Electrochemistry of Self-Assembled Monolayers of Iron Protoporphyrin

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J. Phys. Chem. B 2000, 104, 2868-2877

Electrochemistry of Self-Assembled Monolayers of Iron Protoporphyrin IX Attached to Modified Gold Electrodes through Thioether Linkage Denis L. Pilloud,* Xiaoxi Chen, P. Leslie Dutton, and Christopher C. Moser The Johnson Research Foundation, Department of Biochemistry and Biophysics, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104-6059 ReceiVed: August 5, 1999; In Final Form: January 13, 2000

Iron(III) protoporphyrin IX (Fe(III)PP) and iron(III) hematoporphyrin (Fe(III)HP) were selectively and covalently attached to dimercaptoalkane-modified gold electrodes. Reaction of their vinyl or hydroxyethyl groups with the surface-immobilized thiols produced thioether linkages, reminiscent of the heme macrocycle attachment in c-type cytochromes. Cyclic voltammetry revealed reversible electrochemistry of self-assembled monolayers (SAMs) of FePPs and FeHPs on the thiol-modified gold substrates. The surface coverage estimated from the charges transferred corresponds to 30% of a monolayer. The heterogeneous rate constant of electron transfer between the Fe(III)PPs and the gold substrate decreases exponentially with the length of the spacer, demonstrating a value of 1.0 Å-1 for the tunneling length coefficient, β. At pH 8, a linear increase of the formal redox potential (E°′) with the length of the linker was also observed. This suggests that in the film, there is a close contact between the porphyrins and the alkane SAM: the E°′ is affected by the drop of the electrostatic potential from the electrode to the surface of the alkane SAM, and also that there is a strong ion pairing of the Fe(III)PPs in the film with the anions of the solution. The E°′ of Fe(III)PPs in the SAM shows a strong and complex dependency on the pH of the solution, explained by variations in the coordination of the iron, involving hydroxyl ions, water, and eventually dioxygen molecules. Interactions of the iron with either functional groups present at the surface of the substrate or with the propionate groups attached to the porphyrin ring, do not appear to be involved in the electron-proton transfer coupling mechanisms.

Introduction Until recently, the study of metalloporphyrins in chemical and biochemical redox catalysis1,2 relied on dissolving the metalloporphyrin catalysts. Lately, metalloporphyrins have been immobilized to exploit multiple advantages: (i) The catalyst is no longer lost but can be reused. (ii) The metalloporphyrin redox state can be controlled electrochemically by securing it to an electrically conductive substrate. (iii) The catalytic activity of immobilized porphyrins can be optimized, for example, by creating a favorable environment, such as incorporation in the film of various ligands (imidazole derivatives, CO, ...), or by controlling the surface coverage to affect the interactions between adjacent porphyrins. (iv) Continuous flow reactions permit real time monitoring of reaction completion. Metalloporphyrins are most often immobilized on ion-exchange resins, zeolites, silica, clays, and polymers.1 However, it is now possible to self-assemble catalytic monolayers of metalloporphyrins,3 attaching them to solid substrates by chemically bonding functional groups of the porphyrin macrocycle to the substrate4 or by coordinating the metal to a ligand covalently bound to the support.2c-e,5 In this present work, we will explore the first option. Iron protoporphyrins are self-assembled on modified gold electrodes through the reaction of their vinyl groups with the thiols present at the surface. The selective formation of SAM of free base, metalloproto- and hematoporphyrins on quartz * Address correspondence to this author at 422 Stellar-Chance, Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, Philadelphia, PA 19104-6059. Tel: (215) 898-5668. Fax: (215) 573-2235. E-mail: [email protected].

substrate pretreated with (3-mercaptopropyl)trimethoxysilane (MPS) was recently described in terms of reactions:6

PP and HP are the protoporphyrin and hematoporphyrin macrocycles respectively, which are attached to the thiol through the R-carbon atom of the vinyl or hydroxyethyl groups. The optically transparent quartz substrate allowed study by UV/vis absorption and fluorescence spectroscopy. But to investigate the electrochemical properties of such films, we construct analogous SAMs on gold electrodes, using the same reaction:

Dimercaptoalkanes replaced the silanes as anchor for the porphyrins to the gold substrate. To our knowledge, this is the

10.1021/jp992776w CCC: $19.00 © 2000 American Chemical Society Published on Web 03/04/2000

Self-Assembled Monolayers of Iron Protoporphyrin IX first time dimercaptoalkanes are used as linkers to attach redox centers to an electrode; moreover, this is the first report of a spontaneous formation under mild experimental conditions of thioether bond through the reaction between the vinyl (or hydroxyethyl) groups of Fe(III)PP (or Fe(III)HP) and thiols.7 By cyclic voltammetry, we will characterize the FePPs linked to modified gold electrode, examining in particular the surface coverage, the film homogeneity (kinetic dispersion), the effect of the length of the linker on the rate of electron transfer (ET) between the immobilized FePPs and the electrode, and the coupling between electron and proton transfer (pH effect). Experimental Methods Chemicals and Solvents. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from the Sigma Chemical Co. (St. Louis, MO). Gold (99.99%), iron(III) protoporphyrin IX chloride (Fe(III)PP), dimethyl sulfoxide (DMSO), and (3-mercaptopropyl)trimethoxysilane (MPS) as well as the mercapto- and the dimercaptoalkanes (1,2-dimercaptoethane, 1,3-dimercaptopropane (DMP), 1,4-dimercaptobutane (DMB), 1,5-dimercaptopentane (DMP), 1,6-dimercaptohexane (DMH), 1,8-dimercaptooctane (DMO), and 1,9dimercaptononane (DMN)) were purchased from the Aldrich Chemical Co. (Milwaukee, WI), except for the 1,10-dimercaptodecane (DMD) obtained from Lancaster (Windham, NH). Fe(III)hematoporphyrin IX chloride (Fe(III)HP), Fe(III)protoporphyrin IX dimethyl ester (Fe(III)PPDME), Fe(III)mesoporphyrin (Fe(III)MP), Fe(III)coproporphyrin I (Fe(III)CP), and Fe(III)uroporphyrin I (Fe(III)UP) were obtained from Porphyrin Products, Inc. (Logan, UT) and 2-propanol (HPLC grade) from J. T. Baker (Phillipsburg, NJ). Water was purified using a Milli-Q Water System from the Millipore Corp. (Bedford, MA). Preparation of Solutions of Iron Porphyrins. The preparation of the solutions of porphyrins was previously described.6 The solutions consisted of 0.1 mM of iron porphyrin dissolved in aqueous solution of 100 mM KCl and 10 mM HEPES pH 8, containing less than 0.3% (v/v) DMSO. For FePPDME, the solution consisted of water with 1% DMSO. Preparation of the Gold Substrate. After sonication for 30 min in a detergent solution, glass slides (0.8 × 1.5 cm) were immersed in freshly prepared “Piranha” solution (4/1 (v/v) 9598% sulfuric acid/30% hydrogen peroxide) at room temperature for about 1 h, rinsed with H2O, followed by flushing with argon. Caution: Piranha solution is highly oxidizing and should be handled with extreme care. The slides were then silanized with MPS as previously described6 and immediately placed into a high vacuum evaporator (Denton, Cherry Hill, NJ). Gold was deposited onto the freshly thiolated glass by evaporation under a vacuum of 10-6 Torr at a rate of 2 Å/s until a thickness of 1500 Å was reached. The rate of deposition was monitored with a thickness monitor (Maxtek, Torrance, CA). The gold-coated slides were then used immediately for further treatment. Formation of SAM of Mercapto- and Dimercaptoalkanes on Gold Substrates. Immediately after coating with gold, the slides were immersed in a fresh solution of 2-propanol and 10 mM mercapto- or dimercaptoalkane for 5 h at room temperature. After washing thoroughly the slides with 2-propanol, they were dried with argon. Degradation of the surface of the thiolated substrate coated with dimercaptoalkanes was observed after several hours (revealed by an increase of the hydrophilicity of the surface) when the slides were left in contact with air or immersed in aqueous solution. This is due possibly to the oxidation of the immobilized thiols to sulfonates.8 Therefore,

J. Phys. Chem. B, Vol. 104, No. 13, 2000 2869 the dimercaptoalkane-coated gold slides were always used immediately for measurement or for further coating, and their immersion time in aqueous solution was minimized as much as possible. Formation of SAM of Iron Porphyrins on Prepared Gold Substrates. The gold electrodes freshly coated with dimercaptoalkanes were immersed for 5 h in the aqueous solution of porphyrin maintained anaerobic by bubbling with argon during the coating process. The slides were immersed 15 h in DMSO directly after coating. This step is essential to obtain a monolayer of iron porphyrins at the surface. The slides were then immersed in 2-propanol for at least 1 h. Cyclic Voltammetry. The reference electrode, the counter electrode, and the working electrode were respectively Ag/AgCl NaCl 3 M, a platinum foil, and the gold slide coated with iron porphyrins. The supporting electrolyte solutions were 100 mM KCl, 10 mM HEPES, pH 8, and for the pH dependency measurements 100 mM KCl, 10 mM potassium phosphate. The geometric area of the working electrode was typically 0.8 cm2. The potentials are all converted relative to the normal hydrogen electrode (NHE) (E0′ Ag/AgCl ) +196 mV vs NHE at 25 °C). Cyclic voltammograms (CVs) were recorded on a workstation BAS 100B (Bioanalytical Systems Inc., W. Lafayette, IN) at a resolution of 1 data point/mV. The third scan was used for analysis. The potential scan rate, V, ranged from 0.1 to 297 V/s. The acquisition of the data was performed without electronic filtering for V higher than 1 V/s. The values of the internal resistance of the solution ranged between 10 and 70 ohms depending on the coating of the working electrode. The internal resistance was compensated manually. Calculations of Voltammograms and Rates of Electron Transfer. The procedure for determination of standard rate constants of ET and for the linear sweep voltammetry we used is based on the work of Tender et al.9 Calculations were performed using a personal computer and a locally written program. Electron-Transfer Kinetic Theory. The behavior of cyclic voltammetry of SAM of Fe(III)PP on modified gold electrode is described by the electron transfer (ET) kinetic theory for immobilized electrode reactants based on the Marcus theory. Since this theory was previously discussed in detail,9 we will describe it briefly. For a simple, reversible ET reaction between redox centers covalently attached to the electrode, the forward and reverse half reaction rate constants, kred,η and kox,η depend on the overpotential, η. The overpotential is the difference between the applied potential to the electrode, E and the formal redox potential of the redox species, E°′ (η ) E - E°′). The dependency of kred,η and kox,η on η can be expressed by the Butler-Volmer equations:

(

kred,η ) k° exp kox,η ) k° exp

η 2kBT

( ) η 2kBT

)

(5) (6)

kB is the Boltzmann constant and k° is the standard rate constant. The transfer coefficient is assumed to be 0.5. The ButlerVolmer formulation predicts a continuous, exponential increase of the rate of ET with η. The Marcus relations also predicts an ET rate increase with η when η/λ , 1 (λ is the reorganization energy). However, the rate reaches a maximum at η ) (λ and even decreases at larger η (the inverted region). According to Chidsey et al.,10 for ET involving a metal interface, the electronic distribution about the Fermi level has to be taken into account,

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and the Marcus relations can be expressed as follows:

kred,η ) µFkBT

∫-∞∞

kox,η ) µFkBT

∫-∞∞

exp{-(x - (λ + η)/kBT)2(kBT/4λ)} 1 + exp(x)

dx (7)

exp{-(x - (λ - η)/kBT)2(kBT/4λ)} dx 1 + exp(x) (8)

x is the electron energy relative to the Fermi level, µ is the distance dependent electronic coupling between electrode and redox centers, and F is the density of electronic states in the metal electrode. The electronic coupling, µ depends exponentially on distance, d:

µ ) µ0 exp(-βd)

(9)

β is the decay coefficient for electronic tunneling and depends on the structure of the medium through which the electron tunneling occurs. The current for the first-order reaction of diffusionless electroactive species is

I ) nFA(kox,ηΓred,η - kred,ηΓox,η)

(10)

where Γred,η and Γox,η are the instantaneous surface coverage of the reduced and oxidized redox centers, respectively. Summation of Γred,η and Γox,η gives the total surface coverage (Γ). The calculations of linear sweep voltammetry were performed using eqs 7, 8, and 10. Results and Discussion Confirmation of Integrity of SAMs of Mercapto- and Dimercaptoalkanes on Gold by Double Layer Capacitance. The quality and homogeneity of the SAM coating on gold electrodes are conveniently monitored by cyclic voltammetry measurements of the specific double layer capacitance (per unit area, abbreviated CS) and compared with literature values,11,12 using eq 11:

CS )

Inta,c VA2Escan

(11)

Inta,c is the sum of the integrated currents of both the cathodic and the anodic scans (in A‚V), V is the scan rate (in V‚s-1), A is the geometric area of the electrode (in cm2), and 2Escan is the range of potential scanned (from + 0.5 to - 0.3 V, 2Escan ) 1.6 V). Porter et al.11 and Miller et al.12 model a gold electrode derivatized with n-alkanethiols as a capacitor with the Au electrode surface and the electrolyte solution forming the two conducting plates. Considering the alkane SAM as an ideal capacitor in Helmholtz theory, the reciprocal of the specific capacitance can be described:

CS-1 )

d 0

(12)

d is the distance between the plates of the capacitor,  is the dielectric constant of the separation medium, and 0 is the permittivity of free space (8.85 × 10-14 F/cm). This simple analysis views the dielectric between the electrode and the electrolyte simply as a hydrocarbon chain and neglects the

Figure 1. Reciprocal of the capacitance of gold electrodes derivatized with mercaptoalkanes (b) and dimercaptoalkanes (9) measured at 1 V/s, in 0.1 M KCl and 10 mM potassium phosphate. The lines drawn in the figure correspond to the linear best fit taking into account all data points, with the exception of the two points corresponding to n ) 2 and 3 for dimercaptoalkanes. The values of the slopes of the best-fit line for alkanethiols and alkanedithiols monolayers were 0.053 cm2/ µF per CH2 and 0.051 cm2/µF per CH2, respectively. Standard deviations are of the same size as the symbols.

capacitance of the double layer of the thiol bound to Au and of the mercapto group exposed to the solution. Figure 1 shows that CS-1 for both monolayer assemblies of mercapto- and dimercaptoalkanes increases linearly with the length of the hydrocarbon chain (e.g., the number of methylene groups, n). Numerous studies suggest these monolayer assemblies have fully extended all-trans alkyl chains packed at 30° angle relative to the surface normal.13 Assuming an appropriate thickness increase of 1.12 Å per CH2 group, then eq 12 and the slope in Figure 1 provide an estimate for the dielectric constant  of the hydrocarbon chain in these monolayers. The values of 2.4 and 2.5 for SAMs of mercapto- and dimercaptoalkanes, respectively, compare reasonably well with 2.3 for the dielectric constant of polyethylene,14 as well as 2.3 for SAM of mercaptoalkanes on gold11 and 2.7 for SAM of ω-hydroxythiols on gold12 (assuming a thickness increase of 1.12 Å per CH2 group). CS is independent of the scan rate (from 0.1 to 300 V/s) for monolayers of dimercaptoalkanes with long alkyl chains (n > 4) and for mercaptoalkanes (n > 2). This observation suggests that these monolayers are relatively free of defects, exhibiting a low permeability to the ions of the electrolyte. The shorter dimercaptoalkanes (n < 4) depart from the linear fit of CS-1 vs n (Figure 1) apparently because of formation of multilayers through the formation of disulfide bonds between the immobilized thiol groups and mercaptoalkanes in solution. These same films show an increasing CS with decreasing scan rate, indicating heterogeneity and permeability to the solution. Capacitive current also increases when the dimercaptoalkane SAM are coated with porphyrins (Figure 2) or immersed for several hours in aqueous phase. This may reflect an increased permeability of the alkyl SAM to the solution as well as to the oxidation of the thiols exposed to the surface to sulfonates, as observed recently by X-ray studies of SAM of DMO on gold.8a Preparing the SAM of Iron Hemato- and Protoporphyrins. Previous studies have shown that proto- and hematoporphyrins will physisorb on alkylthiolated surfaces.6 Sonication effectively removes noncovalently bound porphyrins from thiolsilanized quartz substrates, but degrades the gold substrates. A 15 h DMSO soak effectively removes physisorbed hemes through solvation and coordination to the Fe center through the sulfoxide oxygen.15 Residual adsorbed DMSO, seen by fluorescence spectroscopy with quartz substrates,6 is removed by immersion for at least 1 h in 2-propanol. After this treatment, the films no longer show the broad cathodic and anodic CV waves, similar to the ones observed for pure monolayers of

Self-Assembled Monolayers of Iron Protoporphyrin IX

J. Phys. Chem. B, Vol. 104, No. 13, 2000 2871

Figure 2. Cyclic voltammograms of gold electrode coated with DMH only (- - -) as well as with DMH and various iron porphyrins such as (A) Fe(III)PP, (B) Fe(III)MP, (C) Fe(III)HP, (D) Fe(III)UP, (E) Fe(III)PPDME, and (F) Fe(III)CP (s). The CVs were measured in 0.1 M KCl and 10 mM HEPES, pH 8, at a scan rate of 10 V/s. The currents are corrected for the geometrical area of the electrode.

ferrocene-terminated thiol on gold,16 indicating interactions between adjacent redox centers. Chemistry of Attaching Iron Hemato- and Protoporphyrins to Modified Gold Electrodes. The suggested formation of thioether linkage between iron proto- and hematoporphyrins and thiols immobilized at the surface of the gold electrodes was investigated by CV. Gold electrodes precoated with DMH and treated with Fe(III)PP or Fe(III)HP revealed the following redox process (Figure 2A,C): kred,η

Fe(III)PP or Fe(III)HP + e- y\ z Fe(II)PP or Fe(II)HP kox,η (13) In contrast, no detectable faradaic current was observed when gold electrodes precoated with dimercaptoalkanes were treated with Fe(III)MP and Fe(III)UP (Figure 2B,D), both of them lacking vinyl or hydroxethyl groups. The only difference between Fe(III)PP and Fe(III)MP is the replacement of the vinyl groups by ethyl groups. Concerning Fe(III)CP, a small faradaic current was observed (Figure 2F), probably due to the strong physisorption of this particular porphyrin. Also as previously observed on silanized quartz,6 Fe(III)PPDME self-assembles on modified gold with approximately 50% less coverage than for Fe(III)PP. The esterification increases the hydrophobicity of the porphyrin and decreases the character amphiphilic of the protoporphyrin macrocycle. The expected increase in physisorption apparently interferes with chemisorption. Stability and Electrochemical Reversibility of Fe(III)PP Films. Typical cyclic voltammograms of SAM of Fe(III)PPs on modified gold electrode are shown in Figures 2, 4, 5, and 7. The reduction (cathodic) and oxidation (anodic) waves are well formed and similarly shaped. They exhibit an average full peak width at half-maximum (EFWHM) of 125 mV and a cathodicanodic peak separation (∆Ep) less than 3 mV at low V (e0.2 V/s). A small ∆Ep indicates reversible ET for redox centers covalently attached to the electrode; theory predicts a ∆Ep value of 0 mV. In contrast, ∆Ep ) RT/nF (59.2 mV at 298 K, n ) 1)

Figure 3. Representation of cathodic (b) and anodic (0) peak current (A) and charge density (B) as well as cathodic full peak width at halfmaximum (C) vs the scan rate for Au/DMO/Fe(III)PP. (A) The limiting slopes of Ip (- - -) for V < 20 V/s are 22 × 10-6 C cm-2 V-1 (cathodic) and -24 × 10-6 C cm-2 V-1 (anodic). The Ip was calculated from the heterogeneous Marcus kinetics at T ) 293 K with Γav ) 25 × 10-12 mol/cm2, λ ) 0.2, 0.8, and 2.0 eV, and log(k°) ) 3.3 (s). (B) The determination of the charge densities by integrating the cathodic/anodic wave and assuming n ) 1, gives an average value of 26 × 10-6 C/cm2 (cathodic, - - -) and 25 × 10-6 C/cm2 (anodic, s). (C) Comparison between EFWMH observed (- O -) and EFWMH calculated (- 4 -) from the heterogeneous Marcus kinetics with λ ) 0.9 eV, log(k°) ) 3.3 and T ) 293 K.

is expected for an electrochemical process controlled by diffusion. The discrepancy between the observed value for EFWHM and the predicted value (EFWHM ) 90.1 mV, at 298 K, n ) 1) is explained by kinetic dispersion and will be discussed later. The monolayers are stable for at least a week if stored away from light in alcoholic solution (2-propanol). The Fe(III)PPs as SAM showed a remarkable electrochemical stability from pH 2.5 to 13.6 but rapid degradation was observed for films of Fe(III)PPDMEs at pH > 9, revealed by the irreversible disappearance of the cathodic and anodic waves. At pH > 12, an increase of the capacitive current was observed, attributed to the etching of the gold from the glass support.17 Complete removal of the gold coating was observed after 30 min of immersion of the electrodes in buffer, pH 13. Thus, a new slide was used for each measurement above pH 9 and was kept immersed in the aqueous solution for less than 15 min. Surface Coverage of SAM of Iron(III) Protoporphyrin IX on Modified Gold. The average Fe(III)PP coverage of the gold av was 25 × 10-12 mol/ slides coated with DMH or DMO, ΓFePP 2 2 cm . This corresponds to 665 Å /FePP, which means about onethird of a monolayer.18 This value was obtained from the peak currents (Figure 3A) and by determination of the charges transferred at the electrode (Figure 3B). Figure 3A shows that the peak current (Ip) increases linearly with V (for V < 10 V/s). This behavior is expected for iron protoporphyrins covalently

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Figure 5. Cyclic voltammograms of SAM of FePP on gold modified with dimercaptoalkanes at different potential scan rates, V: The FePPs are attached to the gold through DMO. The current, I is normalized by A and V. Following the direction of the arrows, V ) 0.2, 2, 20, 50, 100, 200, and 300 V/s. All other experimental conditions were the same as in Figure 2.

Figure 4. (A) CVs of FePPs attached to gold through different linkers are, according to increasing thickness: DMB, DMP, DMH, DMO, DMN, and DMD. The potential scan rate was 250 V/s. The current I is normalized by the electrode area, A. (B) Representation of E°′ (0) and ΓFePP (b) vs the length of the spacer, n. Experimental conditions were the same as in Figure 2.

attached to the electrode 19 and allows eq 14 to be used to av : estimate the average density, ΓFePP

Ian Icat p p n2F2 Γ )- ) V A A 4RT

(14)

in which n is the number of electron transferred per FePP (n ) 1), F is the Faraday constant, R is the gas constant, and T is the temperature. Equation 15 provides a parallel estimate of the surface coverage from the charges transferred at the electrode.20

Γ)

Q AVneNA

(15)

e is the charge of the electron and NA the Avogadro constant. The charge density (Q/A) was determined by integration of the cathodic/anodic waves over the potential range scanned divided by V and A. The highest surface coverage for SAMs of Fe(III)PP on gold coated with DMH and DMO was 50% lower than the density observed for SAMs of Fe(III)PPs on silanized quartz.6 Modified gold slides could not be sonicated during coating to maximize chemisorption, without damaging the substrate. Furthermore, in the case of electron-proton coupling, the current measured not only reflects the redox process but also the proton transfer. If the reduction of Fe(III)PP is associated with protonation for example, the current observed will be higher than expected for uncoupled ET. We will come back on this point when we will investigate the effect of the pH on CV. Surface coverage is linker length sensitive (Figure 4B); medium length linkers (C6, C8) read almost twice the coverage as do shorter (C3, C4) and longer linkers (C9, C10). The reactivity of the vinyl groups of the Fe(III)PPs toward the thiols immobilized at the surface no doubt depends on the hydrophobicity of the substrate and accessibility of the thiol groups at the surface. Effect of the Scan Rate on the Peak Potentials: Determination of the Rate of Heterogeneous Electron Transfer. Figure 5 shows the conspicuous increase in the splitting of the peak potentials of FePP monolayers with increasing V.

Figure 6. Standard rate constant of ET in function of the length of the spacer for gold coated with dimercaptoalkanes and Fe(III)PP. Subtraction of the formal redox potential (E°′ ) [Ep,c + Ep,a]/2) to the cathodic peak potential (Ep,c) gives the overpotential: η ) Ep,c - E°′. The rate of ET, k°, is then calculated from the Marcus equations. (A) Semilogarithmic plot of the cathodic voltammetric overpotentials vs V for the following spacers: DMD (O), DMN (9), DMO (]), and DMH (2), and calculation of η from eq 7 and 8 with λ ) 0.9 eV, T ) 293 K and from top to bottom, log(k°) ) 2.2, 2.8, 3.3, and 4.1 (s). (B) Comparison of log(k°) of SAMs of Fe(III)PP on modified gold electrodes (0) with log(k°) of SAMs of mixed monolayers of ferrocene on gold electrodes measured by CV (b) 13 and by ILIT (O) methods.24 The number of methylene groups in the spacer alkyl chain is given by n. The distance corresponds to n times 1.12 Å/CH2. In both cases, the linear fits (s) give a β value of 1.0 Å-1. For (A) and (B), the standard deviation is smaller than the size of the symbols.

For immobilized redox centers at the surface of the electrode, this behavior allows k° to be calculated21 from the splitting of the cathodic and anodic waves observed when V is increased (Figure 5). Figure 6A compares experimental η (points) as well as calculated η (curves) as a function of V from eqs 7 and 8, for SAMs of Fe(III)PP on gold modified with dimercaptoalkanes of various lengths, from DMH to DMD. For DMB and DMP, the splitting of the peak potentials was not sufficient to determine k° (14

E°′acids is the formal redox potential at low pH, when all acid/ base groups are protonated. All the results are listed in Table 1. Notice that titration curves of monolayers of Fe(III)PP and Fe(III)PPDME have similar shape. So far we have explained the pH dependency of the formal potentials of films of iron protoporphyrins as a coupling between electron and proton transfer due exclusively to changes in the metal ligation. However, other processes may be involved. For instance, propionic groups attached to the macrocycle of the porphyrins as well as the functional groups present at the surface of the substrate (thiols, sulfonates) could interact directly or indirectly with the iron and therefore influence the pH dependency of E°′. Protonation/deprotonation of the propionic groups has no effect on E°′ since upon esterification, for Fe(III)PPDME as SAM, a very similar titration curve as for Fe(III)PP as SAM was observed (Figure 8A, Table 1). Esterification of the propionates indeed affects the E°′ by increasing it by 20 mV at acidic pH to 40-50 mV at basic pH. This is in agreement with the study of the redox midpoint potential in solution of synthetic hemopeptides reported by Shifman et al.31 In this case, the Fe(III)PPs are bis-coordinated to histidines and their redox potentials show a pH dependency due the glutamates present at the vicinity of the heme. When Fe(III)PPDME replaced Fe(III)PP, a similar pH titration curve was observed (from pH 4 to 10). However, the redox potential was increased by approximately 50 mV, independently of the pH. Corroborating this result, Reid et al.32 observed at pH 7 an increase of 64 mV of the redox potential of FePPDME bound to apo-cytochrome b5 comparatively to FePP. Similarly, but for apo-myoglobin, Lim et al.33 reported an increase of 39 mV for the redox potential, also at pH 7. Acid-base reaction of the functional groups presents at the surface of the substrate, mainly thiols and eventually sulfonates, does not appear to influence the E°′ of the FePPs as SAM. Indeed, from several previous works on thin films of Fe(III)PP adsorbed on a different substrate, graphite,30 the majority show comparable pH dependency curves for E°′ to the ones shown in Figure 8A. The charge density measured for the cathodic and anodic voltammetric waves is low at extreme values of pH and shows a maximum near neutral pH (Figure 8B). This trend is also followed by the peak currents (after subtraction of the capacitive current). Assuming that the charge transferred at the electrode is proportional to the concentration of the electroactive Fe(III)PPs, the Henderson-Hasselbach plot of the cathodic charge density vs pH (not shown) gives two pKa values: 4.2 ( 0.2 and 8.5 ( 0.2.34 The n values of the plots are 0.9, consistent with the involvement of one proton per electron in the reaction. These pKa values correlate well with the pKa1(ox) and pKa2(ox), respectively, for SAM of FePP (Table 1). The low current observed at acidic pH is associated with uncoupled ET as described by eq 18. From acidic to neutral pH, the increase of current reflects a proton-electron coupling:

(H2O)(OH)Fe(III)PP + e- + H+ h (H2O)2Fe(II)PP (26) During reduction of the Fe(III)PP, the electron transfer from

the electrode to the iron and proton intake contributes both to the cathodic faradaic current observed. However, the decrease in current from neutral to basic pH is more difficult to understand. The pH titration curve (Figure 8A) and the Henderson-Hasselbach treatment of the charge density vs pH show that electron and proton are still coupled. Formation of monohydroxo complex upon reduction of the bis(hydroxo) Fe(III)PP does not explain the decrease of the current:

(OH)2Fe(III)PP + e- + H+ h (H2O)(OH)Fe(II)PP

(27)

According to eq 27, the current should remain constant from neutral to basic pH. Therefore, more complex mechanisms are probably responsible for the observed decrease of the charge density. This is another indication of the complexity of the mechanisms involved in the pH dependency of FePP as SAM, as we already explained previously. The pH dependency through variation of the ligation of the iron through water, hydroxy ions, and oxygen is probably a too simplistic explanation, and possibly interactions between adjacent porphyrins or between the iron and the substrate can play a role in the pH dependency. Conclusions Electrochemical investigation reveals not only that novel films using dimercaptoalkanes as spacers between electrode and redox centers are composed of a single monolayer, but that they also are very homogeneous, having close-packed alkyl chains impermeable to water or ions. The inverse linear dependency of double layer capacitance on the length of the chains and the dielectric constant (2.5) close to polyethylene (2.3) indicate that indeed such films are homogeneous monolayers, at least for dimercaptoalkanes with alkyl chains with n > 4. In addition, the exponential decrease of k° for ET between the chemisorbed FePPs and the electrode with the increase of the length of the alkyl chains is another strong indication that dimercaptoalkanes (n > 4) form homogeneous single monolayers at the surface of gold. The value we found for β and the reported value for ET in alkyl medium, are in agreement: 1.0 ( 0.1 Å-1. In addition, recent studies of the structure and the reactivity of film of dimercaptoalkanes on gold show that dimercaptolkanes form structured and homogeneous monolayers on gold.8 The formation of monolayers of iron(III) proto- and hematoporphyrins at the surface of thiolated gold proceeds almost certainly by chemisorption. No desorption from the surface was observed after immersion for 15 h in DMSO, or after immersion of the SAMs in acidic or basic aqueous solutions. Attachment of the metalloporphyrins to the substrate through ligation between the iron and the substrate is excluded. Imidazole (Im) was observed to bind cooperatively and reversibly to the Fe(III)PPs as SAM to form (Im)2Fe(III)PP. Moreover, carbon monoxide also coordinates reversibly to bis(imidazole)-ligated Fe(II)PPs as SAM, by replacing one of the imidazole ligand to form (CO)(Im)Fe(II)PP on both thiolated quartz6 and modified gold electrode.35 Incorporation of exogenous ligands can be very useful in principle to investigate potential and kinetic heterogeneity, since it gives more control on the coordination of the iron and this contributes to a better homogeneity of the SAM, at the condition however that they did not introduce themselves heterogeneity. We did not observe any change in the heterogeneity of the film by switching ligand, if we consider the broadening of the voltammetric waves. Imidazole ligation can possibly introduce heterogeneity, such as dispersion in the orientation of the porphyrins at the surface of the substrate. Linear dichroism of SAM of (Im)2FePP on quartz revealed that

2876 J. Phys. Chem. B, Vol. 104, No. 13, 2000 the average tilt angle vs the surface shifted from 30° to 45° when imidazole was added to FePP as SAM.6 Clearly imidazole influences the orientation of the hemes at the surface. Furthermore, Ksenzhek and Petrova30c studied the pH effect on thin films (not monolayers) on graphite of FePP alone and bound to cyanide. They observed that the use of cyanide as exogenous ligand creates a system which was more complicated electrochemically than that of pure heme, with different monocyanide complexes of iron protoporphyrins. We can reasonably compare their results with ours since the redox titration curve of the film of heme is very similar to the one shown in Figure 8A. Nevertheless, significant differences were observed between films of FePP and (Im)2FePP: (1) The formal redox potential (E°′) of FePP dropped from -0.20 to -0.25 V by adding imidazole. Imidazole titration showed similar binding constant as the one obtained by optical measurements,6 with also a cooperative binding of imidazole to the FePPs. (2) The formal redox potential of (Im)2FePP as SAM is linearly dependent on the length of the spacer (alkanedithiol) as for SAM of FePP, but this dependency is more pronounced. (3) The rate constant for electron transfer, log(k°) for Au/DMD/(Im)2FePP was found to be too high to be measured with our instrument, equal to or higher than 4.4. A detailed study of the binding of Im and CO to FePP as SAM will be the subject of a future publication. The propionates of the porphyrin are certainly not essential to the adsorption process, but nonetheless play an important role: Surface coverage of Fe(III)PPDME was observed to be 50% lower than of Fe(III)PP, on both silanized quartz and modified gold. After removal of physisorbed porphyrins, the slides coated with iron proto- and hematoporphyrins show significant faradaic current in contrast with the other porphyrins lacking the vinyl and hydroxyethyl groups. Furthermore, Theorell7c observed that both free base proto- and hematoporphyrins when heated at 100 °C in concentrated HCl solution for several hours react with cysteine in solution to form thioether linkages. In contrast, mesoporphyrin does not show any reactivity. Sano et al.7d demonstrated that hemato- as well as protoporphyrinogens readily form in solution thioether linkages with thiol compounds while no reaction was observed with other porphyrinogens such as meso-, copro-, or uroporphyrinogen. With the information collected from electrochemical and spectroscopic experiments, a model describing the SAM of FePP can be proposed. From the average density determined from the peak currents and from the charge densities, the distance, edge-to-edge, between neighboring porphyrins is 5 Å, assuming homogeneous distribution of the porphyrins at the surface. By analogy to SAM on silanized quartz,6 the porphyrin planes are probably oriented almost parallel to the substrate surface. It is clear that many questions remain unanswered, and further work needs to be done, for example, to pinpoint more precisely the nature of the binding, especially to determine whether one or both of the vinyl or hydroxyethyl groups at the positions 2 and 4 react with the thiols. The attachment of the iron proto- and hematoporphyrins to the substrate through the spontaneous reaction between the vinyl or hydroxyethyl groups and the immobilized thiols is of biological importance. In c-type cytochromes, the axial ligation between the heme iron and amino acids can be augmented by covalent binding of the heme macrocycle to cysteines by thioether linkage. We know no other reports7 of successful thioether linkages between vinyl or hydroxyethyl groups and thiols under physiological conditions and without enzymatic catalysis either in proteins, in synthetic peptides or with the metalloporphyrins alone. Indeed, even in the biosynthesis of

Pilloud et al. cytochrome c, the mechanism of reaction between the cysteine and the vinyl groups of the heme is poorly understood.36 Electrochemistry of FePP on modified gold substrate is reversible. The redox processes occur by electron tunneling through the alkane spacer, the rate constant of heterogeneous electron transfer (k°) decreasing exponentially with the length of the linker. The increase of E°′ with the length of the linker suggests that in the film, the FePPs are in close contact with the underlayer of alkane SAM and are sensitive to the electrostatic potential existing at the surface of the SAM rather than the potential of the bulk solution. The value of E°′ extrapolated at n ) 0, E°′(n)0), of -0.25 V suggests that the [Fe(III)PPs]+ in the film form ion pairs with the ions in solution. Reducing ion pairing by changing the supporting electrolyte is expected to raise E°′(n)0) toward the value of E°′ in solution (-0.2 V) as well as to decrease the slope of E°′ ) f(n) (Figure 4B), since a weak ion pairing would probably reduce the interaction between [Fe(III)PP]+ and the apolar alkane SAM. The pH dependency of E°′ of FePP as SAM is due to changes in the ligation of the metal. In the acidic and neutral pH range, the iron is coordinated to water molecules and hydroxyl ions. At basic pH, it is difficult to assign a mechanism for the proton/ electron transfer coupling. If we consider the low surface coverage of the FePPs in the film, we can assume that there is very little interaction between adjacent porphyrins. In this case, the formation of monomeric (OH)2FePPs would explain the coupling between electron and proton transfer (eqs 21 and 22). In solution, Davies37 for Fe(III)PP, Kaaret et al.38 for iron tetraphenylporphyrins, and more recently Rodgers et al.39 for porphinatoiron complexes report values of 6.5, 6.5, 4.7 for pKa1(ox), and ≈13, 10.5, and 11.0 for pKa2(ox), respectively. The possibility of formation of µ-peroxo complex between the iron centers of two proximal porphyrins and dioxygen dissolved in solution cannot be excluded (eqs 23 and 24). Even if the monolayer of FePP appears to be dilute, the film can still be composed of islands of close-packed µ-peroxo complexes of iron porphyrins surrounded by uncoated areas. This method presents several advantages for the attachment of porphyrins on solid substrate: (i) The requirements for the film formation are minimal: a solid substrate with surfaceimmobilized thiols and porphyrins with vinyl or hydroxyethyl groups stable in neutral aqueous phase. Free base as well as metalloporphyrins can be chemisorbed in a single step. After coating and washing, no further treatment of the film is necessary. (ii) The Fe(III)PPs are attached to the substrate through the porphyrin macrocycle. The availability for ligation of the two coordination sites at the iron allows to study the interaction of FePP as SAM with different ligands, and to study also the effect that these ligands can have on the catalytic activity of the SAMs of metalloprotoporphyrins. We have already observed that imidazole binds cooperatively to the iron of FePP on modified gold, similarly to silanized quartz.6 We observed also that CO binds to (H2O)2Fe(II)PP as SAM to form (CO)(H2O)2Fe(II)PP as well as to (Im)2Fe(II)PPs as SAM to give (CO)(Im)Fe(II)PP, corroborating also our previous observations on thiolated quartz.6 In the future, we intend to study the interaction of the chemisorbed FePPs with water-soluble synthetic peptides40 as well as with the apo-form of natural hemoproteins. As discussed in the Introduction, chemical catalysis can be a potential application for SAMs of metalloprotoporphyrins. Consequently, we will also investigate the catalytic activity of these films for different reactions: alkene epoxidation, hydrocarbons and alcohol oxidation, alkane hy-

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