.19 Grazing angle FTIR was used to verify assembly of phenylmaleimide molecules on lithographically defined gold substrates and retention of the maleimide functional group. The FTIR spectra from a 5 mm diameter gold disk electrode prepared from a 10 cyclic voltammetry cycle deposition of phenylmaleimide diazonium is compared to the spectra of N-phenylmaleimide20 in Figure 2. Of note is the sharp peak at 1726 cm-1 common to both the N-phenylmaleimide and the phenylmaleimide diazonium assembled electrode spectrum. This absorption stretch is characteristic of the two carbonyl groups of the maleimide. Also in common to both spectra are the peaks at 1636, 1519, and 1385 cm-1, the last of which is characteristic of the aryl C-N stretch vibration. This provides strong evidence that the phenylmaleimide diazonium assembled onto the gold substrate with the vast majority of the maleimide functional groups intact. The electrochemical protocol used to assemble diazonium salts has been reported to affect assembled film order,21 to affect density,7 and lead to the formation of multilayers22 with all properties being shown to be significantly influenced by the substituent functional group.23 Therefore, the assembly of phenylmaleimide onto gold was investigated using three different electrochemical protocols: (1) chronoamperometry (CA, step to -1 V), (2) linear sweep voltammetry (LS, 0 to -1 V at 100 mV s-1), and (3) cyclic voltammetry (CV, 0 to -1 to 0 V at 100 mV s-1). Following assembly, the phenylmaleimide film thickness was measured via ellipsometry. Table 1 shows the average thickness for electrodes prepared from these different methods. These results show that film thickness using potential sweep methods grow at a faster rate and results in thicker films than for fixed-potential methods, which has been previously reported for diazonium salts with other substituent functional groups.21 The ellipsometric data also indicated that film growth reaches (19) Laforgue, A.; Addou, T.; Be´langer, D. Langmuir 2005, 21, 6855-6865. (20) The Aldrich Library of FT-IR Spectra, 2nd ed.; Sigma-Aldrich Co.: St. Louis, 1997. (21) Uetsuka, H.; Shin, D.; Tokuda, N.; Saeki, K.; Nebel, C. E. Langmuir 2007, 23, 3466-3472. (22) (a) Brooksby, P. A.; Downard, A. J. Langmuir 2004, 20, 5038-5045. (b) Anariba, F.; DuVall, S. H.; McCreery, R. L. Anal. Chem. 2003, 75, 3837-3844. (23) (a) Adenier, A.; Barre´, N.; Cabet-Deliry, E.; Chausse´, A.; Griveau, S.; Mercier, F.; Pinson, J.; Vautrin-Ul, C. Surf. Sci. 2006, 600, 4801-4812. (b) Vase, K. H.; Holm, A. H.; Norrman, K.; Pedersen, S. U.; Daasbjerg, K. Langmuir 2007, 23, 3786-3793.
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Table 1. Electrodeposition Method Affect on Phenylmaleimide Film Thickness
electrodeposition method 15 s CA 1 min CA 5 min CA 10 min CA 1 LS 1 CV 5 CVs 10 CVs
film thickness (Å)
standard deviation within electrodesa
standard deviation between electrodesb
equivalent monolayerc
6.60 20.6 46.4 48.6 32.8 34.7 41.5 42.0
1.9 4.0 0.9 1.6 0.9 0.3 0.3 0.8
2.8 4.4 2.3 1.9 1.2 1.8 0.4 0.8
0.7 2.3 5.1 5.3 3.6 3.8 4.5 4.6
Scheme 3. Functionalization of Electrode Surfaces Employing N-Phenylmaleimide Diazoniuma
a Standard deviation of measurements from 10 or more different locations on an electrode surface, three electrodes sampled. b Standard deviation of measurements obtained from three different electrodes. c Calculated thickness of a phenylmaleimide monolayer with 70° tilt is ∼9.1 Å.
Scheme 2. Electroaddressable Deposition of N-Phenylmaleimide Diazoniuma
a (1) Electroreduction of diazonium cation at a conductive substrate with subsequent loss of nitrogen, (2) covalent bonding of phenyl radical to conducting or semiconducting substrate, (3) formation of submonolayer to multilayer films dependent upon electrodepostion protocol.
saturation near five equivalent layers of phenylmaleimide (∼4.5 nm) which is lower than film thicknesses reported for phenyldiazonium molecules with other functional groups (10-100 nm).24 This can be explained using a possible mechanism for the assembly of the phenyl-maleimide diazonium as proposed in Scheme 2. Step 1 shows the electrochemical reduction of the diazonium salt forming an aryl radical, with subsequent loss of dinitrogen. This is followed by covalent attachment to a conducting or semiconducting substrate, step 2.25 Step 3 shows two possible locations on the initially deposited phenylmaleimide layer for subsequent attachment of other aryl radicals. It is commonly accepted that multilayer growth occurs via attachment to previously deposited phenyl rings, as shown in step 3. However, as the maleimide group is an excellent electron acceptor and is not as sterically hindered as the deposited phenyl ring, it serves (24) (a) Adenier, A.; Combellas, C.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. Chem. Mater. 2006, 18, 2021-2029. (b) Bernard, M. C.; Chausse’, A.; CabetDeliry, E.; Chehimi, M. M.; Pinson, J.; Podvorica, F.; Vautrin-Ul, C. Chem. Mater. 2003, 15, 3450-3462. (c) Kariuki, J. K.; McDermott, M. T. Langmuir 2001, 17, 5947-5951. (25) The nature of the bond to Au surfaces is not yet completely understood. Some evidence has been presented showing the possible existence of Au-C and Au-NdN-C bonds. However, our results and those reported by other groups show that the assembled films strongly adhere to gold surfaces resisting ultrasonication, application of extreme potential under basic conditions, and potential cycling under acidic conditions (see refs 12 and 19).
a (A) 6-ferrocenyl-1-hexanethiol functionalization and (B) cytochrome c protein functionalization of phenylmaleimide thin film modified GCEs, (C) direct functionaliztion of a GCE with phenylmaleimide diazonium conjugated to ferrocene labeled ssDNA.
as an additional location for subsequent grafting, as also shown in step 3. However, grafting at the maleimide produces a less conductive path than grafting at the phenyl group, forming a sp3 vs a sp2 bond. This comparatively less conductive path would inhibit multilayer formation and may account for the low saturation thickness. The maleimide group is a powerful tool commonly used for crosslinking of biological molecules and for functionalizing surfaces. The remainder of this paper will demonstrate the utility of N-phenylmaleimide diazonium for functionalization of electrode surfaces. Scheme 3 shows two general methods used to functionalize electrodes with biological and redox-active molecules. In the first method a thin film of phenylmaleimide is deposited onto the electrode followed by treatment in a solution containing the thiol tagged molecule of interest, as shown in Scheme 3A and B. In the second method, N-phenylmaleimide diazonium is reacted with the thiol-tagged molecule, forming a diazonium-activated conjugate. This is followed by direct electrodeposition of the molecule onto the electrode surface, Scheme 3C. A thin film of phenylmaleimide deposited onto a GCE was used to functionalize the electrode surface with redox-active ferrocene molecules. Figure 3 shows a cyclic voltammogram of the phenylmaleimide thin film modified GCE (red) after a 2 h treatment in 500 µM 6-ferrocenyl-1-hexanethiol, followed by rinsing and an additional 15 s sonication in ACN. A reversible redox wave with formal potential, E0′ ) 45 ( 2 mV is observed. This formal potential is negatively shifted by 57 mV from
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Figure 3. Cyclic voltammograms of 6-ferrocenyl-1-hexanethiol treated phenylmaleimide (red) and phenyl (blue) modified GCEs in 0.1 M NaClO4 in ACN, V ) 100 mV s-1. Each electrode was prepared from a 2 CV cycle electrodeposition of the respective diazonium salt followed by a 2 h treatment in 500 µM 6-ferrocenyl-1-hexanethiol in ACN.
ferrocene in solution under similar conditions and is near that reported for other ferrocene modified electrodes.26 The peak separation, ∆EP, at 100 mV s-1 was 58 mV. This ∆EP is greater than the 0 mV separation expected for fully reversible surface confined species, but is consistent with other reports in which electrons must pass through thin films.27 The surface concentration, Γ, of the immobilized ferrocene was calculated from the area of the reduction wave and is 1.9 × 10-10 mol/cm2. This corresponds to 42% of a hexagonal close packed monolayer of the ferrocene moiety (assuming 6.6 Å spherical diameter)28 and is similar to that obtained from self-assembling monolayer based immobilization methods on gold electrodes.29 A control electrode, shown in Figure 3 (blue), was prepared from a similar electrodepostion of phenyl diazonium followed by a 2 h treatment in 500 µM 6-ferrocenyl-1-hexanethiol, rinsing, and 15 s sonication in ACN. A positively shifted redox wave is observed. However, the surface concentration was 12% of that obtained from the phenylmaleimide modified electrode and is attributed to nonspecific binding of the amphiphilic ferrocene to the GC.30 These results demonstrate that a phenylmaleimide surface is robust to brief sonication and can serve as an effective method for functionalizing electrodes with redox-active molecules and that facile electron transfer can be obtained through the phenylmaleimide thin film. Conjugation to cytochrome c through free surface amines does not easily lead to direct electron transfer due to the relative distance of the free amines from the redox center of the enzyme and to the many possible orientations of the immobilized molecule. However, coupling to one of the two thiol groups near the heme cofactor of cytochrome c is more effective to obtain direct electron transfer and results in a more uniform orientation of immobilized enzyme. A thin film of phenylmaleimide was also used to (26) The Ag/AgNO3 (0.01 M in acetonitrile) reference electrode is ∼300 mV vs SCE per Meites, L. Handbook of Analytical Chemistry; McGraw Hill: New York, 1963. (a) Kawaguchi, T.; Tada, K.; Shimazu, K. J. Electroanal. Chem. 2003, 543, 41-49. (b) Rowe, G. K.; Creager, S. E. Langmuir 1991, 7, 23072312. (27) (a) Wang, C.-L.; Mulchandani, A. Anal. Chem. 1995, 67, 1109-1114. (b) Uosaki, K.; Sato, Y.; Kita, H. Langmuir 1991, 7, 1510-1514. (28) (a) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. Soc. 1990, 122, 4301-4306. (b) Seiler, P.; Dunitz, J. D. Acta Crystallogr., Sect. B 1979, B35, 1068-1074. (29) (a) Lee, L. Y. S.; Sutherland, T. C.; Rucareanu, S.; Lennox, R. B. Langmuir 2006, 22, 4438-4444. (b) Rowe, G. K.; Carter, M. T.; Richardson, J. N.; Murray, R. W. Langmuir 1995, 11, 1797-1806. (30) Peng, W.; Zhou, D.-L.; Rusling, J. F. J. Phys. Chem. 1995, 99, 69866993.
Harper et al.
Figure 4. Cyclic voltammogram of a cytochrome c treated phenylmaleimide modified GCE electrode in 0.1 M phosphate buffer, pH 7.4, V ) 150 mV s-1, under argon. Electrode prepared from a 30 s chronoamperometric deposition of N-phenylmaleimide diazonium followed by a 2 h treatment with 500 µM cytochrome c in 0.1 M phosphate buffer, pH 7.4. Inset: Plot of Ip,a and Ip,c vs scan rate.
functionalize a GCE surface with the redox active protein, cytochrome c. Figure 4 shows the cyclic voltammogram of a phenylmaleimide modified GCE following a 2 h treatment in 500 µM cytochrome c and thorough rinsing. Direct electron transfer between the redox-active site of the proteins and the electrode surface was observed. An anodic peak at 22 mV (vs Ag/AgCl) and the corresponding cathodic peak at -95 mV (V ) 150 mV s-1), are attributed to the FeIII/FeII redox couple of the electroactive heme center of cytochrome c. A bare GCE control prepared under the same cytochrome c treatment conditions showed no redox currents. The E0′ of the couple was -24 ( 3 mV, which is near the 18 mV reported for cytochrome c immobilized to gold via an alkanethiol31 and -11 mV for cytochrome c immobilized onto pyrolytic graphite in silver nanoparticle containing films32 (potentials adjusted to a Ag/ AgCl, 3 M NaCl reference). This formal potential is ∼70 mV more negative than that reported for native cytochrome c in solution.33 This may be due to partial structural shifting/unfolding as portions of the protein may be in contact with the maleimide functionalized thin film or the GCE surface. The effect of increasing scan rates is shown in the inset of Figure 4 which plots the cathodic and anodic peak currents versus scan rate. The linear relationship indicates that the electroactive species is confined to the electrode surface. A heterogeneous reaction rate constant, ks, of 3.1 ( 0.2 s-1, and the dependence of potential peak on scan rate categorize this reaction as quasireversible. These results are similar to previous studies of direct electron transfer to cytochrome c immobilized via maleimide to gold surfaces which report ks values between 1 and 22 s-1.34 Integration of the reduction peak indicates an electroactive surface coverage of 8.82 × 10-12 mol/cm2. This is equivalent to 93% of a hexagonally close packed monolayer with the average diameter of 45 Å for cytochrome c.35 The peak width at half peak height, ∆Ep,1/2, is 177 mV, which is larger than the ideal of 90.6 mV for a one-electron-transfer reaction. However, similar ∆Ep,1/2 values for immobilized proteins are commonly reported and may be due to a distribution of orientations of the immobilized protein (31) Jin, W.; Wollenberger, U., Ka¨rgel, E.; Schunck, W. and Scheller, W. J. Electroanal. Chem. 1997, 433, 135-139. (32) Liu, T.; Zhong, J.; Gan, X.; Fan, C.; Li, G.; Matsuda, N. Chemphyschem 2003, 4, 1364-1366. (33) Nelson, D.; Cox, M. Lehninger Principles of Biochemistry; Worth Publishers: New York, 2000. (34) Pardo-Yissar, V.; Katz, E.; Wilner, I.; Kotlyar, A. B.; Sanders, C.; Lill, H. Faraday Discuss. 2000, 116, 119-134. (35) Goodsell, D. S.; and Olson, A. J. Trends Biochem. Sci. 1993, 18, 65-68.
Maleimide-ActiVated Aryl Diazonium Salts
Figure 5. Cyclic voltammogram of a GCE after direct elecrodeposition of a ferrocene tagged ssDNA-maleimide diazonium conjugate, in 20 mM TRIS-HCl buffer, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, V ) 100 mV/s.
to the electrode surface providing a distribution of energy barriers for electron transfer.13,36 This distribution may arise via immobilization from different binding sites on cytochrome c, which contains two cysteine residues each of which may bind to the maleimide surface, and from variance in the surface topology of the phenylmaleimide thin film leading to variance in the path length from the electrode to the cytochrome c. The utility of phenyl maleimide toward the direct electrically addressable immobilization of DNA was also demonstrated. Ferrocene-modified thiol-terminated single stranded DNA was modified with the diazonium, using maleimide-thiol conjugation, and electrically deposited onto a GCE (Scheme 3C). Cyclic voltammetric characterization of the DNA functionalized surface in biological buffer is presented in Figure 5. A quasi-reversible redox wave with E0′ ) 87 ( 2 mV vs Ag/AgCl and ∆EP of 36 mV at 100 mV s-1 was observed and is attributed to the presence of ferrocene. A control electrode prepared with a solution of ferrocene-modified DNA (without the phenylmaleimide diazonium conjugation) under the same deposition conditions did not yield redox currents indicating the response is due to immobilized DNA and not to nonspecific adsorption. The surface density of the immobilized DNA was calculated from the ferrocene oxidation wave measured in ACN and was 3.38 × 1013 molecules/cm2. This surface density is similar to the 3.75 × 1013 molecules/cm2 estimated from X-ray photoelectron spectroscopy for an electrode functionalized with 4-aminobenzylamine-DNA that was diazotized immediately prior to electrodepostion.37 This surface area is also within range of that reported for immobilization of thiol terminated DNA on maleimide functionalized supports.38 This (36) Zhang, Z.; Nassar, A.-E. F.; Lu, Z.; Schenkman, J. B.; Rusling, J. F. J. Chem. Soc., Faraday Trans. 1997, 93, 1769-1774. (37) Corgier, B. P.; Laurent, A.; Perriat, P.; Blum, L. J.; Marquette, C. A. Angew. Chem., Int. Ed. 2007, 46, 4108-4110.
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surface, however, did not prove effective for DNA hybridization (complementary strand) or protein detection (aptamer sequence against the protein, Thrombin). Reaction of diazonium species with nucleic acid bases in solution has been reported in the literature.39 It is therefore probable that diazonium molecules would interact with nucleic acid bases inactivating the DNA strand toward hybridization (see Scheme 3A and B). A related method required the simultaneous diazotination and deposition of the DNA which prevented the diazonium group from reacting with the DNA and allowed for hybridization.37 However, this method might not be suitable for selectively immobilizing multiple DNA probes to the same electrode array due to the harsh reaction conditions. Functionalization of surfaces with ssDNA probes using phenylmaleimide diazonium active toward hybridization is possible using the method employed for ferrocene and cytochrome c immobilization. Specifically, initial deposition a phenylmaleimide film followed by treatment with thiolated DNA yielded surfaces that demonstrated effective hybridization with the biotin-labeled complimentary strand as detected by subsequent labeling with avidin-modified HRP. We are currently investigating less reactive electro addressable surface chemistries (such as sulfonium and iodonium species) for bias-assisted DNA probe immobilization.
Summary This is the first study characterizing phenylmaleimide diazonium electrodeposition onto gold and carbon substrates and its use as a tool for functionalizing electrode surfaces. Assembly and retention of the maleimide functional group on gold substrates was verified using grazing angle FTIR. The effect of electrodeposition protocol was measured via ellipsometry and demonstrated control of film formation from submonolayer to multilayer. Finally, the utility of phenylmaleimide diazonium as an effective means for functionalizing surfaces with redox-active groups, diverse biological molecules, and diazonium conjugates was demonstrated. This versatile and simple tool for producing maleimide surfaces for electrode functionalization may facilitate the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays. Acknowledgment. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract No. DE-AC04-94AL8500. LA702613E (38) (a) Vaidya, A. A.; Norton, M. L. Langmuir 2004, 20, 11100-11107. (b) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205-1209. (c) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044-8051. (39) Dolan, P. L.; Wu, Y.; Ista, L. K.; Metzenberg, R. L.; Nelson, M. A.; Lopez, G. P. Nuclei. Acids Res. 2001, 29, e107.
