A Multifunctional Thin Film Au Electrode Surface Formed by

Jan 27, 2009 - Jason C. Harper, Ronen Polsky, David R. Wheeler, DeAnna M. Lopez, Dulce ... Thicker nitrophenyl films led to diminished PBA-PE diazoniu...
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Langmuir 2009, 25, 3282-3288

A Multifunctional Thin Film Au Electrode Surface Formed by Consecutive Electrochemical Reduction of Aryl Diazonium Salts Jason C. Harper, Ronen Polsky, David R. Wheeler, DeAnna M. Lopez, Dulce C. Arango, and Susan M. Brozik* Biosensors & Nanomaterials, Sandia National Laboratories, PO Box 5800, MS-0892, Albuquerque, New Mexico 87185 ReceiVed September 30, 2008. ReVised Manuscript ReceiVed December 10, 2008 A multifunctional thin film surface capable of immobilizing two diverse molecules on a single gold electrode was prepared by consecutive electrodeposition of nitrophenyl and phenylboronic acid pinacol ester (PBA-PE) diazonium salts. Activation of the stacked film toward binding platinum nanoparticles (PtNPs) and yeast cells occurred via chemical deprotection of the pinacol ester followed by electroreduction of nitro to amino groups. FTIR spectral analysis was used to study and verify film composition at each stage of preparation. The affect of electrodeposition protocol over the thickness of the nitrophenyl and PBA-PE layers was explored and had a profound impact on the film properties. Thicker nitrophenyl films led to diminished PBA-PE diazonium reduction currents during assembly and decreased phenylboronic acid (PBA) layer thickness while allowing for higher PtNP loading and catalytic currents from PtNP-mediated peroxide reduction. Multilayer PBA films could be formed over the nitrophenyl film; however, only submonlayer PBA films permitted access to the underlying layer. The sequence of functional group activation toward binding was also shown to be significant, as perchlorate used to remove pinacol ester also converted aminophenyl groups accessible to the solution to nitrophenyl groups, preventing electrostatic PtNP binding. Finally, SEM images show PtNPs immobilized in close proximity (nanometers) to captured yeast cells on the PBA-aminophenyl-Au film. Such multibinding functionality films that maintain conductivity for subsequent electrochemical measurements hold promise for the development of electrochemical and/or optical platforms for fundamental cell studies, genomic and proteomic analysis, and biosensing.

Introduction Functionalization of conducting and semiconducting surfaces is a vital component in the fields of bioelectronics, molecular electronics, clinical diagnostics, and chemical and biological sensing.1 Of particular interest is the ability to conjugate a surface with two or more different biological, redox active, and/or photo/ chemical sensitive molecules. Such multifunctional surfaces would facilitate collection of complicated data sets that would be relevant to cell signaling studies, genomic and proteomic analysis, and the proof-positive identification of biological organisms.2 Several methods have been devised and employed to allow surface conjugation including photolithography,3 selfassembling monolayers (SAMs),4 silanes,5 stamping,6 mechanical and/or electrochemical7 removal of material, and electropoly* Corresponding author. Telephone: (505) 844-5105. Fax: (505) 8458161. E-mail: [email protected]. (1) (a) Willner, I.; Baron, R.; Willner, B. Biosens. Bioelectron. 2007, 22, 1841–1852. (b) Oh, S. J.; Hong, B. J.; Choi, K. Y.; Park, J. W. OMICS 2006, 10, 327–343. (c) Byrne, R.; Diamond, D. Nat. Mater. 2006, 5, 421–424. (d) Gooding, J. J. Anal. Chim. Acta 2006, 559, 137–151. (2) (a) Medintz, I. Nat. Mater. 2006, 5, 842. (b) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Brozik, S. M. Electroanalysis 2008, 20, 671–679. (c) Rowe-Taitt, C. A.; Hazzard, J. W.; Hoffman, K. E.; Cras, J. J.; Golden, J. P.; Ligler, F. S. Biosens. Bioelectron. 2000, 15, 579–589. (d) Rowe, C. A.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.; MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem. 1999, 71, 3846–3852. (e) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Dirk, S. M.; Brozik, S. M. Langmuir 2007, 23, 8285–8287. (3) (a) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. BioMaterials 2006, 27, 3044–3063. (b) Delamarche, E.; Juncker, D.; Schmid, H. AdV. Mater. 2005, 17, 2911–2933. (c) Yap, F. L.; Zhang, Y. Biosens. Bioelectron. 2007, 22, 775–788. (4) (a) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. (b) Gooding, J. J.; Mearns, F.; Yang, W.; Liu, J. Electroanalysis 2003, 15, 81–96. (c) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877–1881. (5) (a) Senaratne, W.; Andruzzi, L.; Ober, C. K. Biomacromolecules 2005, 6, 2427–2448. (b) Chaki, N. K.; Vijayamohanan, C. K. Biosens. Bioelectron. 2002, 17, 1–12.

merization.8 Of these techniques SAMs based on alkanethiol-gold surfaces have been the most widely used. Several studies utilizing SAMs to form mixed surfaces demonstrating multifunctionality have been published.9 However, the low enthalpy of the Au-S bond, mobility of the SAMs on gold, tendency of the Au-S bond to oxidize in air and media, and a low potential stability window have limited the usefulness of this chemistry.10 Electrode surface modification by the electrochemical reduction of aryl diazonium salts is a promising alternative to conventional electrode modification schemes.11 Electroreduction of the diazonium produces an aryl radical that can then graft to a conducting or semiconducting surface forming a stable covalent bond. This approach has several advantages over alkanethiol-gold chemistry including ease of surface modification, a wider potential window (6) (a) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363–2376. (b) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550–575. (7) (a) Jang, J.-W.; Sanedrin, R. G.; Maspoch, D.; Hwang, S.; Fujigaya, T.; Jeon, Y.-M.; Vega, R. A.; Chen, X.; Mirkin, C. A. Nano Lett. 2008, 8, 1451–1455. (b) Liu, G.; Xu, S.; Qian, Y. Acc. Chem. Res. 2000, 33, 457–466. (c) Schoer, J. K.; Crooks, R. M. Langmuir 1997, 13, 2323–2332. (d) Schoer, J. K.; Zamborini, F. P.; Crooks, R. M. J. Phys. Chem. 1996, 100, 11086–11091. (8) (a) Stern, E.; Jay, S.; Bertram, J.; Boese, B.; Kretzschmar, I.; TurnerEvans, D.; Dietz, C.; LaVan, D. A.; Malinski, T.; Fahmy, T.; Reed, M. A. Anal. Chem. 2006, 78, 6340–6346. (b) Kim, K.; Hwang, J.; Seo, I.; Youn, T. H.; Kwak, J. Chem. Commun. 2006, 4723–4725. (c) Cosnier, S. Anal. Bioanal. Chem. 2003, 377, 507–520. (9) (a) Choi, S.; Murphy, W. L. Langmuir 2008, 24, 6873–6880. (b) Lamb, B. M.; Barrett, D. G.; Westcott, N. P.; Yousaf, M. N. Langmuir 2008, 24, 8885– 8889. (c) Boozer, C.; Yu, Q.; Chen, S.; Lee, C.-Y.; Homola, J.; Yee, S. S.; Jiang, S. Sens. Actuators, B 2003, 90, 22–30. (d) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. Langmuir 2000, 16, 6927–6936. (e) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548–6555. (f) Patel, N.; Davies, M. C.; Hartshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485–6490. (10) (a) Willey, T. M.; Vance, A. L.; van Buuren, T.; Bostedt, C.; Terminello, L. J.; Fadley, C. S. Surf. Sci. 2005, 576, 188–196. (b) Flynn, N. T.; Tran, T. N. T.; Cima, M. J.; Langer, R. Langmuir 2003, 19, 10909–10915.

10.1021/la803215z CCC: $40.75  2009 American Chemical Society Published on Web 01/27/2009

Multifunctional Thin Film Au Electrode Surface

for subsequent electrochemistry, and high stability under longterm storage in air and during potential cycling under acidic conditions.12 Diazonium salts with a wide range of substituent groups useful for surface functionalization have been reported including biotin,13 maleimide,14 carboxyl,15 amine16 thiol,17 boronic acid,18 and azide or alkyne for click chemistry.19 Another significant advantage of diazonium electrodeposition chemistry over alkanethiol surfaces is that the surface coverage and density of the resulting film can be controlled by the experimental conditions yielding submonolayer to multilayer films.11f,20 Because it may not always be synthetically straightforward to incorporate the various substituents into a single molecule we investigated the results from grafting different diazonium molecules to provide a multifunctional surface. The electrodeposition of aryl diazoniums with differing substituent groups should yield stable films that are mixed or stacked in structure capable of multiple functionalities on a single surface. Indeed, two-component films prepared from aryl diazonium salts have been reported. In these pioneering works twocomponent films are formed by simultaneous assembly of two diazonium compounds in a single solution resulting in mixed surfaces,12b,21 or consecutive deposition leading to stacked structures.22 Poly(dimethylsiloxane) PDMS molds have also been used to pattern assembly of two differing diazoniums via fill-in or consecutive assembly.23 These works employ electrochemistry, XPS, AFM, and other techniques to elucidate the chemical composition of the resulting binary films and the dependence of film properties on the deposition conditions. To date, only one study has utilized diazonium electrodeposition for formation of a multifunctional film toward a specific application. In this work, Liu and Gooding prepared a two-component carbon surface by electroreduction of a mixture containing diazoniums with oligo(phenylethynlene) and poly(ethylene glycol) (PEG) functionality.21a Oligo(phenylethynlene) served as a conductive path to the electrode allowing direct electron transfer to surface(11) (a) Delamar, M.; Hitmi, R.; Pinson, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1992, 114, 5883–5884. (b) Allongue, P.; Delamar, M.; Desbat, B.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1997, 119, 201–207. (c) Downard, A. J. Electroanalysis 2000, 12, 1085–1096. (d) Bernard, M.-C.; Chausse´, A.; Cabet-Deliry, E.; Chehimi, M. M.; Pinson, J.; Podvorica, F.; VautrinUl, C. Chem. Mater. 2003, 15, 3450–3562. (e) Kuo, T.-C.; McCreery, R. L.; Swain, G. M. Electrochem. Solid State 1999, 2, 288–290. (f) Anariba, F.; DuVall, S. H.; McCreery, R. L. Anal. Chem. 2003, 75, 3837–3844. (12) (a) Liu, G.; Bo¨cking, T.; Gooding, J. J. J. Electroanal. Chem. 2006, 600, 335–344. (b) Liu, G.; Liu, J.; Bo¨cking, T.; Eggers, P. K.; Gooding, J. J. Chem. Phys. 2005, 319, 136–146. (13) Dequaire, M.; Degrand, C.; Limoges, B. J. Am. Chem. Soc. 1999, 121, 6946–6947. (14) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Brozik, S. M. Langmuir 2008, 24, 2206–2211. (15) (a) Corgier, B. P.; Marquette, C. A.; Blum, L. J. J. Am. Chem. Soc. 2005, 127, 18328–18332. (b) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Arango, D. C.; Brozik, S. M. Biosens. Bioelectron. 2008, 23, 757–764. (c) Polsky, R.; Harper, J. C.; Dirk, S. M.; Arango, D. C.; Wheeler, D. R.; Brozik, S. M. Langmuir 2007, 23, 364–366. (16) (a) Lee, C. S.; Baker, S. E.; Marcus, M. S.; Yang, W.; Eriksson, M. A.; Hamers, R. J. Nano Lett. 2004, 4, 1713–1716. (b) Ruffien, A.; Dequaire, M.; Brossier, P. Chem. Commun. 2003, 912–913. (c) Shabani, A.; Mak, A. W. H.; Gerges, I.; Cuccia, L. A.; Lawrence, M. F. Talanta 2006, 70, 615–623. (17) Nielsen, L. T.; Vase, K. H.; Dong, M.; Besenbacher, F.; Pedersen, S. U.; Daasbjerg, K. J. Am. Chem. Soc. 2007, 129, 1888–1889. (18) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Arango, D. C.; Brozik, S. M. Angew. Chem., Int. Ed. 2008, 47, 2631–2634. (19) Evrar, D.; Lambert, F.; Policar, C.; Balland, V.; Limoges, B. Chem. Eur. J. 2008, 14, 9286–9291. (20) Brooksby, P. A.; Downard, A. J. Langmuir 2004, 20, 5038–5045. (21) (a) Liu, G.; Gooding, J. J. Langmuir 2006, 22, 7421–7430. (b) Louault, C.; D’Amours, M.; Be´langer, D. ChemPhysChem 2008, 9, 1164–1170. (22) (a) Adenier, A.; Combellas, C.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. Chem. Mater. 2006, 18, 2021–2029. (b) Brooksby, P. A.; Downard, A. J. J. Phys. Chem. B 2005, 109, 8791–8798. (c) Brooksby, P. A.; Downard, A. J. Langmuir 2005, 21, 1672–1675. (d) Solak, A. O.; Eichorst, L. R.; Clark, W. J.; McCreery, R. L. Anal. Chem. 2003, 75, 296–305. (23) Downard, A. J.; Garrett, D. J.; Tan, E. S. Q. Langmuir 2006, 22, 10739– 10746.

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immobilized horseradish peroxidase or myoglobin, while PEG served to decrease nonspecific adsorption of bovine serum albumin and components of blood serum onto the electrode surface. In this study we demonstrate for the first time the utility of bias-assisted diazonium assembly for modification of Au electrodes with a thin film possessing dual binding functionality. Consecutive deposition of nitrophenyl and phenylboronic acid pinacol ester diazonium salts on a single Au electrode allowed for the dual immobilization of citrate-capped platinum nanoparticles via electrostatic interactions with electro-generated aminophenyl groups and yeast cells via cyclic ester formation between chemically deblocked boronic acid groups and saccarides present in yeast membranes. This study explores the impact of the consecutive electrodeposition protocols on the functionality of the resultant stacked surface and was shown to affect film thickness, binding affinity, solution access to the underlying layer, and electrocatalytic properties. Significantly, the order of activation of the nitrophenyl and phenylboronic acid pinacol ester groups toward binding was also shown to affect surface functionality. This versatile and simple method for forming multifunctional surfaces proved to be an effective means for immobilization of diverse molecules at intimate proximities on the same electrode surface without the requirement of printing, lithography, or discrete individually addressable electrodes and serves as a compliment to other methods.

Experimental Section Materials. Aqueous solutions were prepared with 18 MΩ water using a Barnstead Nanopure water purifier (Boston, MA). Sodium periodate (NaIO4), anhydrous acetonitrile (ACN), and tetrahydrofuran (THF) were purchased from Acros Organics (Beel, Belgium). Sodium phosphate monobasic, sodium phosphate dibasic, sodium citrate, and 30% H2O2 were purchased from Sigma (St. Louis, MO). 4-Nitrophenyl diazonium tetrafluoroborate, 4-aminophenylboronic acid pinacol ester (97%), nitrosonium tetrafluoroborate (NOBF4), diethyl ether, and tetrabutylammonium tetrafluoroborate (Bu4NBF4) were obtained from Aldrich. Dihydrogen hexachloroplatinate (PtCl62-) was from Alfa Aesar (Ward Hill, MA). Sulfuric acid, ethyl alcohol (95% denatured), and potassium chloride were purchased from Fischer Scientific (Pittsburgh, PA). Tris-HCl was from Fluka (Buchs, Switzerland). All reagents were used as received. Synthesis of Phenyl Boronic Acid Pinacol Ester (PBA-PE) Diazonium. Phenyl boronic acid pinacol ester diazonium was prepared as previously reported.18 Briefly, 0.315 g (2.69 mM) of NOBF4 was dissolved in 5 mL of anhydrous acetonitrile under nitrogen and then cooled to -40 °C. A 0.53 g (2.42 mM) amount of 4-aminophenylboronic acid pinacol ester (97%) was dissolved in 12 mL of anhydrous acetonitrile under nitrogen. The amine solution was added slowly by cannula to the stirred -40 °C solution of NOBF4. The resulting solution was stirred for approximately 1 h at -40 °C and then allowed to warm to 0 °C and stirred for an additional 10 min. This solution was then cannulated into 800 mL of rapidly stirred cold diethyl ether in air. The resulting white precipitate was collected on a Bu¨chner funnel and dried under vacuum yielding 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenediazonium, 0.64 g (2.0 mmol), 83%. Preparation of Platinum Nanoparticles (PtNPs). Platinum nanoparticles were prepared by heating 100 mL of 1 mM PtCl62in nanopure water to reflux with stirring followed by slow addition of 10 mL of a 38.8 mM aqueous sodium citrate solution. This solution was stirred under reflux for approximately 1 h during which the solution turned from light yellow to black in color. The heat was removed and the solution was allowed to cool to room temperature while stirring. The solution was then passed through a 100 000 MW cutoff Centricon centrifugal filter (Millipore, Billerica, MA) and washed twice with water.

3284 Langmuir, Vol. 25, No. 5, 2009 Electrochemical Instrumentation. All electrochemical measurements were performed on a PGZ100 Voltalab potentiostat (Radiometer Analytical, Lyon, France) and were measured versus a 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). Gold disk electrodes, 5 mm diameter, were prepared via thermal evaporation of a 200 Å Cr 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 10 min, washed with nanopure water, and dried under a stream of nitrogen. Ellipsometry, FTIR Spectroscopy, and Microscopy. 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. FTIR measurements were obtained with a Nicolet 6700 Fourier transformed infrared spectrometer with a liquid nitrogen cooled mercury-cadmium-telluride (MCT) detector. An external specular reflectance attachment (SMART SAGA) was used to obtain an incidence angle of 80° with unpolarized light. A total of 1024 scans 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. Transmission electron microcope (TEM) images were taken on a Tecnai F-30ST TEM/ STEM operated at 300 kV. Scanning electron microscope (SEM) images were taken by a Zeiss Supra 55VP field emission gun scanning electron microscope using a 5 kV accelerating voltage directly from Au electrodes that were allowed to dry. Optical microscope imaging was obtained using an Olympus IX70 microscope and recorded using an Olympus DP71 camera. Electrode Functionalization. Nitrophenyl thin films were assembled onto clean gold electrodes using chronoamperometry, linear sweep, or cyclic sweep methods in a solution of 1 mM nitrophenyl diazonium and 0.1 M Bu4NBF4 in acetonitrile (ACN). After electrodeposition, the electrodes were briefly rinsed with ACN, followed by an ethanol rinse and a 15 s sonication in ethanol. After sonication, the electrodes were again rinsed in ethanol and dried under a stream of nitrogen. A stacked, multifunctional thin film was then formed by consecutive assembly of phenyl boronic acid pinacol ester diazonium onto nitrophenyl-modified gold electrodes using cyclic voltammetry in a solution of 1 mM phenyl boronic acid pinacol ester diazonium and 0.1 M Bu4NBF4 in ACN. Following electrodeposition, the electrodes were again rinsed, sonicated, and dried as described above. Nitrophenyl/phenyl boronic acid pinacol estermodified electrodes were treated with 100 µL of a 50 mM NaIO4 solution (4:1 Water:THF) for 30 min to remove the pinacol blocking ester and rinsed thoroughly in water. The surface was conditioned for 1 h in 100 mM Tris-HCl, pH 8.5, yielding a nitrophenyl/phenyl boronic acid thin film. Conversion of nitrophenyl to aminophenyl groups was achieved by cyclic voltammetry from -300 to -1300 mV in an ethanol:water (1:9) solution with 0.1 M KCl as electrolyte. This surface was then treated with 25 µL of the washed PtNP solution (pH ∼5) for 10 min followed by rinsing with water and drying with nitrogen. Finally, 50 µL of yeast cells (∼1 × 107 cells/mL, S. cereVisiae strain INVSc1, Invitrogen, Carlsbad, CA) in 0.1 M sodium phosphate buffer, pH 7.4, were placed onto the electrodes for 5 min and followed by gentle washing of the electrodes three times with buffer.

Results and Discussion Preparation of the multifunctional thin film gold electrode surface is shown in Scheme 1. First, a nitrophenyl thin film was assembled onto the electrode via bias-assisted grafting of nitrophenyl diazonium (1). This was followed by the consecutive electrodeposition of phenyl boronic acid pinacol ester (PBA-

Harper et al. Scheme 1. Assembly of a Multifunctional Thin Film Electrode Surfacea

a Preparation of the stacked multifunctional thin film occurred via (1) electrodeposition of nitrophenyl diazonium onto a Au substrate; (2) electrodeposition of pinacol ester phenylboronic acid diazonium forming a stacked thin film; (3) chemical deprotection of phenylboronic acid functional groups; (4) electrochemical reduction of nitrophenyl to aminophenyl functional groups; (5) electrostatic immobilization of citrate capped PtNPs to aminophenyl groups; (6) immobilization of yeast cells via cyclic ester formation between saccarides in the yeast cell membrane and phenylboronic acid groups.

PE) diazonium onto the nitrophenyl film, forming a thin film with both nitro and boronic acid functional groups (2). These two functional groups were then activated toward binding by chemically removing pinacol ester blocking groups (3) followed by electrochemical reduction of nitro to amino groups (4). Positively charged aminophenyl groups (pKa 4.6)24 served to electrostaticly immobilize negatively charged citrate-capped PtNPs25 (5) while boronic acid groups were used to immobilize whole cells through the formation of cyclic esters with saccarides26 present on yeast cell membranes. Electrodeposition of nitrophenyl diazonium films onto gold surfaces has been the subject of several recent studies which show that the electrodeposition protocol can have a profound affect on the order, thickness, and electron transport kinetics of the resulting nitrophenyl film.27 As the nitrophenyl film in this work serves as a conducting layer for the bias-assisted assembly of the PBA-PE film, its properties can have a significant affect on the subsequent assembly of the boronic acid film. Cyclic voltammetry (CV) and ellipsometry were used to characterize the effect of the nitrophenyl film thickness on the subsequent electrodeposition of PBA-PE. Three different electrodeposition (24) Braude, E. A.; Nachod, F. C. Determination of Organic Structures by Physical Methods; Academic Press: New York, 1955. (25) (a) Shipway, A. N.; Lahav, L.; Gabai, R.; Willner, I. Langmuir 2000, 16, 8789–8795. (b) Shustak, G.; Shaulov, Y.; Domb, A. J.; Mandler, D. Chem. Eur. J. 2007, 13, 6402–6407. (26) (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem., Int. 1995, 35, 1910–1922. (b) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2002, 124, 12486–12493. (c) Pavey, K. D.; Olliff, C. J.; Paul, F. Analyst 2001, 126, 1711–1715. (d) Soh, N.; Sonezaki, M.; Imato, T. Electroanalysis 2003, 15, 1281– 1290. (27) (a) Harper, J. C.; Polsky, R.; Dirk, S. M.; Wheeler, D. R.; Brozik, S. M. Electroanalysis 2007, 19, 1268–1274. (b) Uetsuka, H.; Shin, D.; Tokuda, N.; Saeki, K.; Nebel, C. E. Langmuir 2007, 23, 3466–3472. (c) Haccoun, J.; VautrinUl, C.; Chausse´, A.; Adenier, A. Prog. Org. Coat. 2008, 63, 18–24.

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Table 1. Effect of Diazonium Electrodepostion Method on Stacked Surface Film Thickness nitrophenyl diazonium nitrophenyl film equivalent monolayers film thickness after 2 CV B(OH)2 equivalent electrodeposition method thickness (Å)a (nitrophenyl)b Γ (mol NO2/cm2)c diazonium electrodepostion (Å)a monolayers (B(OH)2)b 1 min CA 1 LS 2 CV

4.0 ( 0.4 7.9 ( 1.3 10.3 ( 1.8

0.6 ( 0.06 1.2 ( 0.20 1.5 ( 0.27

0.90 × 1011 3.0 × 1011 1.1 × 1010

8.0 ( 0.6 10.9 ( 1.6 12.0 ( 1.0

0.4 ( 0.06 0.3 ( 0.16 0.2 ( 0.10

a Standard deviations were calculated from eight or more independent measurements on each of three electrodes sampled per electrodeposition technique. Calculated thickness of a nitrophenyl and a phenylboronic acid pinacol ester monolayer with 70° tilt is approximately 6.7 Å and 10.0 Å, respectively. c Surface concentration calculated from the total charge transferred during the six-electron nitro to amine reduction.

b

Figure 1. Cyclic voltammograms of 1 mM phenylboronic acid pinacol ester diazonium on nitrophenyl thin film-modified gold disk electrodes prepared under various electrodeposition techniques. The second CV cycle for each electrode is omitted for clarity. Nitrophenyl diazonium electrodepostion method: Blue, 1 min CA; green, 1 LS; red 2 CVs. 0.1 M Bu4NBF4 in ACN, V ) 100 mV · s-1.

protocols were used to deposit nitrophenyl films onto Au electrodes: (1) 1 min chronoamperometric deposition (CA, step to -1 V), (2) linear sweep (LS, 0 to -1 V at 100 mV · s-1), and (3) 2 CVs (0 to -1 to 0 V at 100 mV · s-1). Following assembly, the nitrophenyl film thickness was measured using ellipsometry. Table 1 shows the average thickness for electrodes prepared from these different methods resulting in submonolayer, monolayer, and 1.5 monolayer nitrophenyl films for the three deposition protocols, respectively. In agreement with previous reports, potential sweep methods led to thicker and less ordered films than fixed potential depositions.27a,b The nitrophenyl-modified electrodes were subsequently modified with PBA-PE using a 2 CV (0 to -1 to 0 V at 100 mV · s-1) electrodeposition from the corresponding diazonium salt. Cyclic voltammograms for the PBA-PE diazonium deposition on the nitrophenyl-modified electrodes with differing film thicknesses are shown in Figure 1. Submonolayer nitrophenyl-modified electrodes (blue) showed a reductive peak shoulder at -105 mV that is attributed to the electroreduction of the diazonium functional group (see Supporting Information). This shoulder is not as sharp and is shifted -25 mV compared to that obtained from electrodeposition of PBA-PE diazonium onto a clean Au electrode (Figure S1, Supporting Information). The higher overpotential required for reaction and lower relative currents are a manifestation of the higher resistance to electron transfer through the nitrophenyl thin film. Quasireversible peaks centered near -550 and -820 mV are also present in CV measurements of the PBA-PE diazonium precursor, 4-aminophenylboronic acid pinacol ester (0.1 M Bu4NBF4 in ACN) and may be redox reactions of pinacol ester hydrolysis products or impurities in the starting

material. The irreversible reduction wave at -220 mV remains unassigned but is not associated with diazonium grafting (see Supporting Information). A trend of decreasing PBA-PE diazonium electroreduction currents with increasing nitrophenyl film thickness is observed for the samples prepared from single LS (Figure 1, green) and 2 CV (Figure 1, red) nitrophenyl diazonium depositions. This was expected, as heterogeneous electron transfer through a nitrophenyl film has been shown to decrease as film thickness increases.27a Electron transfer from electrodes with 1.5 monolayer nitrophenyl films was suppressed such that no PBA-PE diazonium reduction peak was observed. However, absence of the diazonium reduction peak does not indicate that assembly has not occurred. Studies combining electrochemical quartz crystal microbalance (EQCM) and cyclic voltammetry showed that although a diazonium reduction peak is not observed, consistent growth of the diazonium film with subsequent potential sweeps can occur.28 Assembly of PBA-PE onto the nitrophenyl films was verified via ellipsometry measurements following PBA-PE diazonium deposition and is reported in Table 1. As expected, the thickest PBAPE films were deposited onto the thinnest nitrophenyl films because of higher electron transfer kinetics through the nitrophenyl film, enhancing PBA-PE diazonium electroreduction and grafting. Equivalent monolayers reported in Table 1 were calculated directly from ellipsometry measurements. As this method averages surface topography over the laser spot size, these values accurately account for grafting onto the topography of the existing film and/or the exposed gold surface. These values, however, do not describe the percent composition of a given slice of film with respect to nitrophenyl or PBA-PE species. The functional groups of the stacked diazonium film were activated toward binding by first removal of the pinacol ester group via chemical deprotection (see Experimental Section). This was followed by electrochemical reduction of nitro groups to amines. The first reductive sweep in aqueous solution produced a sharp wave corresponding to the six-electron reduction of nitrophenyl to aminophenyl. This peak was not observed in subsequent sweeps, indicating complete conversion of all electrically accessible nitro groups. The area of the reduction waves corresponds to the total charge transferred during the reaction and hence the number of nitro groups electrically accessible to the electrode and the electrolyte solution. Nitro surface concentrations, Γ, are reported in Table 1. The greatest surface concentration of NO2 corresponds to the nitrophenyl film electrode assembled with 2 CVs and is followed by electrodes prepared from 1 LS, and 1 min CA. This trend agrees with the average nitrophenyl film thickness data obtained from ellipsometry. These surface concentrations are similar to values obtained from nitrophenyl films on Au with similar thicknesses as we have reported earlier.27a Grazing-angel FTIR spectra analysis was further used to study and verify film composition at each stage of preparation. The spectrum measured from a Au surface prepared from a 2 CV (28) Laforgue, A.; Addou, T.; Be´langer, D. Langmuir 2005, 21, 6855–6865.

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Figure 3. Cyclic voltammograms of 1 mM H2O2 in 50 mM NaPB, pH 7.4, V ) 100 mV · s-1. Grey, clean Au electrode; red, nitrophenyl Au; blue, aminophenyl Au; green, PtNP-treated nitrophenyl Au; black, PtNPtreated aminophenyl Au. All films were formed via 2 CV electrodeposition of nitrophenyl diazonium. Inset: Catalytic H2O2 reduction peak currents from PtNP-treated Au surfaces with (white) varying nitrophenyl diazonium electrodeposition procedure, and (black) phenylboronic acid aminophenyl stacked Au surfaces with varying nitrophenyl diazonium and constant 2 CV phenylboronic acid pinacol ester electrodeposition. Figure 2. Grazing-angle FTIR spectroscopy measurements for gold electrodes prepared according to Scheme 1: Black, nitrophenyl surface; red, PBA-PE nitrophenyl surface; green, PBA-nitrophenyl surface; blue, PBA-aminophenyl surface. All surfaces were prepared from 2 CV electrodepositions of the respective diazonium salt(s).

nitrophenyl diazonium electrodeposition is shown in Figure 2 (black). Of note are two peaks at 1534 cm-1 and 1351 cm-1 (labeled peaks 1 and 2) characteristic of the asymmetric and symmetric stretch modes, respectively, of the nitro group.29 Upon subsequent treatment with a 2 CV PBA-PE diazonium electrodeposition, the stacked surface exhibits properties of both boron and nitro functionalities (Figure 2, red). The FTIR spectrum shows a clear B-phenyl stretch mode at 1417 cm-1 (peak 3) and retains the band at 1351 cm-1 (peak 4) which may be the overlap of both the symmetric nitro and B-O stretches. Also present is the asymmetric nitro stretch which has shifted slightly to 1536 cm-1 (peak 5). This peak is not present in the FTIR spectra of PBA-PE deposited alone on Au,18 verifying that the thin film contains both nitro and PBA functional groups. A broad O-H stretch at 3400 cm-1 (peak 6) is indicative of some inadvertent hydrolysis. FTIR spectroscopy following chemical deprotection of the pinacol ester group (Figure 2, green) showed significant enhancement in the O-H stretch modes at 3400 cm-1 (peak 7). Still present are the asymmetric nitro stretch at 1521 cm-1 (peak 8) and the symmetric nitro and B-O stretches at 1351 cm-1 (peak 9). The FTIR spectrum following electrochemical reduction of nitrophenyl to aminophenyl groups is shown in blue (Figure 2). The peak at 1351 cm-1 (peak 10) is not as pronounced as in previous spectra, indicative of loss of the symmetric nitro stretch while the peak at 1276 cm-1 (peak 11), characteristic of the C-N stretch, has increased in intensity. The peak at 1351 cm-1 is not completely lost because of remaining B-O stretching. Further evidence of nitro to amine conversion arises from the loss of the asymmetric nitro stretch at 1521 cm-1 (peak 8) and (29) The Aldrich Library of FT-IR Spectra, 2nd ed.; Sigma-Aldrich Co.: Milwaukee, WI, 1997.

the appearance of a peak at 1634 cm-1 (peak 12) characteristic of the NH2 deformation band. Use of PtNPs in this study served as a means to determine the accessibility of the aminophenyl groups following PBA-PE deposition, to measure the conductive properties of the stacked thin film, and allowed for visualization of PtNP binding in relation to immobilized yeast cells via microscopy. The affinity of the aminophenyl film toward electrostatic immobilization of PtNPs was initially investigated without subsequent assembly of the PBA-PE film. Cyclic voltammograms for various Au electrode surfaces in a 1 mM peroxide solution are shown in Figure 3. A clean gold electrode (gray), nitrophenyl film-modified electrode (red, 2CV electrodeposition), and aminophenyl film-modified electrode (blue, 2 CV electrodeposition) showed no significant electrocatalytic peroxide reduction currents and only moderate background currents attributed to reduction of dissolved oxygen in the electrolyte solution. Similarly, a nitrophenyl film prepared from a 2 CV electrodeposition followed by a 10 min treatment with PtNPs (green) showed no electrocatalytic currents. In contrast, an aminophenyl surface treated with PtNPs followed by thorough rinsing (black, 2 CV electrodepostion) yielded a cathodic current wave near -70 mV characteristic of PtNP catalyzed H2O2 reduction.30 This NP-catalyzed H2O2 reduction is highly dependent on the NP size with optimal currents obtained from 2-4 nm diameter particles.31 TEM imaging was used to determine the size distribution of the synthesized PtNP, as shown in Figure 4A. As expected from the efficient peroxide reduction currents, the PtNPs were 2.7 ( 0.4 nm in diameter. The PtNP(30) (a) Chow, K.-F.; Mavre´, F.; Crooks, R. M. J. Am. Chem. Soc. 2008, 130, 7544–7545. (b) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2268–2271. (31) (a) Antoine, O.; Bultel, Y.; Durand, R. J. Electroanal. Chem. 2001, 499, 85–94. (b) Kinoshita, K. J. Electrochem. Soc. 1990, 137, 845–848. (c) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Rawlings, J. A.; Brozik, S. M. Chem. Commun. 2007, 2741–2743.

Multifunctional Thin Film Au Electrode Surface

Figure 4. (A) TEM image of citrate-capped catalytic PtNPs. SEM images of (B) PtNP-treated aminophenyl Au surface, (C) PtNP-treated phenylboronic acid-nitrophenyl stacked surface, (D) PtNP-treated phenylboronic acid-aminophenyl stacked surface. All films were formed via a 2 CV electrodeposition of nitrophenyl diazonium. Samples C and D were additionally treated with a 2 CV electrodeposition of BPA-PE diazonium.

modified aminophenyl surface was also stable to successive rinses, drying, and treatment with 300 mM sodium citrate solution. Additionally, these currents demonstrate that the deposited thin film remains conductive, allowing for subsequent electrochemical measurements. Both the number of aminophenyl groups and deblocked phenylboronic acid (PBA) groups were shown to affect PtNP immobilization. The inset of Figure 3 shows the electrocatalytic peak currents obtained from H2O2 reduction at aminophenyl Au surfaces prepared from 1 min CA, 1 LS, and 2 CV nitrophenyl depositions (white bars). A strong correlation is observed between the ellipsometry data, NO2 surface concentrations, and electrocatalytic peak currents showing that increasing film thickness and density lead to higher PtNP loading. The most significant increase in current was obtained between the surfaces prepared from 1 min CA and 1 LS deposition. This indicates that increasing the aminophenyl surface coverage from submonolayer to monolayer had a greater impact on PtNP loading than increasing the aminophenyl group surface density (1 LS and 2 CV samples). Formation of PBA films onto aminophenyl films negatively impacted the affinity of the underlying aminophenyl film toward PtNP immobilization (see inset to Figure 3, black bars). All stacked PBA-aminophenyl films showed lower catalytic peak currents than aminophenyl films prepared under identical nitrophenyl electrodeposition protocols. This is likely because of blocking of portions of the aminophenyl film onto which the PBA was assembled. Interestingly, the decrease in catalytic currents between PBA-aminophenyl and aminophenyl films prepared from a 1 min CA nitrophenyl deposition was half that obtained between PBA-aminophenyl and aminophenyl films prepared from 1 LS or 2 CVs nitrophenyl depositions. As aminophenyl films prepared from 1 min CA electrodepositions are submonolayer, a substantial portion of PBA presumably assembled onto the free Au surface, providing a less detrimental impact on PtNP immobilization. Despite the relatively lower

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PtNP loading and catalytic currents on PBA-aminophenyl surfaces, submonolayer PBA films permitted access to a substantial portion of the underlying aminophenyl layer, providing significant catalytic peak currents that increase for electrodes with higher aminophenyl film thickness and lower PBA thickness. PBA-PE films thicker than submonolayer could be obtained by changing the conditions of electrodeposition. For example, a 2 CV nitrophenyl diazonium electrodeposition followed by a 5 CV PBA-PE diazonium electrodeposition yielded film thicknesses of 9.3 ( 2.5 Å and 26.0 ( 0.9 Å, respectively, forming a 1.7 equiv monolayer PBA-PE thin film. Following removal of the pinacol ester blocking group and electrochemical reduction of nitro to amino groups, the surface was treated with PtNP solution. CVs in peroxide solution revealed that these stacked surfaces were not effective at capturing PtNPs. This could be because of the approximately 30% lower (8.0 ( 2.0 × 1011 mol NO2 · cm-2) nitro to amine conversion compared to surfaces prepared from a 2 CV nitrophenyl and 2 CV PBA-PE diazonium electrodeposition. It is also likely that the thicker PBA films have sterically hindered PtNP access to the underlying reduced nitrophenyl film. Deblocked PBA surfaces (pKa ∼ 8)32 do show minor nonspecific binding of citrate-capped PtNPs, generating a peroxide catalytic reduction peak that is 23% of that obtained from a similarly treated aminophenyl surface. These results are also depicted in the SEM images presented in Figure 4. PtNPs are found at high density on the aminophenyl film (Figure 4B, 2 CV nitrophenyl diazonium electrodeposition) while the deblocked PBA-nitrophenyl film shows very few immobilized PtNPs (Figure 4C, 2 CV nitrophenyl, 2 CV PBA-PE diazonium electrodeposition). The PtNP-treated PBA-aminophenyl stacked surface shows a large number of immobilized particles (Figure 4D, 2 CV nitrophenyl, 2 CV PBA-PE diazonium electrodeposition); however, the density is not as high as that obtained from the aminophenyl surface alone. As access to the underlying aminophenyl surface was demonstrated by electrostatic interactions with citrate-capped PtNPs, the potential exists for the use of common amine conjugation methods including carbodiimide chemistry or homo/heterobifunctional cross-linkers to immobilize other diverse molecules to the aminophenyl surface. The order in which the functional groups are activated is important, as IO4-, used to remove the pinacol ester, can also oxidize amine groups, forming hydroxylamine or nitro groups. Treatment of an aminophenyl surface with the IO4- pinacol ester deprotection solution for 30 min converted 22% of amino groups back to nitro groups (assuming complete oxidation) as determined by integration of the nitro reduction peak before and after IO4treatment. The percentage of reacted amine groups may be higher if incomplete oxidation occurred forming hydroxylamine groups. These reacted amines are likely those most accessible to the IO4solution on the surface of the thin film. Following IO4- treatment, the partially oxidized aminophenyl surface was ineffective at capturing PtNPs as determined by CV in H2O2 solution. Presumably, loss of these surface amino groups was sufficient to prevent electrostatic binding of the citrate-capped PtNPs to the Au electrode surface. The affinity for yeast cell adhesion to the stacked thin film was determined for the blocked PBA-PE-aminophenyl film and the deblocked PBA-aminophenyl film after a brief treatment with yeast cells in buffer. As presented in the microscope images in Figure 5, the blocked PBA-PE surface (Figure 5A) had very few cells nonspecifically bound to the surface while many yeast cells (32) Yan, J.; Springsteen, G.; Deeter, S.; Wang, B. Tetrahedron 2004, 60, 11205–11209.

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on a deblocked PBA-aminophenyl functionalized Au electrode. All electrodes were prepared from a 2 CV nitrophenyl and 2 CV PBA-PE diazonium electrodeposition. Although not visible through the yeast cell, PtNPs are expected to exist beneath the cell at a density similar to that observed surrounding the cell. The close proximity of captured PtNPs and yeast cells on this surface demonstrate ideal immobilization conditions for placement of cell monitoring probes that may detect chemicals, signaling molecules, or proteins excreted from the cell(s).

Conclusions

Figure 5. White light images of (A) pinacol ester blocked phenylboronic acid-aminophenyl and (B) deblocked phenylboronic acid-aminophenyl Au electrodes treated with yeast in nanopure water and rinsed with 0.1 M Tris-HCl, pH 8.5. (C) SEM image showing PtNPs near the edge of an immobilized yeast cell (dark shadow on the right) on a deblocked phenylboronic acid-aminophenyl Au electrode. All films were formed via a 2 CV electrodeposition of nitrophenyl diazonium followed by a 2 CV electrodepostion of BPA-PE diazonium.

adhered to the deblocked PBA-aminophenyl surface (Figure 5B). This is consistent with previous studies showing capture of sugars and cells at boronic acid-modified surfaces18,26 and shows that the underlying aminophenyl layer has no significant effect on yeast cell adhesion. Figure 5C demonstrates the utility of the multifunctional thin film for immobilization of both PtNPs and the capture of yeast cells. This SEM image shows PtNPs in close proximity to an immobilized yeast cell, seen as a dark shadow,

This report detailed the formation of a thin film with dual binding functionality on a single Au electrode surface via consecutive electroreduction of diazonium salts with two distinct substituent groups. FTIR analysis and simultaneous immobilization of PtNPs and yeast cells demonstrates that both aminophenyl and boronic acid groups in the thin film are accessible and functional. The electrodeposition protocol and order of functional group activation have profound effects on the properties of the resultant thin film. Electrocatalytic currents from peroxide reduction at immobilized PtNPs show that the thin film remains conductive, facilitating subsequent electrochemical measurements. In addition to electrostatic interactions, the aminophenyl surface can potentially be used with common carbodiimide chemistry or homo/heterobifunctional cross-linkers to conjugate other diverse molecules at intimate proximities to immobilized cells. Such surfaces potentially allow for the immobilization of nanoparticles, antibodies, DNA probes, and whole cells on a single electrode surface without the requirement of printing, lithography, or discrete individually addressable electrodes having significant implications for platforms allowing real-time analysis of cell signaling, cell-cell and host-pathogen interactions, genomic and proteomic analysis, and biosensing. Acknowledgment. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under contract DEAC04-94AL8500. Supporting Information Available: Cyclic voltammograms and analysis of the PBA-PE diazonium assembly at a clean Au electrode. This material is available free of charge via the Internet at http://pubs.acs.org. LA803215Z