Concentration Dependence of Aggregate Formation upon Adsorption

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Concentration Dependence of Aggregate Formation upon Adsorption of 5-(Octyldithio)-2-nitrobenzoic Acid on Gold Electrodes M. Darder,† E. Casero,† D. J. Dıáz,‡ H. D. Abrunã,*,‡ F. Pariente,† and E. Lorenzo*,† Departamento de Quı´mica Analı´tica y Ana´ lisis Instrumental, Universidad Auto´ noma de Madrid, Canto Blanco 28049, Madrid, Spain, and Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301 Received February 17, 2000. In Final Form: September 21, 2000 The adsorption of 5-(octyldithio)-2-nitrobenzoic (O-DTNB) onto gold electrode surfaces has been investigated with a quartz crystal microbalance, and the process appears to follow the Freundlich adsorption model. Atomic force microscopy images of adsorbed films obtained from 20 µM and 10.0 mM ethanolic solutions of O-DTNB exhibited dramatically different morphologies which were ascribed to the formation of multilayer equivalent aggregates. Similar to adsorbed films derived from 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), cycling the potential past -0.55 V for adsorbed films of O-DTNB resulted in the generation of a reversible, pH-dependent, redox couple ascribed to formation of the hydroxylamine from the nitro group. For films obtained from a high concentration (10.0 mM) of O-DTNB, generation of the reversible redox couple did not appear to result in desorption of the additional adsorbed material as ascertained from EQCM data. The cyclic voltammetric profiles of [Fe(CN)6]3-, hydroxymethylferrocene, and [Co(phen)3]2+ at gold electrodes modified with O-DTNB was pH dependent, and this behavior was ascribed to the acid/ base behavior of the carboxylic acid substituent.

Introduction The preparation and characterization of ordered thin films on solid substrates, ranging in thickness from a monolayer to multilayers, have been the subject of numerous investigations and reports, especially over the past decade. Such interest is due, at least in part, to the fact that such deliberately designed interfacial structures show considerable technological promise in numerous applications including electronic and optical devices, sensors, protective layers, model biological membranes, and patternable materials for resists and for information storage1-7 among others. We have had a long-standing interest in the modification of electrode surfaces, in general, and in their applications especially as they relate to electrocatalytic and analytical applications. In the latter, we have paid particular attention to amperometric biosensor applications. In this regard we have employed a variety of strategies for surface modification including the electrodeposition of films derived from 3,4-dihydroxybenzaldehyde and related materials,8 electrodeposition and electropolymerization of transition metal complexes,9 and most recently through adsorption, onto gold surfaces, of sulfur-containing mol† ‡

Universidad Autońoma de Madrid. Cornell University.

(1) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Isreaelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932-950. (2) Ulman, A. An introduction to Ultrathin Organic Films from Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (3) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87-96. (4) Gaines, G. L., Jr. Insoluble Monolayers; Interscience: New York, 1966. (5) Sagiv, J. J. Am. Chem. Soc. 1980, 102, 92-98. (6) Murray, R. W. Molecular Design of Electrode Surfaces; Wiley: New York, 1992. (7) Bard, A. J. Integrated Chemical Systems; Wiley: New York, 1994. (8) Pariente, F.; Tobalina, F.; Lorenzo, E.; Abrunã, H. D. Anal. Chem. 1996, 68, 3135-3142.

ecules (such as DTNB, 5,5′-dithiobis(2-nitrobenzoic acid)) with the subsequent generation of redox active (hydoxylamine) groups via electrochemical triggering.10 The adsorption of sulfur-containing materials such as thiols,11 sulfides,12 and disulfides13on gold (silver and other) surfaces is now very well established.2,14,15 It has been generally found that in numerous cases such molecules form highly ordered monolayers upon adsorption. Moreover, because of the strength of this interaction, these materials can be adsorbed onto such surfaces even in the presence of other functional groups thus allowing for the preparation of surfaces deliberately modified with a wide array of functional groups.16 Gold surfaces modified with alkanethiols (as well as other sulfur-containing groups) have been used as anchors to immobilize biological molecules in order to construct both enzyme biosensors17-22 and immunosensors.23 (9) Wu, Q.; Maskus, M.; Pariente, F.; Tobalina, F.; Fernańdez, V. M.; Lorenzo, E.; Abrunã, H. D. Anal. Chem. 1996, 68, 3688-3696. (10) Casero, E.; Darder, M.; Takada, K.; Abrunã, H. D.; Pariente, F.; Lorenzo, E. Langmuir 1999, 15, 127-134. (11) (a) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569. (b) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155-7164. (c) Walczak, M. M.; Chung, C.; Stole, S. M.; Widrig, C. A.; Porter, M. D. J. Am. Chem. Soc. 1991, 113, 23702378. (12) (a) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (b) Rubinstein, I.; Steinberg, S.; Tor, Y.; Shanzer, A.; Sagiv, J. Nature 1988, 332, 426-429. (c) Katz, E.; Borovkov, V. V.; Evstigneeva, R. P. J. Electroanal. Chem. 1992, 326, 197-212.13. (13) (a) Hickman, J. J.; Ofer, D.; Zou, C.; Wrighton, M. S.; Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 1128-1132. (b) Bain, C. D.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1989, 5, 723-727. (c) Katz, E. J. Electroanal. Chem. 1990, 291, 257-260. (14) Bain, C. D.; Whitesides, G. M. Science 1988, 240, 62-63. (15) Pan, W.; Durning, C. J.; Turro, N. J. Langmuir 1996, 12, 44694473. (16) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358-2368. (17) Kajiya, Y.; Okamoto, T.; Yoneyama, H. Chem. Lett. 1993, 21072110. (18) Willner, I.; Lion-Dagan, M.; Marx-Tibbon, S.; Katz, E. J. Am. Chem. Soc. 1995, 117, 6581-6592.

10.1021/la0002381 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/11/2000

Concentration Dependence of Aggregate Formation

We present herein a study of the adsorption process of 5-(octyldithio)-2-nitrobenzoic acid (O-DTNB) onto gold electrodes and on the characterization of the resulting films. O-DTNB adsorbs onto gold surfaces, and the resulting coverage and structural details of the deposit are strongly dependent on the solution concentration of O-DTNB from which the adsorption is carried out. The kinetics and thermodynamics of adsorption have been investigated by electrochemical and in-situ quartz crystal microbalance (QCM) techniques. The kinetics appear to follow an activation mechanism while the adsorption thermodynamics appear to be best described by the Freundlich isotherm. The structure/topography of the deposited layers has been studied by atomic force microscopy (AFM). In a manner analogous to the behavior exhibited by films of DTNB on gold, we find that films derived from O-DTNB also exhibit the generation of redoxactive hydroxylamine/nitroso groups by electrochemical triggering. The blocking characteristics of the deposited layer, in terms of the permeability of various redox couples, have also been investigated with particular emphasis on the pH dependence of the response. The potential utility of gold surfaces modified with O-DTNB in amperometric biosensor applications is discussed. Experimental Section Materials. O-DTNB was purchased from Fluka and stored at 4 °C. Although most experiments were carried out with the asreceived material which was of at least 97% purity, indistinguishable results were obtained using material that had been purified by column chromatography. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Aldrich (at least 95% purity) and used as received. Hydroxymethylferrocene and 1-decanethiol were purchased from Fluka and used as received. Potassium ferricyanide was purchased from Carlo Erba. [Co(phen)3](PF6)2 (phen is 1,10-phenanthroline) was synthesized as previously described.24 Sodium phosphate (Sigma Chemical Co.) was used in the preparation of buffer solutions. Water was purified with a Millipore Milli-Q-System. Apparatus. Voltammetric Measurements. Cyclic voltammetric studies were performed with an Autolab/PGSTAT10 potentiostat from Eco-Chemie. The electrochemical experiments were carried out in three-compartment electrochemical cells with standardtaper joints so that all three compartments could be hermetically sealed with Teflon adapters. Gold disk electrodes (2 mm diameter, 0.031 cm2 geometric area, and 0.090 cm2 microscopic area), sealed in soft glass were used as working electrodes. A large area of coiled platinum wire was employed as a counter electrode, and all potentials are reported against a sodium-saturated calomel electrode (SSCE) without regard for the liquid junction. All solutions were deaerated with nitrogen gas for at least 30 min before use, and the gas flow was kept over the solution during experiments. AFM Experiments. Contact-mode atomic force microscopy (AFM) images were obtained in 0.1 M HClO4 (Fisher Scientific) using a Molecular Imaging 6 µm scanner (D scanner), Molecular Imaging fluid cell, Molecular Imaging Isolation Chamber, and Digital Instruments Nanoscope E controller. A Au(111) disk electrode (grown from the melt, cut, and polished at the Materials Preparation Facility of the Cornell Center for Materials Research, CCMR) was mounted on the cell using a custom (homemade) Teflon mount. All images shown are unfiltered and no off-line (19) Imamura, M.; Haruyama, T.; Kobakate, E.; Ikariyama, Y.; Aizawa, M. Sens. Actuators 1995, B24-25, 113-116. (20) Creager, S. E.; Olsen, K. Anal. Chim. Acta 1995, 307, 277-289. (21) Dong, X. D.; Lu, J.; Cha, C. Bioelectrochem. Bioenerg. 1995, 36, 7-76. (22) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052-2066. (23) Marksich, M.; Whitesides, G. M. Annu. Rev. Biomol. Struct. 1996, 25, 55-78. (24) Goss, C. A.; Abrunã, H. D. Inorg. Chem. 1985, 24, 4263-4267.

Langmuir, Vol. 16, No. 25, 2000 9805 zoom was used. Values for the detector setpoints used were kept low (in the range of 0.0-1.0 V) to avoid excessive pressure by the tip against the film. Scan rates between 1 and 5 Hz at 512 samples resolution were used in order to obtain more detailed images. Standard Si3N4 AFM tips were used without further modification. Grain analysis was carried out using the standard Nanoscope Software algorithms. A low-pass filter was applied prior to the algorithm. QCM Measurements. AT-cut quartz crystals (5 MHz) of 25 mm diameter with Au electrodes deposited over a Ti adhesion layer (Maxtek Co.) were used for electrochemical quartz crystal microbalance (EQCM) measurements. An asymmetric keyhole electrode arrangement was used, in which the circular electrode geometrical areas were 1.370 cm2 (front side) and 0.317 cm2 (backside). The electrode surfaces were overtone polished. Prior to use, the quartz crystals were cleaned by immersion in piranha solution, H2SO4/H2O2 (3:1). Caution: Piranha solution is extremely reactive! They were subsequently rinsed with water and acetone and dried in air. The quartz crystal resonator was set in a probe (TPS-550, Maxtek) made of Teflon in which the oscillator circuit was included, and the quartz crystal was held vertically. The probe was connected to a cell by a homemade Teflon joint which was immersed in water-jacketed beaker thermostated at 25.0 ( 0.1 °C with a Haake F6 digital temperature controller. The frequency was measured with a plating monitor (PM-740, Maxtek Inc.) and simultaneously recorded by a personal computer. To study the mass changes associated with the adsorption of O-DTNB onto a gold electrode, a QCM probe was immersed in ethanol and the frequency of the quartz crystal was monitored versus time. After the temperature and frequency had stabilized (typically 15-20 min), an aliquot of an ethanolic stock solution of O-DTNB was added so that the final concentration of O-DTNB was either 20 µM or 2.0 mM. Procedures. Electrode Conditioning and Adsorption of ODTNB. Polycrystalline disk gold electrodes were polished with 1.0 µm diamond paste (Buehler), rinsed with water, and sonicated for 10 min in distilled water. The electrodes were activated by holding the potential at +2.0 V for 5 s in 0.1 M H2SO4 and then at -0.35 V for 10 s, followed by potential cycling from -0.35 to +1.5 V at 4 V/s for 1 min. Finally the cyclic voltammogram characteristic of a clean polycrystalline gold electrode was recorded (from -0.2 to +1.5 V) at 100 mV/s and used to calculate the microscopic area by integration of the cathodic peak associated with the reduction of the gold oxide. The electrode was subsequently rinsed with water and ethanol and used immediately in the preparation of the monolayer. The conditioned electrode was immersed for 3 h at room temperature in a (20 µM to 10.0 mM) solution of O-DTNB in ethanol. Afterward, the electrode was thoroughly rinsed with ethanol, water, and finally with 0.1 M phosphate buffer, pH 7. Electrodes were employed immediately after preparation. AFM Imaging of DTNB and O-DTNB. Prior to modification, the Au(111) disk electrode was flame annealed for 5 min and quenched in Milli-Q water (which had been previously degassed for 15 min with prepurified nitrogen) and subsequently placed in ethanol solutions of DTNB or O-DTNB. Studies were carried out at 1.0 and 10.0 mM solutions of DTNB and O-DTNB. The freshly annealed electrode was immersed in the respective solution for 2.5 h, rinsed with fresh ethanol, and mounted on the microscope cell for analysis.

Results and Discussion Adsorption of O-DTNB. Quartz Crystal Microbalance (QCM) Studies. We have employed the QCM technique to study the thermodynamics and kinetics of adsorption of O-DTNB on gold. The QCM technique allows measurement of mass changes at surfaces associated with adsorption through the accompanying changes in the resonant frequency of the quartz crystal. Thus, decreases in mass correspond to increases in frequency and vice versa. The frequency and mass changes are related by the Sauerbrey equation25

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∆f ) -Cf ∆m

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(1)

where ∆f is the change in frequency (Hz), ∆m is the mass change (ng cm-2), and Cf (17.7 ng Hz-1 cm-2) is a proportionality constant for the 5.0 MHz crystals used in this study. Figure 1 shows the frequency changes as a function of time when the resonator was in contact with the (A) 20 µM and (B) 2.0 mM O-DTNB solutions, respectively. As can be seen, following the addition of O-DTNB, the frequency decreased rapidly during the first 10 min, especially at the higher concentration, followed by a more gradual decrease until a steady state was reached (Figure 1). In the case of the 20 µM solution of O-DTNB, the final decrease in frequency was 7 Hz (Figure 1A). From this change in frequency and using eq 1, the mass deposited (through adsorption) on the electrode surface was determined to be 124 ng, which, if due only to adsorbed O-DTNB, would correspond to about of 3.5 × 10-10 mol cm-2. This value is close to that which would be expected (ca. 4 × 10-10 mol cm-2) for a compact monolayer of O-DTNB on a gold surface. When the solution concentration of O-DTNB was 2.0 mM (Figure 1B), the final decrease in frequency was 287 Hz. Following the same analysis as presented above, the mass deposited on the electrode surface was determined to be 4797 ng, which, if due only to adsorbed O-DTNB, would correspond to a surface coverage of about 1.4 × 10-8 mol cm-2. This value is not only dramatically higher (by a factor of 40) than that presented above but also well above that of a compact monolayer. This would suggest that adsorption of O-DTNB from the more concentrated (2.0 mM) solution results in the formation of a multilayer equivalent film, perhaps in the form of aggregates. In fact, differences in the topology of the films obtained upon adsorption of O-DTNB from high and low solution concentrations were evident in AFM images (vide infra). However, we should also mention that the formation of multilayer equivalent deposits (aggregates) does not imply, nor do we suggest, that those aggregates are present in solution. We are not aware of any measurements of the critical micelle concentration (cmc) of O-DTNB. However, taking as a point of reference the values for C8-C10 hydrocarbons with aromatic substituents which are of the order of 10-20 mM (in aqueous solution), we do not believe that such aggregates are present in solution. The shape of the frequency-time profile can be employed in order to study the kinetics of adsorption. In general, an adsorption process can be controlled either by transport (diffusion) or by kinetics (activation controlled), which predict time dependencies that are t1/2 and exp(t), respectively. Fits of the data to both transport and kinetic control models were attempted, and for both concentrations the fit to an activation-controlled model was significantly better than that for a transport-controlled model. The solid lines in parts A and B of Figure 1 represent exponential fits to the data (open circles) from which rate constants of the order of 0.12 and 0.49 min-1 were obtained, respectively. We have also employed the QCM technique to study the thermodynamics of adsorption of O-DTNB on gold. In the case of monolayer adsorption onto an electrode surface, the adsorption equilibrium can be described in a general fashion as26

Msol + nSads a Mads + nSsol (25) Sauerbrey, G. Z. Phys. 1959, 1555, 206. (26) Trasatti, S. J. Electroanal. Chem. 1974, 53, 335-363.

(2)

Figure 1. Time dependence of the frequency changes of a quartz-crystal resonator in (A) 20 µM and (B) 2.0 mM O-DTNB ethanolic solutions at 25.0 ( 0.1 °C. Solid lines represent fits to a first-order kinetic equation.

In order for a solution species (Msol) to adsorb (Mads) onto an electrode surface, surface sites must be available; hence the displacement of preadsorbed solvent (Sads) molecules and/or other species (e.g., supporting electrolyte ions) from the electrode surface is necessary. The equilibrium relationship between the concentration of M in the bulk solution, C*, and its concentration on the surface (Γ) is described by an adsorption isotherm. Various types of adsorption isotherms have been proposed,26,27 differing mainly in the type of adsorbate-adsorbate interactions allowed and the number of layers adsorbed. The Langmuir adsorption isotherm

βC* ) Θ/1 - Θ

(3)

(where β is the adsorption coefficient and Θ is the fractional coverage defined as Γ/Γs, where Γs is the saturation coverage), which is the simplest, describes the adsorption process when the only interaction present is that due to size. This isotherm also assumes that only a single layer (a monolayer) of adsorbate forms on the surface, hence the ability to determine a saturation coverage. The fact that coverage values derived from QCM measurements were in the multilayer regime indicated that the Langmuir model could not appropriately describe our system. There have been previous reports of self-assembling systems that give rise to coverages in excess of a monolayer. For (27) Gregg, S. J.; Sing, K. S. W. Adsorption, Surface Area and Porosity; Academic Press: London, 1982.


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Figure 3. Plot of kobs (min-1) vs solution concentration (mM in ethanol) for O-DTNB at 25 ( 0.1 °C.

obtained, which is clearly not the case for our system (at least over the range of concentrations studied), as seen in Figure 2A. An adsorption isotherm which does not require a saturation or maximum coverage was proposed by Freundlich

Γ ) k(C*)1/n

Figure 2. (A) Plot of surface coverage vs solution concentration (M in ethanol) for O-DTNB at 25 ( 0.1 °C after 90 min of deposition onto a gold electrode of a quartz crystal resonator. (B). Plot of log (surface coverage) vs log concentration of the data presented in part A. Lines are fits to two linear segments following the Freundlich isotherm. The dashed lines are the projections of the intersection of the two lines onto the surface coverage and concentration axes.

example Bard and co-workers28 have imaged, using AFM, multilayers of n-octadecanethiol on gold substrates and determined the adsorbate coverage vs time profile using QCM. The adsorption isotherm for O-DTNB deposited onto a polycrystalline gold electrode is shown in Figure 2 A. As is evident from both the amount of deposited material and the shape of the isotherm, the Langmuir model is not appropriate for this system. Deviations from the Langmuir adsorption model are often observed. These may be caused by surface heterogeneities, adsorbate-adsorbate interactions, or adsorption of more than one layer (i.e., multilayer adsorption), which is a common phenomenon in the adsorption of gases and vapors on solid surfaces.27,29 The Slyging and Frumkin adsorption isotherm

Θ ) 1/f ln(aC*)

(4)

where f and a are constants and C* is the solution concentration of the adsorbate, can sometimes be used to describe multilayer adsorption phenomena.27 However, this isotherm requires that a maximum coverage be (28) Kim, Y. T.; McCarley, R. L.; Bard, A. J. Langmuir 1993, 9, 19411944. (29) Brunauer, S. The adsorption of gases and vapors; Princeton University Press: Princeton, NJ, 1945.

(5)

where k is related to the adsorption capacity and n characterizes the energy distribution of the adsorption sites.30 The Freundlich equation does not become linear at low concentrations nor does it show a saturation or limiting value. The values of k and n may be obtained from a plot of log Γ versus log C* (bulk concentration of adsorbate). The analysis of adsorption based on the Freundlich model provides the adsorbent capacity from the intercept, and the slope (1/n) provides a measure of the intensity of adsorption. However, we also recognize that the use of this approach does not provide the level of insight that the use of the Langmuir and/or Frumkin isotherms do. A plot of log Γ vs log C* for the data in Figure 2A is presented in Figure 2B. As can be ascertained, the plot yields two straight segments with very good correlation (>0.99). Although somewhat speculative on our part, we believe that the change in slope might indicate the transition (onset) from monolayer to multilayer adsorption (or aggregate formation). In fact, the extrapolation of the intersection onto the surface coverage axis yields a value of 4.6 × 10-10 mol/cm2, which is very close to that estimated for a compact monolayer. Clearly, an increase in the surface coverage above this value is in excess of a monolayer. From the data at concetrations below 1 mM, a plot of kobs vs [O-DTNB] (Figure 3) yields a straight line with a slope (kads) of 1.7 mM-1 min-1 and a positive intercept (kdes of 0.17 min-1). This indicates that over this concentration range the desorption process plays an important part and that the system is at equilibrium. AFM Imaging of DTNB and O-DTNB Films on Au(111). To further characterize the nature of the adsorbed layer of O-DTNB on gold, especially its concentration dependence, we carried out contact mode AFM studies on Au(111) electrodes as described in the Experimental Section. Images were obtained in 0.1 M HClO4 of (30) Freundlich, H. Colloid & Capillary Chemistry; E. P. Dutton & Co.: New York, 1926.

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Figure 4. AFM imaging: (A) 1750 nm × 1750 nm image of a freshly annealed Au(111) electrode; (B) 1000 nm × 1000 nm image of a DTNB film on the Au(111) electrode, prepared by placing the Au electrode in contact with a 1.0 mM ethanolic solution of DTNB for 2.5 h; (C) 2500 nm × 2500 nm image of a DTNB film prepared by placing the Au electrode in contact with a 10.0 mM ethanolic solution of DTNB for 2.5 h; (D) 1000 nm × 1000 nm image of an O-DTNB film prepared by placing the Au electrode in contact with a 1.0 mM ethanolic solution of O-DTNB for 2.5 h; (E) 1000 nm × 1000 nm image of an O-DTNB film prepared by placing the Au electrode in contact with a 10.0 mM ethanolic solution of O-DTNB for 2.5 h; (F) 350 nm × 350 nm image (closer inspection) of an O-DTNB film prepared as in Figure 3E.

the freshly annealed Au(111) electrode as well as after modification at high (10.0 mM) and low (1.0 mM) concentrations with both O-DTNB as well as with DTNB, the latter serving as reference. Images of the freshly annealed Au(111) surface exhibited very large flat terraces as is evident in Figure 4A. After such an electrode was placed in contact with a 1.0 mM solution of DTNB, the images obtained had a noticeably grainy structure as is evident in Figure 4B. Molecularly resolved images were not attainable, possibly due to the strong interactions between the tip and the adsorbed film and other tip effects. It is important to note that the underlying structure of the gold surface is clearly evident suggesting that the adsorbed layer of DTNB is quite thin. When the adsorption of DTNB was carried out from the 10.0 mM solution, the resulting images were also quite grainy (Figure 4C), and although the underlying gold substrate surface was not as well defined, it was still evident. Thus, although there appears to be a somewhat higher coverage of DTNB, the resulting structure/topology of the deposit does not appear to depend strongly on the solution concentration of DTNB from which adsorption was carried out.

Darder et al.

Figure 5. Molecular structures of DTNB (A) and O-DTNB (B) obtained from Hyper-Chem.

Turning our attention to the case of O-DTNB, we observed a rather different concentration-dependent behavior. Figure 4D shows the image of a Au(111) surface after exposure to a 1.0 mM solution of O-DTNB. Although the Au(111) structure is still evident, the features are much less clearly defined when compared to the images of DTNB obtained under similar conditions (Figure 4B). Molecularly resolved images were also unattainable, as was the case for DTNB films, and the tip appeared to interact somewhat more strongly with these films. In stark contrast to the DTNB films, when the adsorption of O-DTNB was from the 10.0 mM solution, a dramatic change in surface morphology was evident. It can be observed in Figure 4E that the surface now has what appears to be larger aggregates of O-DTNB. Closer inspection (Figure 4F) shows that the surface is covered by regular aggregates. This is consistent with the QCM results (vide supra), which suggest the formation of O-DTNB aggregates at the higher concentrations. A careful grain-size analysis shows features of an average radius of 2.4 ( 0.2 nm. The length of fully extended O-DTNB was estimated from molecular modeling calculations (HyperChem; HyperCube, Ltd.) to be about 1.97 nm (Figure 5). The fact that the observed aggregates have an average radius of 2.4 ( 0.2 nm would suggest that these might be forming micelles. The size of O-DTNB mentioned above (Figure 5) would be qualitatively consistent with such an interpretation. Thus, both the QCM and AFM data indicate that at high solution concentrations, O-DTNB appears to form multilayer aggregates on the surface. Redox Activity of O-DTNB Layers. We have also investigated the redox properties of adsorbed layers of


Figure 6. Cyclic voltammograms at 100 mV/s in pH 7.0 phosphate buffer (0.1 M) over the potential range of -0.30 to +0.20 V for: (A) (solid line) a bare Au electrode, (dotted line) a gold electrode modified with an O-DTNB layer (from a 1.0 mM ethanolic solution); (B) a gold electrode modified with an O-DTNB layer after applying a potential of -0.55 V for 3 s.

O-DTNB. In these studies, close parallels will be drawn to the closely related molecule DTNB whose adsorption and redox behavior (of the adsorbed films) we have previously studied.10 In that study we showed that upon potential cycling past -0.55 V the nitro groups of adsorbed DTNB are transformed into the corresponding hydroxylamine which exhibited a well-behaved pH-dependent redox couple centered at -0.04 V at pH 7.0. To ascertain if gold electrodes modified with O-DTNB exhibited an electrochemical behavior similar to that previously observed for DTNB, cyclic voltammetric studies were carried out in 0.1 M phosphate buffer (pH 7.0) at 100 mV/s over the potential range of -0.3 to 0.2 V. It should be noted that in all of these studies O-DTNB was not present in solution. Figure 6 shows the cyclic voltammograms obtained for O-DTNB modified electrodes, before (A, dotted line) and after (B) the potential was held at -0.55 V for 3 s. As can be seen, before the potential was held at -0.55 V, no Faradaic processes were observed over the potential range studied for either the bare (Figure 6A, solid line) or the O-DTNB modified gold electrode (Figure 6A, dotted line). However, in the latter case, there is a significant decrease in the capacitative current as would be anticipated for an electrode covered with a low dielectric layer. When the potential was held at -0.55 V for 3 s and then scanned over the range of -0.3 to +0.2 V, a well-behaved redox couple centered at -0.04 V at pH 7.0 was observed (Figure 6B). This electrochemically triggered transformation is ascribed to the reduction of the nitro group to the corresponding arylhydroxylamine in a way analogous, and as would be anticipated, to the mechanism previously proposed for DTNB. Virtually the same electrochemical behavior was observed for gold electrodes modified with either 20 µM or 10.0 mM O-DTNB solutions. From an integration (at slow sweep rate) of the charge under the voltammetric wave,

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average surface coverage values of 2.6 × 10-11 and 4.2 × 10-11 mol/cm2 were obtained for gold electrodes modified with 20 µM and 10.0 mM O-DTNB solutions, respectively. These values are to be compared to and contrasted with those derived from QCM measurements which were 3.5 × 10-10 and 1.4 × 10-8 mol/cm2, respectively. In the case of DTNB, we had previously determined that the efficiency of the surface redox transformation (i.e., the ratio of the coulometric charge under the wave centered at -0.04 V to the charge associated with its generation by holding the potential of the DTNB modified electrode at -0.55 V) was of the order of 50%. Assuming a similar efficiency for O-DTNB, the predicted coverages for the wave at -0.04 V would be of the order of 1.7 × 10-10 and 7.0 × 10-9 mol/cm2, for modification from the 20 µM and 10.0 mM O-DTNB solutions, respectively. However, the experimentally measured values, as mentioned above were 2.6 × 10-11 and 4.2 × 10-11 mol/cm2. Although there are significant differences in both instances, for the case of the 10.0 mM solution, the difference is of the order of a factor of about 170. This could be due to either (i) desorption of the aggregates once formation of the arylhydroxylamine species has taken place on the molecules that are in direct contact with gold substrate or (ii) that only that fraction of the adsorbed O-DTNB which is in direct contact with the electrode can undergo the redox transformation. EQCM measurements on surfaces modified from the 10.0 mM O-DTNB solution exhibited only a very small (ca. 7 Hz) increase in frequency (mass decrease) upon cycling of the electrode potential past -0.55 V and generation of the arylhydroxylamine suggesting that the latter and not the former is likely responsible for the observed response. Blocking Characteristics of O-DTNB Multilayer Films. To further characterize the physicochemical properties of O-DTNB films on gold, we have carried out cyclic voltammetric studies of various redox probes including [Fe(CN)6]3- (1.0 mM), hydroxymethylferrocene (1.0 mM), and [Co(phen)3]2+ (phen is 1,10-phenanthroline) (1.0 mM) on bare and O-DTNB modified electrodes. These molecules were chosen so as to be able to assess the effects of charge (negative, positive, neutral) of the redox probe with the charge of the adsorbed layer controlled by pH given the presence of the carboxylic acid group on the O-DTNB molecule. We also explored the effects of modifying the surface with 20 µM and 10.0 mM O-DTNB solutions since the previous experiments indicated very different morphologies for the adsorbed layers prepared from such solutions. Moreover, some experiments were carried out with gold electrodes modified with decyl mercaptan (DM), which was employed as an analogue of the octyl chain in O-DTNB. Note that in all of these studies the potential excursions were never past -0.55 V where the above mentioned generation of the nitro groups to the hydroxylamine take place. Thus, the nitro groups of the O-DTNB are present. As anticipated, the permeability of the O-DTNB layer to the various probes was pH dependent. At pH 1.7, where the carboxylic acid in O-DTNB is largely protonated (the pKa of O-DTNB is estimated to be similar to that of nitrobenzoic acid which is of the order of 2.2), the electrochemical responses of all of the redox probes, for a gold electrode modified from a 10.0 mM solution of O-DTNB (Figure 7), were virtually identical to those observed with bare gold electrodes (data not shown). The pH dependence of the response was manifest at pH 7.0 where the carboxylic groups are assumed to be completely deprotonated. In this case, the cyclic voltammetric response for [Fe(CN)6]3- was significantly attenuated with

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Figure 7. Cyclic voltammograms at 100 mV/s in pH 1.7 and 7.0 phosphate buffers for gold electrodes modified by immersion for 3 h in a 10 mM O-DTNB ethanolic solution: (A) 1.0 mM [Fe(CN)6]3-; (B) 1.0 mM hydroxymethylferrocene; (C) 1.0 mM [Co(phen)3]2+.

a very large ∆Ep value (Figure 7A). We attribute such behavior to a strong electrostatic repulsion arising from the carboxylate group. The response of the electrically neutral hydroxymethylferrocene remained virtually unchanged (Figure 7B) suggesting little, if any, change in the permeability, as would be anticipated. On the other hand, the response for [Co(phen)3]2+ exhibited an increase in peak current and sharpening of the voltammetric profile (Figure 7C). We ascribe this to an electrostatic attraction between the negatively charged carboxylate group and the positively charged metal complex. That is, the immobilized layer exhibits ion-exchange properties. In fact, if an electrode that had been modified with O-DTNB and exposed to a [Co(phen)3]2+ solution was rinsed and placed in pure supporting electrolyte, the voltammetric response for [Co (phen)3]2+ was still evident, demonstrating that the deprotonated O-DTNB layer retained the metal complex. Moreover, if the electrode was subsequently rinsed with pH 1.7 buffer (where all the carboxylate groups would be protonated), all the redox activity was lost, again consistent with the above arguments. We also carried out similar studies for electrodes modified with 20 µM O-DTNB as opposed to 10.0 mM as described above. In the case of [Fe(CN)6]3-, and at pH 7.0, the voltammetric response was attenuated somewhat, but not nearly as much as in the previous case. Similarly, although there was an increase in ∆Ep it was not nearly

Darder et al.

as large as before. For hydroxymethylferrocene there was essentially no change in the behavior, as would be anticipated. For the positively charged probe, [Co(phen)3]2+, there was a slight increase in the peak current values at high pH (7.0) relative to low (1.7) pH values. Again, as before, the effects were less dramatic than those observed for electrodes modified with 10.0 mM O-DTNB. These observations would suggest that for low concentrations of O-DTNB (20 µM) the adsorption gives rise to a layer that is more permeable than that obtained at high (10.0 mM) O-DTNB concentrations, which we ascribe to the formation of surface aggregates in the latter but not the former. These results are, again, in good agreement with those derived from AFM and QCM studies as indicated above. Finally, we carried out a study of the blocking behavior toward hydroxymethylferrocene of gold electrodes modified with decyl mercaptan (DM). This material was used in order to assess the blocking effects of an alkyl chain thiol and to compare it against O-DTNB, particularly with regards to the octyl chain present in O-DTNB. For gold electrodes modified with DM (from 1.0 mM ethanolic solution for 3 h), the cyclic voltammetric response for hydroxymethylferrocene exhibited peak currents that were significantly diminished and ∆Ep values that were greatly increased when compared to the behavior exhibited at O-DTNB modified (under similar conditions) gold electrodes. This would indicate that the DM monolayer is much more tightly packed than the O-DTNB monolayer. This would be the anticipated result since in DM the alkyl chains are very close together so that chain/chain interactions are strong and dominant. In O-DTNB, the alkyl chains are significantly further apart due to the presence of the aromatic (nitrobenzoic acid) substituent so that chain/chain interactions are significantly reduced. The combination of a long alkyl chain with a short aromatic (benzoic acid) substituent could provide an environment conducive to the immobilization of enzymes with retention of activity and allowing for easy transport (permeation) of redox-active probes used as mediators. This was one of the motivations in the choice of O-DTNB as a surface modifier. In fact, we have recently shown31 that electrodes modified with O-DTNB are capable of incorporating membrane-bound enzymes such as gluconate dehydrogenase, fructose dehydrogenase, and Cyt B2 with retention of very high enzymatic activity. On the other hand, non-membrane-bound enzymes such as glucose oxidase and peroxidase did not exhibit much of a propensity to bind to O-DTNB modified surfaces as ascertained form quartz crystal microbalance measurements. Although, this represents a limited number of examples, the O-DTNB layers appear to be “membranemimetic” and we ascribe this property to their “mixedlayer” character. Conclusions 5-(Octyldithio)-2-nitrobenzoic (O-DTNB) adsorbs onto gold electrode surfaces, and the process follows the Freundlich adsorption model. The QCM technique was employed to follow the adsorption process. For solution concentrations of O-DTNB above 1.0 mM, the frequency/ mass changes are consistent with the adsorption of multilayer equivalents. AFM images of adsorbed films obtained from 1 mM and 10 mM ethanolic solutions of O-DTNB exhibited dramatically different morphologies which were ascribed to the formation of surface aggregates. (31) Darder, M.; Casero, E.; Pariente, F.; Lorenzo, E. Anal. Chem. 2000, 72, 3784.


From an image analysis, the aggregates were found to have an average radius of 2.4 ( 0.2 nm, consistent with the estimated molecular dimensions of O-DTNB. Cycling the potential past -0.55 V for adsorbed films of O-DTNB resulted in the generation of a reversible, pH-dependent, redox couple ascribed to formation of the arylhydroxylamine from the nitro group in a manner analogous to 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). For films obtained from a high concentration (10.0 mM) of O-DTNB, generation of the reversible redox couple (by scanning the potential past -0.55 V) did not appear to result in desorption of the additional adsorbed material as ascertained from EQCM data. The cyclic voltammetric profiles of [Fe(CN)6]3-, hydroxymethylferrocene, and [Co(phen)3]2+ at gold electrodes modified with O-DTNB was pH dependent, and this behavior was ascribed to the acid/base behavior of the carboxylic acid substituent. At high pH values (e.g., 7.0) where the carboxylic acid group is deprotonated, the electrochemical response of [Fe(CN)6]3was greatly attenuated and the wave was much more irreversible when compared to the behavior at low (1.7) pH. The redox response of the neutral probe hydroxy-

Langmuir, Vol. 16, No. 25, 2000 9811

methylferrocene remained largely unaffected. For the positively charged probe, [Co(phen)3]2+, the peak currents increased at high pH, and this was ascribed to the films acting as an ion exchange. Given that the morphology of the deposit as well as their hydrophobicity and acid/base properties can be modulated, these materials appear as promising candidates for the immobilization of enzymes and other biologically active materials. We are currently carrying out such investigations, and the results will be presented elsewhere.31 Acknowledgment. This work was supported by the Comunidad Autońoma de Madrid through the Grant 07M/ 0016/1999 and by the CICyT of Spain through the Grant PB98-0082 and by the Cornell Center for Materials Research (CCMR). D.D. acknowledges support by the Ford Foundation and a R. L. Sproull fellowship from the CCMR. M.D. and E.C. acknowledge support by the Comunidad Autońoma de Madrid. LA0002381

Concentration Dependence of Aggregate Formation upon Adsorption

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