Aqueous

© Copyright 2002 by the American Chemical Society

VOLUME 106, NUMBER 6, FEBRUARY 14, 2002

FEATURE ARTICLE Electronic Properties of Functional Biomolecules at Metal/Aqueous Solution Interfaces J. Zhang,† Q. Chi,† A. M. Kuznetsov,‡ A. G. Hansen,† H. Wackerbarth,† H. E. M. Christensen,† J. E. T. Andersen,† and J. Ulstrup*,† Building 207, Department of Chemistry, Technical UniVersity of Denmark, DK-2800 Lyngby, Denmark, and The A.N. Frumkin Institute of Electrochemistry of the Russian Academy of Sciences, Leninskij Prospect 31, 117071 Moscow, Russia ReceiVed: August 2, 2001; In Final Form: NoVember 5, 2001

Monolayers of molecules, which retain their function in the adsorbed state on solid surfaces, are important in materials science, analytical detection, and other technology approaching the nanoscale. Molecular monolayers, including layers of functional biological macromolecules, offer new insight in electronic properties and stochastic single-molecule features and can be probed by new methods which approach the single-molecule level. One of these is in situ scanning tunneling microscopy (STM) in which single-molecule electronic properties directly in aqueous solution are probed. In situ STM combined with physical electrochemistry, single-crystal electrodes, and spectroscopic methods is now a new dimension in interfacial bioelectrochemistry. We overview first some approaches to spectroscopic single-molecule imaging, including fluorescence spectroscopy, chemical reaction dynamics, atomic force microscopy, and electrochemical single-electron transfer. We then focus on in situ STM. In addition to high structural resolution, in situ STM offers a singlemolecule spectroscopic perspective. This emerges most clearly when adsorbate molecules contain accessible redox levels, and the tunneling current decomposes into successive single-molecule interfacial electron transfer (ET) steps. Theories of electrochemical ET and in situ STM of redox molecules as well as specific cases are addressed. Two-step in situ STM represents different molecular mechanisms and even new ET phenomena, related to coherent many-electron transfer. A number of systems are noted to accord with these views. The discussion is concluded by attention to one of the still very few redox proteins addressed by in situ STM, the blue copper protein Pseudomonas aeruginosa azurin. Use of comprehensive electrochemical techniques has ascertained that well-defined protein monolayers in two opposite orientations can be formed and interfacial tunneling patterns disclosed. P. aeruginosa azurin emerges as by far the most convincing case where in situ STM of functional metalloproteins to single-molecule resolution has been achieved. This comprehensive approach holds promise for broader use of in situ STM as a single-molecule spectroscopy of metalloproteins and illuminates prerequisites and limitations of in situ STM of biological macromolecules.

1. Introduction Two-dimensional films of proteins, DNA and DNA fragments, and other biomolecules at solid surfaces in contact with * To whom correspondence should be addressed. Phone: +45 45252359. Fax: +45 45883136. E-mail: [email protected]. † Technical University of Denmark. ‡ The A.N. Frumkin Institute of Electrochemistry of the Russian Academy of Sciences.

aqueous electrolyte solution are broadly important. Recognized technological perspectives, relate to (a) chromatography,1 (b) protein-membrane interactions and pharmaceutical drug delivery,2,3 (c) biologically induced corrosion,4 (d) biocompatibility of metallic implantates,5 (e) support for attached tissue cultures6 (“biofilms”), (f) analytical chemistry involving immobilized enzymes or antibodies and their substrates7,8 (glucose, dioxygen,

10.1021/jp0129941 CCC: $22.00 © 2002 American Chemical Society Published on Web 01/17/2002

1132 J. Phys. Chem. B, Vol. 106, No. 6, 2002

Figure 1. A. Upper figure: Schematic view of ex situ STM configuration. The tip-sample distance is controlled by the bias voltage Vbias and the tunneling current. Lower figure: Schematic view of electrochemical configuration. The electrode-solution potential difference is controlled relative to a reference electrode. B. In situ STM configuration. Both the sample-solution and the tip-sample potential differences are controlled by combining the configurations in A. The tip is insulated except at the very end.

etc.) or antigens9 (e.g., fluorescein), (g) DNA hybridization and screening,10,11 and (h) other areas where, broadly, biological liquids are in contact with solid surfaces.12,13 Protein film technology has been framed broadly by macroscopic concepts and statistically averaged observables, such as adsorption isotherms,12-14 spectroscopy15-18 (UV/vis, ellipsometry, internal reflection absorption and fluorescence, IR and Raman spectroscopy), microcantilever technology,19,20 quartz crystal microbalance21 (QCM), and electrochemistry.22-24 In situ scanning probe microscopies (SPM) such as in situ scanning tunneling (STM)25-27 and atomic force microscopy (AFM)25,28 and single-molecule electromagnetic spectroscopies (SMS)29-31 now offer structural and dynamic detail of individual (bio)molecules. In situ single-molecule notions have been extended to electrochemical32-34 and conductivity behavior34-36 in metallic nanosize structures. The notion in situ here and in the following refers to the adsorbed molecular films directly in contact with aqueous electrolyte solution as the natural reaction medium (Figure 1). In situ SPM systems hold perspectives in two-dimensional protein and other biomolecular film structure and function. They hold a clue to structural mapping and

visualization of adsorbed molecules in situ with unprecedented resolution but, also, new approaches to single-molecule function and spectroscopy. These are represented by AFM forcedistance relations and STM tunnel current/bias Voltage or oVerVoltage relations. In situ nanogap conductance is a related spectroscopic property.34-36 Such correlations reflect singlemolecule electronic properties analogous to single-molecule optical absorption and emission, with crucial information about energetics and electronic-vibrational coupling not otherwise offered. The complementarity of in situ SPMs to macroscopic methods offers another dimension of single-molecule characterization but also discloses limitations. A limitation is that analytical identification at the molecular level is, broadly, elusive. In situ STM/ AFM resolution of soft biological macromolecules is also at best molecular, far from the close to atomic resolution for small or intermediate-size molecules, and the number of well characterized in situ two-dimensional biomolecular systems is still quite small. Combination of in situ SPM with other techniques such as single-crystal voltammetry, X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy is therefore essential. An additional dimension, intermediate between macroscopic and molecular scale is in the context of supramolecular assemblies. Visualization and control of biomolecular function, say of immobilized enzymes, and correlation with macroscopic enzyme kinetics are here one perspective. Two-dimensional assemblies with electronic rectification and other molecular functions are another one. These areas now offer working examples and are much closer to realization than just a few years ago.37-39 In this paper, we address recent theoretical and experimental advances of in situ SPM. Focus is on in situ STM of large molecules and biomolecules, combined with interfacial electrochemical electron transfer (ET), viewed as a single-molecule spectroscopy. In section 2, we overview some approaches to single-molecule spectroscopic properties. These include singlemolecule excitation and emission, chemical dynamics, Coulomb blockade at ultra-small electrodes, and AFM. A brief overview of theoretical notions of interfacial ET is given in section 3 as a reference. In section 4, we discuss the in situ STM process, as a class of single-molecule spectroscopic phenomena and as a new case for nanoscale electrochemical ET. In situ STM is, thus, a special long-range ET process with clear analogies and differences compared with other chemical and biological longrange ET processes.40-44 Current-voltage relations offer spectroscopic information regarding energetics and electronic level broadening and clues to the STM tunneling mechanisms. This is followed by a discussion of recent data which illustrate in situ STM and single-molecule function. Section 4 is concluded by a discussion of a redox metalloprotein, i.e., the blue singlecopper protein Pseudomonas aeruginosa azurin immobilized on Au(111) surfaces.45-47 This is the first case for structural singlemolecule resolution of a documented functional redox protein under potential control. It is shown, particularly, that high-quality single-crystal electrode surfaces and complementary electrochemical and spectroscopic techniques must be combined with in situ STM. Some perspectives are discussed in Section 5. 2. Some Examples of Single-molecule Spectroscopy To put in situ STM in perspective, we consider first some other single-molecule spectroscopies developed over the past decade. 2.1. Single-Molecule Fluorescence Excitation and Emission in Condensed Phases. Single-molecule fluorescence spectra and

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Figure 2. Left: Energy diagram of in situ STM of a redox molecule. Electron tunneling from the negatively (substrate, left) to the positively (tip, right) biased electrode is via the molecular redox level. The equilibrium redox level position is shown as vacant; that is, the substrate electrochemical potential is on the positive side of the equilibrium potential. Fluctuations in the nuclear coordinate(s) q take the vacant redox level close to the Fermi level of the negatively biased electrode where interfacial ET occurs. The occupied level is trapped below the Fermi level of the positively biased electrode by further vibrational relaxation. Renewed thermal activation transmits the electron to the positively biased electrode in the overall two-step process. An analogous sequence of events proceeds when the substrate potential is on the negative side of the equilibrium potential and the redox level at equilibrium is initially occupied and located below the Fermi level of the positively biased (right) electrode. Middle: Nuclear potential (Gibbs free) energy surfaces corresponding to the level configurations to the left. Right: Analogous three-level potential surface configuration corresponding to resonance Raman scattering or hot fluorescence, with absorption of a photon of frequency ν and emission of a photon of frequency ν′ < ν.

excited state lifetimes have provided detailed information about local molecular environments of large organic molecules at high dilution in optically transparent matrices both at cryogenic and room temperatures.30,31,48-51 Optical excitation by laser beams of micrometer lateral extension are focused on resonance with wings of inhomogeneously broadened absorption bands where few molecules are represented. Spatial resolution is thus in the submicrometer range, but optical resolution is at the singlemolecule level. Important data are (a) bursts of fluorescence after laser illumination, (b) emission spectra with singlemolecule photon distributions, (c) single-molecule fluorescence lifetimes, and (d) fluorescence tracking to disclose structural and time fluctuations caused by the local (nanoscale) environment. Single-molecule spectroscopy can resolve molecular components in inhomogeneously broadened spectra, caused by local environment distributions in space and time. Distributions of fluorescence lifetimes can be disentangled, and correlations between single-molecule fluorescence lifetimes and peak frequencies can be constructed. Notable is stochastic time evolution of single-molecule features, spanning time ranges from subseconds to thousands of seconds, caused by configurational fluctuations or by molecular hopping between different environments. This perspective carries over to in situ STM at the solidliquid interface. Vibrationally resolved single-molecule emission spectra have also been obtained.51 The laser is in resonance with the 0-0 transition, and vibrational excitation is monitored by the shift between the laser frequency and the emission peak frequency in a resonance Raman scattering mode. This three-level feature has an analogue in in situ STM of redox molecules (Figure 2).52-54 The ground electronic state in STM is the initial vacant or occupied state of an adsorbed molecule, which is restored after electronic transmission through the molecule. Other single-molecule spectroscopic perspectives relate to photochemical hole burning, site selective spectroscopy, spectral diffusion, and single-molecule magnetic resonance.30,31,49,50,55 2.2. Single-Molecule Chemical Dynamics. Real time enzyme reactivities, resolved at the single-molecule level in aqueous solution or gels, are important cases for chemical reaction dynamics, with a bearing on in situ STM. Two cases are illuminating. Resonance energy transfer and polarization anisotropy were used to investigate the Ca2+-dependent DNA-

cleaving enzyme staphyloccal nuclease, doubly labeled with a donor and an acceptor group or singly labeled.56 The singly labeled enzyme could also be combined with labeled oligonucleotide substrate to investigate enzyme-substrate interactions. Fluctuations in donor and acceptor fluorescence could be assigned to conformational fluctuations in the donoracceptor distance. The duration of the substrate acceptor emission signal reflects the duration of the enzyme catalyzed chemical reaction. Equally detailed mapping of single-molecule enzyme action is provided by a study of the enzyme cholesterol oxidase.57 This enzyme, E, contains flavine adenine dinucleotide (FAD) naturally fluorescent only in the oxidized state.57,58 The enzyme catalyses oxidation of cholesterol by dioxygen to the ketosteroid, followed by isomerization of the latter. Single-molecule FAD emission follows an on-off pattern, directly reflecting the cyclic enzyme oxidation and reduction

E-FAD + S a E-FAD-S f E-FADH2 + P E-FADH2 + O2 a E-FADH2-O2 f E-FAD + H2O2

(1)

where S is the substrate and P is the product. Each blinking corresponds to a single molecular turnover. The autocorrelation function of the single-enzyme population in a given redox state could be determined. This was previously possible only by molecular dynamics simulation. With such molecule-based information, stochastic time evolution of the enzyme activity could be characterized, extending to both static rate heterogeneity among individual enzyme molecules and fluctuations of the single-molecule reaction rates. In situ STM of redox molecules in principle offers comparable spatial and electronic resolution but is far from the time resolution of single-molecule emission spectroscopy and chemical reaction dynamics. 2.3. Approaches to Single-Molecule Interfacial Electrochemical Electron Transfer. Detection of interfacial singlemolecule reactivity has been achieved. Lu and Xie could resolve single-molecule emission spectra of photoexcited cresyl violet adsorbed on indium tin oxide (ITO).59 Subnanosecond fluorescence decay could be assigned to interfacial ET to the ITO conduction band. Single-molecule fluorescence decay displays

1134 J. Phys. Chem. B, Vol. 106, No. 6, 2002

Figure 3. Single-electron current-voltage staircase of [(trimethylammonio)methyl]-ferrocene-ferrocinium at a Ir-Pt 2-3 nm ultramicroelectrode. From ref 62, with permission.

exponential kinetics but 40 different molecules spanned a 7-fold lifetime variation, disclosing molecular stochastic effects. Singlemolecule electrochemical detection has also been based on the scanning electrochemical microscope.60,61 A few molecules (ultimately a single molecule) are trapped in a 10-20 nm pocket under a SPM tip squeezed against a substrate electrode. Singlemolecule currents cannot be directly detected, but if electrochemical conversion of a molecule at the tip is followed by reconversion at the substrate, the current is amplified by many orders of magnitude. Oxidation of a ferrocene derivative (trimethylammonium-methyl-ferrocene) at a Pt-Ir tip followed by reconversion to ferrocene at ITO substrate has illustrated this. Current-time records over several hundred seconds, and statistical analysis could, further, identify 0.2 pA individual molecule fluctuations in the ultramicroscopic solution space under the tip. Other interfacial electrochemical ET systems have shown single-electron tunneling (SET) and Coulomb blockade effects.62 Electrochemical Coulomb staircases are a new phenomenon but a theory of electrochemical SET was reported earlier.63 Currentvoltage characteristics of [(trimethylammonio)methyl]-ferrocene/ ferrocinium in a two-interface ultramicroelectrode system (2.5 and 3.2 nm) was found to exhibit a staircase shape,62 with subfemtoampere currents (Figure 3). The ultrasmall size of the electrodes means that the single-electron charging energy e2/ 2C, where e is the electronic charge and C is the electrode capacitance, exceeds kBT already at room temperature, with kB being Boltzmann’s constant and T being the temperature. A broadly characterized system class with quantized capacitance charging is nanoscale gold clusters covered by variablelength organic thiolates.64-67 Differential pulse voltammetry, double layer capacitances, and charge-transfer resistances show fine-structure corresponding to single-electron charging, with peak separations, ∆V

∆V )

ze CCLU

(2)

CCLU is the capacitance of a single cluster, and the integers, z, represent charging by successive single-electron transfer. The SET behavior of the clusters is illuminated also by conspicuous staircase STM current-bias voltage relations66 (83°K, ultrahigh vacuum environment). The STM and electrochemical capacitive

voltage steps were, moreover, similar. Charge transfer of variable-size, monodisperse metallic clusters thus ranges from single-cluster SET, via single-electron charging in assemblies of clusters, to almost bulk electronic double layer charging. Electrochemically based quantum conductance, in an aqueous electrolyte environment, was studied by Tao and associates.34,35 Ultrathin “nanowires” of copper and gold could be prepared by electrochemical etching with parallel conductance monitoring. Diameters of a few atoms equivalent to the electronic mean free path exhibit stable conductances roughly in integral multiples of the fundamental quantum conductance 2e2/h,68 where h is Planck’s constant. The nanowire conductance also exhibits electrochemically based quantum effects, in the form of molecule-specific adsorption features. These are correlated with the adsorption energy (2,2′-bipyridine and adenine) and ascribed to scattering of the electrons from adsorbate molecules or a change in the atomic wire configuration on adsorption. These observations are other new cases for electronic singlemolecule spectroscopy. Nanometer-scale wire and gap fabrication are also important frames for single-molecule trapping and single-molecule electronic properties in a vacuum and solution.34-36,69,70 2.4. Atomic Force Spectroscopy and Single-Molecule Reactivity. Some observations on AFM put electrochemical in situ STM in another perspective. AFM of adsorbed biomolecules at solid surfaces in contact with an aqueous solution does not involve the requirements for electrochemical potential control as STM, and in some respects, image interpretation is even facilitated by the aqueous environment.26,71-73 AFM does not usually reach the same level of adsorbate image resolution as high-resolution STM26-28 but offers other details regarding single-macromolecule structure and dynamic features. The following observations can be compared with STM addressed in section 4. Atomic force spectroscopy has become an approach to singlemolecule unfolding of tertiary structures of proteins,74,75 polysaccharides,76-78 and polyalcohols.79 Mapping of elastic properties of large protein complexes such as photosystem II80 also belongs to this category. Force-induced unfolding is the single-molecule limit of biomolecular unfolding and complementary to macroscopic approaches based on thermally or chemically, rather than mechanically induced denaturation. Comprehensive data representing single-molecule protein unfolding are available for titin and related molecules.74 These are large muscle proteins composed of units of immunoglobin, Ig, with 89 to 100 amino acids folded in β-barrel structures. Individual domains can be unfolded successively by pulling AFM forces. Peak forces, of 150-300 pN, cause a lowering of the activation free energy of the unfolding process with sequential chain length increases of ≈30 Å and potential widths corresponding to only a single amino acid. This has been viewed as the extension needed to reach the transition state and is indicative of highly cooperative single-domain unfolding. Unfolding rates can be assigned, and refolding can be controlled by relaxing the unfolded molecules under force control. Comparison with chemically induced macroscopic unfolding, finally, assigns converging unfolding rate constants at zero force or denaturant concentration. Comparable detail has been obtained for polysaccharides.76-78 Chemical and mechanical deformation of DNA is important in genetic information transfer, binding to proteins, etc. Doubleand single-strand DNA stretching, and unwinding in aqueous salt solution, have been induced by AFM and related force spectroscopy.81-84 Targets have been whole DNA molecules

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with mesoscopic contour lengths, 15-20 µm or ≈5 × 104 base pairs,81-83 and 10-30 base pair oligonucleotides.84 DNA can be stretched by elastic forces to 1.7 times its contour length at a force of 70 pN.81,82 Longer stretching involves conformational changes in the carbohydrate units. Elastic DNA stretching is controlled also by hydration, ionic strength, and chemical crosslinking, framed by multistate models, with ladder conformations and partial melting as intermediate states. AFM of 10-30 base pair oligonucleotide duplexes have illuminated unwinding to single strands84 by covalent attachment of complementary strands to the tip and substrate. “Zipper-like” unwinding occurs in the 20-50 pN force range, correlated with the number of base pairs, temperature and ionic strength, and macroscopic thermal dissociation rates.84 2.5. Force-Spectroscopy of Ligand-Receptor Interactions. Formation and dissociation of biological ligand-receptor complexes are cases of single-molecule biological reactiVity, quantified by in situ AFM. Avidin-biotin (protein-small ligand)85,86 and antigen-antibody (protein-protein)87-89 association and dissociation have been characterized. Mapping of the forces between biotin (receptor) covalently immobilized on polymer beads and avidins immobilized by tip adsorption provides distributions of single protein-receptor pair interactions.85,86 These correlate with the unbinding enthalpy and binding potential widths in the structural range of the proteinbinding pocket. Single-molecule pair interactions also emerge from AFM of antigen-antibody complexes between fluorescein (antigen) and wildtype and mutant Ig fragments (antibody) on gold-plated substrates. The pair interaction force, F, correlates with the macroscopic dissociation rate constant, koff, as

koff(F) ) k0off exp

( ) Fxβ kBT

(3)

where k0off is a constant and xβ represents the difference between the protein-ligand distance in the bound and transition states. AFM has, finally, been brought to approach single-macromolecule chemical reactivity, but this notion has been assigned to different levels of resolution. Transcription of DNA to RNA by E. coli RNA polymerase on mica, followed by in situ AFM, is close to molecular resolution, and processing of individual DNA molecules through the enzyme could be imaged in real space and time.90 Single-molecule enzyme activity and inhibition of lysozyme on mica91 could be monitored in AFM as reversible height fluctuations in the presence of substrate and inhibitor molecules, respectively. Hydrolysis of immobilized phospholipid catalyzed by phospholipase A2 in solution92 is, finally, a case for AFM of enzyme activity, where the substrate is immobilized and the solute enzyme is freely mobile. Lateral resolution here is, however, in micrometer rather than nanometer ranges. 3. Notions of Interfacial Electrochemical Electron Transfer 3.1. Some General Observations. In situ STM of molecular adsorbates resembles in crucial respects electrochemical interfacial ET, and some theoretical notions on physical electrochemistry constitute part of our discussion of in situ STM. It was noted that chemical and biological ET in homogeneous solution has reached high levels of theoretical and experimental characterization.40-44 Similar detail in ET between molecular redox centers and metal, semiconductor, or semimetal electrodes has been more elusive. Fundamental similarities and differences from ET in solution were recognized early.43,44,93-96 Among

the former, the nature of the ET process as a quantum mechanical transition between two electronic-vibrational states, free energy relations, the notion of “work terms” and “double layer effects”, and the view of ET as a tunneling process96,97 are the most important. Particularly, the Brønsted relation between the rate constant and the reaction free energy of elementary charge transfer (proton transfer) processes in solution98 is equivalent to the Butler-Volmer relation between the electrochemical current, j(η) and the overvoltage η:99

( )

j(η) ) j0 exp -

( )

Reη G0q ; j0 ∝ exp kBT kBT

(4)

This is the simplest electrochemical free energy relation and carries over to in situ STM. j0 is the exchange current. The activation Gibbs free energy, G0q, incorporates all of the nuclear activation features. R () R(η)) is the electrochemical transfer coefficient

R ) -kBT[d ln j(η)/d(eη)]

(5)

A conspicuous difference is that the electrode incorporates a continuous manifold of electronic states, whereas only a single pair of states is involved in homogeneous ET. Consequences of this, such as the absence of an inverted Gibbs free energy region, are recognized.43,44,100 A second difference relates to the nature of the electronic wave functions. These are spatially localized for ET in solution but two-dimensionally delocalized in electrochemical ET. The electrochemical tunneling factor is therefore conveniently approached by invoking electronic densities rather than individual level wave functions.101,102 Theoretical and experimental detail of electrochemical ET has been fraught with the complexity of the nonuniform interfacial environment. New well characterized systems are, however, becoming available, with variable-length pure and functionalized alkanethiolates and related molecules self-assembled on electronically “soft” metals, mostly gold in focus.103-112 Interfacial ET of small redox molecules such as ferrocene exhibits clear features of electron tunneling across the alkylthiolate films. This warrants new theoretical approaches based on electronic density functional theory for the metal surface and statistical mechanical frames for the reacting molecules and the solvent. Combined with in situ SPM, molecular-based views of the electrochemical interface are therefore approaching a new level. Electrochemical ET of adsorbed biological macromolecules, redox metalloproteins in particular,113-115 was long regarded separately from recent achievements of physical electrochemistry such as single-crystal electrode procedures, surface spectroscopies, etc. “Soft” macromolecules such as single- and multicenter redox proteins are, thus, vulnerable to strong electric fields, environmental anisotropy, etc. Focus in interfacial bioelectrochemistry has therefore been different and directed toward conditions for robust voltammetry. Diffusive reversible ET patterns are broadly observed for cytochromes, blue copper proteins, and iron-sulfur proteins, whereas peroxidases, blue oxidases, iron-sulfur enzymes, and flavoenzymes are examples of macroscopically mapped monolayer enzyme electrocatalysis.113-116 Well-defined monolayers of redox proteins at pure or modified electrode surfaces have opened options for methods other than cyclic voltammetry, such as electrochemical impedance,47,117-120 electroreflectance spectroscopy,119,121-123 and surface enhanced resonance Raman spectroscopy,124-126 enabling characterization of both adsorption and interfacial ET.

1136 J. Phys. Chem. B, Vol. 106, No. 6, 2002 is the Fermi function, and F() is the electronic level density at the energy :

f() ) {1 + exp[( - F)/kBT]}-1; F() ) (Vme3/2/p3π3)x2( - b) (7) where me is the mass of the electron, b is the bottom of the conduction band, 2πp is Planck’s constant, and W(;η) is the rate constant (s-1) for ET from the level . Equations 5-7 are a general basis for a range of specific systems. These extend to metal, semiconductor, semimetal, and superconductor electrodes and to harmonic and anharmonic electronic-vibrational interactions, nuclear tunneling, etc.43,44,129,130 The following form applies broadly: Figure 4. Electronic energies (left) and potential (Gibbs free) energy surfaces for interfacial electrochemical ET. Cathodic process.

Introduction of theoretical notions based on the overpotential dependence of the rate constants, electron tunneling, etc. are therefore warranted. Bowden et al.127 addressed, for example, monolayers of horse heart cytochrome c electrostatically adsorbed on self-assembled ω-mercaptoalkanoic acids on polycrystalline gold. Niki et al.122,123 reported rates for ET between cyt c and gold electrodes across variable-length ω-mercaptoalkanoic acids by electro-reflectance spectroscopy. The electrostatic nature of the surface interactions was substantiated by ionic strength and pH effects. Tunneling rates displayed exponential distance decay for carbon chain lengths longer than eight CH2 groups. Similar observations based on potential jump techniques and surface enhanced resonance Raman spectroscopy have been reported.126 A comprehensive study based on several interfacial techniques has disentangled adsorption and interfacial ET features of Pseudomonas aeruginosa azurin at single-crystal gold electrodes covered by variable-length alkylthiolates.47,118 The protein is here adsorbed hydrophobically. This will be in focus in section 4. Multicenter redox metalloenzyme voltammetry has, finally, been brought to a level where intramolecular ET and other mechanistic features can be distinguished. Studies by Armstrong and associates have, for example, identified electrochemical analogues of peroxidase catalytic cycles and disclosed interfacial ET routes through the ET centers of fumarate reductase in the electrocatalytic reduction of fumarate.23,116 Observations such as these warrant stronger efforts toward introducing new physical electrochemical methodology and new theoretical frames, also in interfacial bioelectrochemistry. 3.2. Some Theoretical Frames of Electrochemical and Bioelectrochemical ET. Figure 4 illustrates the fundamental electrochemical (cathodic) ET process.43,44,100,128 The current is composed of contributions from all electronic levels of the (here metal) electrode. The redox level is strongly coupled to the environment, with the initially vacant level in equilibrium well above the Fermi level of the electrode, F. Configurational fluctuations lower the level to match populated levels in the electrode where ET proceeds, with subsequent electron trapping on the molecule. In the diabatic limit of weak electrodemolecule interaction, the current density is, broadly independently on the system properties:

j(η) )

∫d F()f()i(;η);

i(;η) ) eCox(z*)∆zW(;η) (6)

Cox(z*) is the concentration of oxidized molecule at the distance z* from the surface, and ∆z is a narrow z range around z*. f()

{ x

}

ωeff [ER + eη - ( - F)]2 exp 2π 4ERkBT

W(;η) ) κel(;η)

κel(;η) ) [TA(;η)]2

4π3 ,1 ERkBTp2ωeff2

(8)

where ER is the nuclear reorganization Gibbs free energy, and ωeff is the effective vibrational frequency of all the nuclear modes which contribute to ER. κel(;η) is the electronic transmission coefficient, the most important feature of which is the electron exchange factor, TA(;η), which couples the level  with the molecular redox level. Levels around the Fermi level dominate in broad overpotential ranges at metal electrodes, where j(η) takes the quadratic form

[

j(η) ≈ j0 exp -

(eη)2 eη 2kBT 4ERkBT

]

j0 ) j(η ) 0) ) [πeF(F)/p]x2CoxCredkBT/ER ×

(

∆z[TA( ≈ F;η ) 0)]2 exp -

)

ER (9) 4kBT

and Cred is the concentration of the reduced molecule. An alternative form is101,102

[

j(η) ) (πe/2p)δ∆zCox[V(z*)]2M(η) exp -

]

(ER + eη)2 4ERkBT (10)

where δ is a narrow energy range around F, V(z*) is the physical interaction between the electrode and the molecule, and M(η) is the square of the electronic density oVerlap. This form is suited to incorporate interactions between the metal electrode and the interfacial environment. An approximate simple form of M(η) is

M(η) ≈ A exp[-βmet(a - jz)] + B exp[-βmol(a - jz)]

(11)

where a (≈z*) is the distance of the molecular center from the electrode surface and jz ) jz(η) is the front of the metallic electron density. βmet and βmol (Å-1) are the distance decay factors of the electronic densities of the metal and the molecule, respectively, and A and B are weakly distance and electrode potential dependent quantities. Equation 11 discloses lability of the electronic factor induced by variable electrode charge,101,102 but βmet often significantly exceeds βmol, and tunneling is still dominated by the molecular term in eq 11.

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The formalism above refers to the diabatic limit. The validity conditions for this limit are different from ET in homogeneous solution because of the manifold of electronic levels43,131

κel()F()kBT , 1

(12)

The opposite, adiabatic limit is equally appropriate to both electrochemical ET and in situ STM. There are two approaches to this limit. In one approach, the focus is on multiple transitions between two manifolds of electronic levels representing the reactants’ (R) and products’ (P) states.131 The potential surfaces, spanned by the nuclear coordinate(s) q, are

UR(;q) ) UR(q) + ; UP(′;q;η) ) UP(q;η) + ′

(13)

 and ′ are energies for different electron distributions, counted from the energy corresponding to the Fermi distribution. Transitions are described by master equations, with simple solutions in the two limits given by eq 12 and the opposite inequality. The adiabatic current is

jad(η) ) eCox∆zWad(η); Wad(η) )

[

]

ωeff Gqad(η) exp 2π kBT

(14)

The activation free energy is determined by the maximum of the potential free energy surface

{ [

Uad(q) ) -kBT ln exp -

] [

]}

UR(q) UP(q;η) + exp kBT kBT

(15)

The quadratic form in eq 8 or 9 is, however, often appropriate. The electronic factor, κF, is thus absent in the adiabatic limit. The other approach was initiated by Hush,132,133 who recast the electronic manifold into a single effective orbital. This resembles work much later based on chemisorption theory.134 Chemisorption-based formalism applies both to electrochemical135 and electrocatalytic ET processes between a reacting molecule and a chemisorbed atom electronically broadened by the metal.136,137 Such views have also been central to STM. Recent work on adiabatic electrochemical ET based on chemisorption theory was initiated by Schmickler135 and followed by others.138-141 Common to these approaches is the construction of potential surfaces such as eq 16 based on the width of the redox level, ∆. The following form139 is illuminating:

Gqad )

[

]

(ER + eη)2 ∆ ∆2 ln 2 + 4ER 2π ∆ + (E + eη)2 R

(16)

and shows that the electronic coupling, ∆, reduces the diabatic activation barrier (∆ < ER) but recovers the quadratic forms at very small ∆. Electronic diabatic effects appear as electronhole pair excitations which arouse electronic “friction”. The transmission coefficient resembles the transmission coefficient associated with multiple single-level transitions, but the two views are different. The temperature-dependent number of contributing metallic levels is, for example, not inherent in electron-hole pair excitation. Views of adiabatic electrochemical ET carry over to in situ STM. The most important observation is that features of bias and overvoltage spectroscopy are dominated by the nuclear dynamics, but an apparent tunneling factor still appears as the number of contributing levels. 4. In Situ STM As an Interfacial Single-Molecule Spectroscopy In situ STM involves interfacial ET of adsorbed molecules and can be regarded both as a single-molecule spectroscopy

and a class of nanoscale electrochemical processes. We provide some theoretical notions and note cases of in situ STM of redox adsorbate molecules. Emphasis is on tunnel mechanisms when low-lying molecular levels approach the Fermi levels of the substrate and tip and thermally activated ET channels open. 4.1. Electronic Specificity and Spectral Features of Surface Adsorbates. 4.1.1. Electron Tunneling through Liquid Water Layers. In situ STM has been established as a high-resolution technique for topography and dynamics of the electrochemical interface. The resolution of surface reconstruction,142-144 metal underpotential deposition,142,143 adsorption,145-156 phase transitions,144,154,155 and adsorbed biological macromolecules45-47,157-161 ranges from atomic to mesoscopic. This qualifies in situ STM as a single-molecule spectroscopy. Transfer from ultrahigh vacuum (UHV) or air (ex situ STM) to aqueous environment involves more profound technological and fundamental changes than for AFM. The central external parameter of ex situ STM is the bias voltage between the substrate and tip. Electrochemical in situ STM requires independent control of the substrate and tip electrochemical potentials (Figure 1). An additional external parameter is therefore the voltage difference between substrate and solution, expressed by the overvoltage of the substrate electrode.26,162-164 The second major difference is the static and fluctuational effects of an assembly of water molecules in the tunnel gap. Static effects arise from electronic interactions with the electronic solvent polarization and pseudopotential forces. Other effects are associated with inertial polarization fluctuations. Both effects enhance electron tunneling compared to vacuum.164 The following enhancement factor for randomly fluctuating barriers of width a and average height Uav(z) was derived early:165

(

2me2V02l Γ ) exp Γ0 p2

∫0a

dz

x2me[Uav(z) - ]

)

;

V0 ) x〈[U(z) - Uav(z)]2〉 (17)

where l is the correlation length of the barrier height fluctuations, me is the mass of the electron, and V0 is the mean deviation from Uav(z). The ratio between the tunneling amplitudes through the fluctuating barrier, Γ, and the average barrier height, Γ0, always exceeds unity and is one reason for common observation of lower STM barriers in solution than in a vacuum.164 Such views have been used as a frame for noise analysis.166,167 Inertial polarization fluctuations superimposed on a rectangular barrier of height U, for example, recast eq 17 in the form167

(

)

πmekBTcλ Γ ) exp ; c ) ∞-1 - s-1 2 Γ0 pU

(18)

where the temperature and polarization correlation length, λ, appear explicitly. Solvent structural fluctuations have been advanced in studies of bias potential variations across the potential of zero charge (pzc).168 Tunneling through layers of water molecules has been addressed theoretically by schemes where the time dependent Schro¨dinger equation was solved in the fields of the water molecular assemblies preconfigured by molecular dynamics computation.169-171 The electron-water interaction holds a balance between repulsive interactions with the oxygen atoms and attractive interactions with the hydrogen atoms and the electronic polarizability. This leads the electronic density to

1138 J. Phys. Chem. B, Vol. 106, No. 6, 2002 expand three-dimensionally, analogous to electron tunneling in redox metalloproteins and other long-range ET in homogeneous solution. Instantaneous water orientations are crucial and induce both orders of magnitude variations and resonance-like features in individual tunneling routes. 4.1.2. Tunneling through Atomic and Molecular Adsorbates. Theoretical approaches to STM of atomic and molecular adsorbates include the adsorbate-substrate electronic structure and the tunneling current. The former rests on jellium172 or band structure calculations173 and molecular electronic structure calculations. Most approaches to the current rest on perturbation theories, following the formalism of Bardeen174 and Tersoff and Hamann175 and of adsorption by Newns and Anderson134 and Lang.172 This led early to the recognition that the electronic structure rather than molecular shape is imaged.172-177 Tunneling currents are mediated by orbitals which may take significant values also between adsorbate atoms. The currents are also determined by the imaging conditions, and different contrasts emerge as the bias voltage bring different HOMOs and LUMOs close to the two Fermi levels. One illustration of the electronic features is the adsorption of porphyrin on Au(111) modified by preadsorbed iodide in solution.178 Porphyrin is imaged at low currents, but iodide is imaged underneath at high currents. The porphyrin layer reappears on current reversal, showing that the difference is rooted in electronic effects and not in desorption or other irreversibility. Investigations of adenine and functionalized longchain alkanes on highly oriented pyrolytic graphite (HOPG) in a vacuum have provided theoretical frames for such observations and formal conditions for STM contrast behavior,179-181 on the basis of tight-binding and extended Hu¨ckel theory. The substrateadsorbate wave functions at the energy , φsyst(), was recast as a linear combination of the adsorbate, φads, and substrate continuum wave functions, ψ(′)

φsyst() ) aads()φads +

∫ d′ b(′) ψ(′)

(19)

where aads() and b(′) are mixing coefficients. The current is obtained by coupling the tip wave functions, φtip(′′) perturbatively to the broadened adsorbate states

itunn ∝

∫

d′

∫

d′′

[T′,ads]2[Tads,′′] (ads + Λads,substr)2 + [T′,ads]2

(20)

where ads is the adsorbate energy, Λads,substr is the adsorbate energy shift, and T′,ads and Tads, are the electron exchange factors for adsorbate coupling to the tip and substrate, respectively. Equation 20 reflects the superexchange nature of the STM process. Bright contrasts are expected as adsorbate energies approach resonance with the Fermi energy, ads f 0. The same atoms can therefore give different contrasts. For example, N6, N7, and N9 in adenine in given orientations on HOPG do not appear in STM, whereas N1, N3, and all of the carbons do. This can be assigned to the atomic LUMO coefficients and the energy denominators. Sulfur, nitrogen, alkene and alkyne multiple bonds, and iodine give brighter contrasts than the methylene backbone, oxygen, and the other halogens darker contrasts. Functional group contrasts can be related to their ionization potentials, indicative of hole transfer. These studies have not addressed the solvent polarization, which would modify the adsorbate charges and energetics, but approaches to the latter are available,136,137 offering comparison of molecular adsorbates ex situ and in situ.

4.2. In Situ STM of Redox Molecules as a Single-Molecule Spectroscopy. 4.2.1. Mechanisms and ET Scenarios of Redox Adsorbate in Situ STM. We address next some theoretical expectations when adsorbed redox molecules are probed by in situ STM52-54,182-185 and approaches to in situ STM spectroscopy and single-molecule electrochemical ET are offered. Combination with in situ AFM of the same potential controlled target molecules would provide complete mapping of both electronic and topographic adsorbate properties. The presence of two electrodes, moreover, discloses important differences from single-surface electrochemical ET, even to the extent of new interfacial ET phenomena. Real system data, which illuminate these expectations in varying degrees of detail, will be addressed in sections 4.3 and 4.4, but their number is small. The notions could, therefore, also be a heuristic frame in new system design. Figure 2 shows schematically the in situ STM/ electrochemical system.52-54,183-185 A molecule with a discrete electronic redox level is located in the gap between substrate and tip, both represented by continuous distributions of electronic levels. These are populated up to the Fermi levels, FL (substrate) and FR (tip), which are mutually shifted by the bias voltage Vbias. All energies are referred to FL. Other representations of the finite-size tip are also available.53,175 The redox level is electroactive and can exist in an oxidized, ox, and a reduced, red, valence state. The localized electronic states are strongly coupled to the molecular and solvent environment, similar to electrochemical ET. The environmental reorganization Gibbs free energy in the tunnel gap is, however, significantly smaller than in the semiinfinite space in electrochemical ET. The activationless and barrierless overpotential regions are therefore much more important for in situ STM than for electrochemical ET. The reorganization free energy can, for example, easily be expected to approach energies corresponding to the bias voltage. This has strong implications for the nature of the in situ STM process. A consequence of the electronicvibrational interaction is that the redox level energy depends strongly on the level population. As in electrochemical ET, the vacant state energy is above the Fermi energies of the electrodes, cf. Figure 4. The occupied or reduced level energy is low because of excess electronic-vibrational interaction and located below the substrate Fermi level, but because the in situ STM reorganization Gibbs free energy is small, the occupied level may be trapped aboVe the Fermi level of the positively biased electrode (tip). This expands the in situ STM current/voltage patterns.185 The physical interaction between the redox level and the metallic substrate and tip can, further, be either weak or strong. These limits accord with different mechanisms of the STM process. As in electrochemical ET, the in situ STM process is controlled by nuclear configurational fluctuations. These take an initially vacant level above the Fermi level of the negatively biased electrode or an initially occupied level below the Fermi level of the positively biased electrode to levels close to the appropriate Fermi level. The following scenarios can then be envisaged: (a) The redox level can remain above (or below) both Fermi levels. ET or hole transfer between substrate and tip is then mediated by superexchange via the lowered, and fluctuating, off-resonance redox level. This is equivalent to an “indentation” in the tunneling barrier. (b) The vacant (occupied) redox level can be taken across the Fermi level of the negatively (positively) biased electrode where ET from occupied levels in the negatively biased electrode to the vacant redox level (or from the occupied redox

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J. Phys. Chem. B, Vol. 106, No. 6, 2002 1139

ad,max Figure 5. Dependence of normalized in situ STM tunneling current, iad tunn/itunn , on the overvoltage at fixed bias voltage. Fully adiabatic limit. eξη in units of kBT. Vbias ) 0.1 V. The four correlations in each frame represent, from top to bottom, increasing values of the reorganization Gibbs free energy Er. In units of kBT, Er ) 8, 12, 16, and 20. Left: γ ) 0.5. Middle: γ ) 0.75. Right: γ ) 0.25.

level to vacant levels in the positively biased electrode) occurs. The originally vacant (occupied), now temporarily occupied (vacant), level initiates vibrational relaxation, but the second ET step proceeds while the level is still in the “energy tip region” between the two Fermi levels. This can be called vibrationally coherent two-step ET.53,183 The overall process is represented by the three-level potential surface diagrams in Figure 2 where the initial and final states are vertically displaced by the bias voltage, eVbias.52 Relations between this pattern and other electronic-vibrational three-level processes such as resonance Raman scattering and hot fluorescence have been noted.44,52-54,186 (c) Coherent two-step ET requires strong adsorbate interaction with both substrate and tip. If the interaction is weak, the redox level relaxes in the intermediate state, and the process becomes a sequence of two equilibrated ET steps.53 (d) A new tunneling phenomenon arises when the adsorbate interactions with both substrate and tip is strong. Vibrational relaxation is again initiated after the first ET step, but a multitude of individual electron exchange events proceeds underway to intermediate state equilibrium, before the level is trapped below or above the appropriate Fermi level. Many electrons are thus transferred in a single in situ STM event.184 This could be one reason for the high apparent current density per molecule commonly encountered in STM. (e) Figures 5 and 6 represent the two single-molecule spectroscopic current-voltage relations, based on variation of the bias Voltage, at fixed substrate potential, and the substrate overpotential, at fixed bias voltage. The latter entails parallel variation of substrate and tip potential relative to a common reference electrode. Bias voltage variation induces vertical tip Fermi level displacements. As the redox level is exposed to part of the potential drop, features in the current-bias voltage relation are expected when the bias voltage takes the redox level across the substrate Fermi level. Overpotential variation at fixed bias potential is equivalent to a parallel vertical shift of both Fermi levels. The current then first rises as the redox level approaches one of the Fermi levels. Further increase leads the current to decrease, as vacant levels of the positively biased electrode become increasingly thermally inaccessible. (f) The expectations in e apply when eVbias is small compared with the single-electron transfer reorganization Gibbs free energy, eVbias e ER. When eVbias exceeds ER, a new feature appears.185,187 After the first ET step, say from the negatively biased electrode to the vacant redox level, the occupied level relaxes to a position aboVe the Fermi level of the positively biased electrode. Electrons are then transmitted to the latter by activationless interfacial ET (Figure 7), with no bias Voltage dependence. OVerpotential dependence is confined to the region

Figure 6. Dependence of the normalized in situ STM tunneling current (Figure 5) on the bias voltage. Energy quantities in units of kBT. γ ) 0.5. Er ) 12. Top: the current. Bottom: the derivative current.

up to eη f ER with a constant tunneling current above ER. Still further increase eventually takes the relaxed occupied redox level below both Fermi levels where the current drops. The spectroscopic features are thus strongly convoluted with the large bias voltage. As ER in the tunnel gap is small, this could be a common expectation. 4.2.2. Tunneling Current and Two-Step ET Mechanisms of in Situ STM. We provide next analytical formalism for some of the cases listed above, appropriate for reported cases of in situ STM of large redox molecules, considered in sections 4.3 and 4.4. We focus on two-step ET where the redox state population is temporarily changed. Independently of the specific nuclear dynamics, inside the energy tip region two characteristic times, τstL and τstR, characterize electron exchange between the redox level and the negatively (“left”) and positively (“right”) biased electrode

τstL ≈

p p ; τstR ≈ ∆L ∆R

(21)

1140 J. Phys. Chem. B, Vol. 106, No. 6, 2002 kr/o, respectively. The diabatic rate rate constant B kr/o and A constants are

B k o/r ) κLFL B k r/o ) κRFR

( ) [ ( ) [

ωeff 2kBT × 2π RR

exp -

Figure 7. Oxidized and reduced equilibrium energy levels of the redox molecule in the in situ STM configuration when the bias voltage is large and exceeds the reorganization Gibbs free energy Er. The current is large and activationless above a threshold value of the overvoltage approximately corresponding to Er and largely independent of both the bias voltage and overvoltage above this value. Large overvoltages, ≈2Er, eventually make the current drop again.

The energy broadenings, ∆L and ∆R, are given by

∆L ≈ π ∆R ≈ π

∑k (VkL)2δ( - kL) ) π(VkL)2FL

∑k (VkR)2δ( - kR) ) π(VkR)2FR

τst ) τstL + τstR, itunn )

e τst

(23)

In the limit of weak coupling in both molecule-metal contacts, τstL and τstR are long and itunn is small. The equilibrium redox levels are, however, strongly off-resonance with metallic energy levels in the energy tip region when eVbias < ER on which we focus first. Electron tunneling is thus controlled by nuclear configurational fluctuations, which bring the redox level into the tip region. Resonance between the redox level and accessible levels in the two metals is thus confined to a certain vibrational relaxation time, τrel. In the diabatic limit of weak coupling with the metals, this time it is small compared to both τstL and τstR

τrel , τstL and τstR

(

(24)

so that only a single ET step can be accommodated within the time τrel before vibrational relaxation takes the redox level out of the energy tip region. Dynamics of the vibrational system is thus crucial. The nuclear environmental effects cannot be reduced to static inhomogeneous broadening, and the process is in general a stepwise two-electron transfer process.53 A rate constant isomorphous with those for electrochemical interfacial ET can be assigned to each of the ET steps. The rate constant for ET from the “left” metal (negatively biased) to the ko/r. ET from the “right” oxidized redox level, ox, is denoted B metal (positively biased) is assigned the rate constant A ko/r. The second step of the process involves ET from the reduced redox level, red, to either the “left” or the “right” metal, assigned the

]

(ER - eVbias + eξη + eγVbias)2 4ERkBT

)

eξη + eγVbias ; kBT eξη + eγVbias - eVbias k r/o exp (25) A k o/r ) B kBT

A k r/o ) B k o/r exp -

(

)

RL and RR are the respective electrochemical transfer coefficients, ξ is the fraction of the substrate-solution potential drop, and γ is the fraction of the bias potential, at the molecular redox center. The steady-state tunneling current is, in the totally diabatic limit

k r/o - A k o/rA k r/o B k o/rB idiab ) e tunn B k o/r + B k r/o + A k o/r + A k r/o

(22)

where VkL and VkR are the electron exchange integrals coupling the redox level with the left and right metal, respectively. FL and FR are the corresponding metallic electronic densities of state. The steady-state tunneling current through the redox level proceeds in the tunneling time

]

(ER - eξη - eγVbias)2 ωeff 2kBT exp 2π RL 4ERkBT

(26)

Equation 26 reduces to simpler forms at finite bias, eVbias > kBT ≈ 25 mV. For example, A ko/rcan be disregarded at positive bias, giving

k r/o B k o/rB idiab tunn ) e o/r B k +B k r/o

(27)

Equations 26 and 27 show that thermally activated stepwise ET is indeed competitive with superexchange in the fully diabatic limit. idiab tunn thus follows the second, and superexchange the fourth, power of the (small) tunneling factor. The latter mechanism can therefore be disregarded when coupling between the redox level and both metals is weak. Equations 24-27 and the scenario discussed apply to the fully diabatic limit of weak interaction at both metal electrodes. The opposite, adiabatic, limit is represented by a different formalism and even a new ET phenomenon. The adiabatic limit accords with, cf. section 3:

κLFLkBT > 1; κRFRkBT > 1

(28)

The electronic energy range over which ET proceeds is therefore small, i.e., , kBT:

∆L ≈

1 1 ; ∆R ≈ ; ∆L,∆R , kBT κLFL κRFR

(29)

and so are the corresponding relaxation times through ∆L and ∆R, i.e., τ∆L and τ∆R:

τ ∆S )

∆S

; τS , τrel; S ) L and R

V|∂(Ui - Uf)/∂q|

(30)

where Ui and Uf are diabatic potential surfaces spanned by the nuclear coordinate(s) q and V is the velocity for thermal motion along q. Equations 28-30 disclose the following nontraditional nature of the fully adiabatic in situ STM process:

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J. Phys. Chem. B, Vol. 106, No. 6, 2002 1141

(a) Thermal fluctuations again take the oxidized (reduced) redox level across the Fermi level of the negatively (positively) biased electrode. As the redox level crosses into the energy tip region, the average level population, 〈n〉, changes from close to zero above FL, or close to unity below FR, to the following value between zero and unity

∆L 〈n〉 ≈ ∆L + ∆R

(31)

(b) The population is dynamic. The first ET step leading to level population is followed by depopulation prior to vibrational relaxation. This is a consequence of the adiabatic limit of strong interaction and the short electron exchange time compared with the vibrational relaxation time:

τrel . τL ≈

p p ; τrel . τR ≈ ∆L ∆R

(32)

which follows from eqs 28-30. The single-electron transfer cycle is followed by a sequence of subsequent cycles transferring a number of electrons, no/r, in a single STM event before the occupied or vacant level is trapped. no/r is of the order

no/r ≈

eVbias 1 1 ; ∆ ) + , kBT ∆ κLFL κRFR

(33)

This number can be large, up to several orders of magnitude. The fully adiabatic limit is thus physically notably different from the more conventional behavior of the fully diabatic limit by transferring coherently a large number of electrons in the singlemolecule STM process. The steady-state adiabatic tunneling current is

ko/r )

[

ko/rkr/o iad tunn ) 2eno/r o/r k + kr/o

]

ωeff Gqo/r(η,Vbias) exp ; 2π kBT kr/o )

(34)

[

]

ωeff Gqr/o(η,Vbias) exp (35) 2π kBT

Gqo/r(η,Vbias) and Gqr/o(η,Vbias) are the activation Gibbs free energies determined by the barrier height along the coordinate q, cf. section 3.2. The following forms are mostly appropriate: 184,185

Gqo/r

(ER - eξη - eγVbias)2 ) ; 4ER Gqr/o )

(ER - eVbias + eξη + eγVbias)2 (36) 4ER

[

kr/o ) ko/r exp -

]

eξη + eγVbias kBT

(37)

More generally, the activation free energies are given by the effective adiabatic potential along q, i.e., U(q), determined by the average redox level occupation number, 〈n(q)〉188

1 U(q) ) pωq2 2

∫0q dq 〈n(q)〉 x2ERpω

(38)

Equations 34-37 can be given the following simple and broadly valid forms when the bias voltage is small in the sense noted and the redox level contacts with the two metals symmetric, i.e., κLFL ) κRFR ) κF. By eq 33, we can convert eq 34 to

ko/rkr/o iad tunn ) eκF(eVbias) o/r k + kr/o

(39)

The electronic properties of the metals thus appear explicitly even though the rate constants are adiabatic and independent of these properties, cf. eq 35, quite differently from electrochemical ET at a single surface where κF is only apparent in the diabatic limit. This is because κF in eqs 34 and 39 now determines the number of electrons transferred. Equation 39 can be given the following explicit form, using eqs 35-37:

(

)

ωeff ER + eVbias 1 iad exp × tunn ) eκF(eVbias) 2 2π 4kBT (1/2 - γ)eVbias - eξη (40) cosh-1 2kBT

[

]

which represents the dependence of the tunneling current on both the overvoltage and bias voltage when |eVbias| < ER. The at most important implication is a current maximum, iad,max tunn

η ) ηmax )

11 - γ Vbias ξ2

(

)

(41)

located at the equilibrium potential, ηmax ) 0, when the redox site is exposed to half the bias potential drop, γ ) 1/2. This is due to the two-step nature of the process and the confinement of contributing metallic levels to the energy tip. It is not due to onset of the inverted overpotential region. A maximum also prevails for resonance and coherent ET but is shifted to the overpotential |eη| ≈ ER.182,183 The maximum for adiabatic twostep ET is shifted to positive and negative overpotentials when the redox center is closer to the substrate and tip, respectively,184 (Figures 5 and 6). Equations 27 and 39-41, along with eqs 26 and 35-37, represent the “spectroscopic” band shape features in the tunneling current dependence of the overvoltage and bias voltage, determined by level energetics and electronic-vibrational coupling. 4.2.3. Tunneling Spectroscopic Band ShapessA Generalization. The notions above illuminate expectations of in situ STM current-voltage spectroscopy. Inclusion of vibrational fine structure caused by local high-frequency modes is straightforward.189 The following other cases, which we consider briefly, are important: (i) the partially adiabatic limit where one molecule-metallic contact accords with the adiabatic limit and the other one accords with the diabatic limit;187 (ii) large bias voltages where eVbias exceeds the reorganization free energy;187 and (iii) functional molecules with more than one redox center.39,190 4.2.3.1. Partially Adiabatic in Situ STM Processes. This limit applies when the redox center is significantly closer to one of the electrodes than to the other one.184 It could be important, for example, in efforts toward in situ STM mapping of redox metalloproteins where the redox centers are commonly asymmetrically located in the protein structure. Electronic equilibrium is then established at the contact of strong interaction. The current-voltage relations resemble those for the fully adiabatic limit with the current maximum shifted toward negatiVe and

1142 J. Phys. Chem. B, Vol. 106, No. 6, 2002 positiVe potentials when the electronic contacts are strongest at the positiVely and negatiVely biased electrode, respectively. 4.2.3.2. In Situ STM Currents at Large Bias Voltages. It was noted (Figure 7) that a different pattern emerges when the bias voltage exceeds the reorganization Gibbs free energy, |eVbias| > Er. An initially vacant redox level above the Fermi level of the negatively biased electrode is then trapped below this level but above the Fermi level of the positively biased electrode after redox level reduction, rather than below this level such as for small bias voltages, |eVbias| < Er. Similarly, an initially occupied redox level below the Fermi level of the positively biased electrode is trapped above this level but below the Fermi level of the negatively biased electrode after oxidation of the redox level. This pattern, denoted as “nonreactive mode”,185 imposes new features on the current/voltage relationships. Increasing the positive bias Voltage at a given overvoltage above the equilibrium potential first takes the vacant redox level to resonance with the Fermi level of the negatively biased electrode. The current strongly increases around this value of the bias voltage (≈2Er) and then remains independent of eVbias and the temperature, taking the form

inon-react ) tunn

e ∆L∆R p ∆L + ∆R

Figure 8. Rectification and threshold effects in ET between two metals such as an STM substrate and tip, via intramolecular ET in a donoracceptor molecule. Left: Zero bias voltage when the donor level, red D , is occupied and the acceptor level, ox A , is vacant. Middle: A positive threshold bias voltage, Vbias, brings the two levels to resonance where ET occurs. Right: The oxidized donor level, ox D , and the reduced acceptor level, ox D , relax to positions opposite to those in the initial state. Negative bias voltages would increase the donor-acceptor energy gap with insignificant current flow in the opposite direction.

(42)

where ∆L and ∆R are given by eq 22. The current/bias voltage relation thus displays the character of a rectifying “switch” device of in situ STM. Increasing negative oVerVoltage, at given large positive bias voltage, |eVbias| > Er, also first increases the current in a nonreactive mode. When |eη| f Er, an η range of ≈Vbias of constant tunneling current follows, again given by eq 42. A current drop follows this when |eη| has reached such high values that the reduced redox level is trapped below the Fermi level of the positively biased electrode. The current/overvoltage relation therefore resembles a rectangular “pulse” instead of a Gaussian such as for small bias voltage. Similar patterns emerge when the equilibrated redox level is initially in the reduced state below the Fermi level of the positively biased electrode. Work on these cases is in progress.185,187 In section 4.4, we shall use the notions to a specific system. 4.2.3.3. Rectification and Molecular Electronic Function of Multicenter Redox Molecules. Notions and formalism in multiphonon in situ STM have perspectives in the context of multicenter redox molecules. Electronic delocalization in metallic cluster compounds or clusters in metalloproteins would, for example, hold promise for improved electronic overlap and enhanced electron tunneling. Molecular adsorbates may also possess distinct redox sites such as in donor-acceptor molecules of the form D-S-A, where D is a donor group, A is an acceptor, and S is a molecular spacer. D-S-A molecules have long been a focus as prototype molecular scale electronic elements with rectification, threshold, and switch features.37-39,190,191 This perspective has recently strengthened as actual molecular monolayer rectification192,193 and logical circuits based on molecular redox switches194 coming close to qualify as “molecular devices” have been reported. In situ STM is here central and enjoys the unique merit compared with other single-molecule spectroscopies of possessing intrinsically the metallic interfaces needed in contacting any functional molecular device with outer electronic circuits. Figure 8 illustrates molecular D-S-A rectification and switching. The D-S-A monolayer is inserted between two metallic conductors. The donor is closest to the substrate, and

Figure 9. Normalized current-bias voltage relations for the donoracceptor system in Figure 8. The two curves reflect different potential distributions in the tunneling gap as represented by the parameters γD and the acceptor γA. Solid line: γD ) 0.25, γA ) 0.75. Dashed line: γD ) 0.1, γA ) 0.5. Other details in ref 195.

the acceptor is closest to the tip. In the simplest case, the donor level (occupied) must be below and the acceptor level (vacant) above both Fermi levels at zero bias voltage. On application of a positiVe bias voltage, both levels are lowered, but the acceptor level moves faster than the donor level. As the levels cross, electron flow from the negatively to the positively biased electrode, via intramolecular ET, is induced. Application of a negatiVe bias voltage increases the gap between the occupied donor and the vacant acceptor level, enhancing the ET barrier. The scheme in Figure 8 has been incorporated in a theoretical frame on the basis of electron flow as a sequence of electronicvibrational transitions through the D-S-A molecules similar to in situ STM of redox molecules with a single center.195 Figure 9 is representative of computed current-bias voltage relations and illuminates rectification and switch effects, determined by level energies and electronic-vibrational couplings. These observations are a frame for monolayer rectification to be discussed below. 4.3. In Situ STM of Electronic Structures and Reactivity of Large (Bio)Molecules. Mapping of topography, electronic structure, and reactivity of organic aromatics,196-200 sulfur compounds,145-147,201 metalloporphyrins202-206 and -phthalocyanins,207-209 hetero-poly-tungstates,210,211 functional nanoscale assemblies,39,193,194,212 and metalloproteins45-47,157-159,213-216 are exciting achievements of in situ STM.217 Some of these systems can be controlled by the STM bias and overvoltage, disclosing a single-molecule spectroscopic function. The systems interface with broader single-molecule classes such as discussed in section 2. We now bridge observed electronic function in some of these systems with the theoretical frames in Section 4.2.

Feature Article 4.3.1. Orbital-Mediated Electron Tunneling Spectroscopy of Metal Phthalocyanins. STM of several metal phthalocyanins (MPc) on Au(111) in UHV illustrate the nature of STM as an electronic rather than topographic single-molecule mapping.207,208 The molecular structures of CoPc, FePc, CuPc, and NiPc are identical, but CoPc (d7) and FePc (d6) show a bright contrast at the metal center, whereas CuPc (d9) and NiPc (d8) show a “hole”. The drastic difference is caused by CoPc and FePc d orbitals close to resonance with the substrate Fermi level, whereas those of CuPc and NiPc are strongly off-resonance. The CoPc and FePc d orbitals but not those of CuPc and NiPc are therefore favorable superexchange mediators, cf. eq 20. These observations might carry over to in situ STM. This has not been achieved, but inelastic tunneling spectroscopy of the MPc’s in Al-Al2O3-MPc-Pb tunnel junctions points to tunneling via low-lying redox levels, temporarily oxidized or reduced in appropriate bias voltage ranges.209 Observations supporting this view are (a) spectral peaks at positive and negative bias in the temperature range 4-200 °K and (b) correlations between the peaks and the MPc room temperature oxidation potentials. Room temperature redox processes, however, invariably involve strong electronic-vibrational coupling to an environmental low-frequency nuclear continuum. Orbitalmediated transitions in the solid-state junctions are, strikingly, independent of temperature in the whole range. Electronic level broadening must therefore involve distributions of highfrequency modes or temperature-dependent reorganization or driving force parameters. 4.3.2. Current-Voltage Spectroscopy of Organic Redox Molecules. Electrochemical conversion of large organic molecules has been mapped to single-molecule resolution. Tao et al. could follow electrochemical oxidation of xanthine197 and guanine198 on graphite by combining in situ AFM and STM. Significant differences in the two imaging modes of xanthine oxidation to uric acid were observed, reflecting the topographic and electronic properties, respectively. Such differences have been found for other aromatic molecules such as 2,2′-bipyridine.154 Oxidation could be followed by both AFM and STM as reaction zones expanding from surface defects, leaving an ordered uric acid monolayer distinguishable from the reactant monolayer. In combined in situ STM and IR studies, the electrooxidation of adsorbed phenoxide on Au(111) to monomer, dimer, and trimer products could also be followed.200 The reactant molecules were found to be adsorbed end-on through the oxygen atom, whereas the products were oriented parallel to the electrode. Molecular scale imaging of surface monolayer phase transitions, for example, of 2,2′-bipyridin and DNA bases have been addressed by single-crystal voltammetry and in situ STM.154,155 These are other cases for dynamic phenomena, at levels complementary to in situ AFM. Taniguchi et al. could follow changes in high-resolution STM of self-assembled monolayers of bis(2-anthraquinyl)disulfide on Au(100) induced by electrochemical reduction to the hydroquinone.196 New single-molecule features were assigned to different molecular orientations in the oxidized and reduced states but can also be ascribed to different mediating orbitals in the two potential ranges. Snyder and White found symmetric ex situ STM current-bias voltage relations of Fe-protoporphyrin IX (FePP) multilayers on HOPG.205 The data were interpreted as tunneling via FePP. The current could be converted to a rate constant which exhibited a bias voltage dependence resembling the quadratic form expected from ET theory, but the rate constants must be composite in view of the multilayer nature of the adsorbate.

J. Phys. Chem. B, Vol. 106, No. 6, 2002 1143 Data by Lindsay, Tao, and their associates offer more direct accordance with in situ single-molecule spectroscopy and STM as sequential two-step ET.199,203,204 Current-bias voltage relations for several metalloporphyrins covalently linked to Au(111) in mesitylene solution illuminate metal specific behavior and different STM contrasts for redox active and inactive metalloporphyrins.204 The itunn/Vbias relations for the former are strongly asymmetric, with broad derivative current/bias voltage peaks at large negative bias voltage, cf. Figures 5 and 6. Different single molecules displayed distributions of peak potentials and widths, reflecting possibly single-molecule stochastic effects. The formalism in section 4.2 offers options for the two-step mechanism. If the formal potential is close to zero and the fraction of the bias voltage drop at the metalloporphyrin site, γ ≈ 0.4, then reorganization Gibbs free energies of 0.2-0.3 eV and coinciding tunneling factors give the observed peak position and width. Partially or totally diabatic behavior, with the substrate-adsorbate coupling weakest, also shifts the peak derivative potential to negative values with γ values larger than 0.5. The second view accords best with the theoretical conclusions in ref 204. Thiolated carotene embedded in a selfassembled 1-docosanethiol monolayer (straight-chain alkanethiol with 22 carbon atoms) on Au(111) in toluene is another case for single-molecule conductivity.199 Carotene has the same length as the embedding alkanethiol. A conducting AFM tip was used so that both topography and conductivity could be imaged. A new AFM force/bias voltage spectroscopy was established, and the electrostatic nature of the tip-surface interaction was identified. The alkanethiol is, moreover, electronically intransparent, so that both molecules are imaged in AFM, but only carotene is imaged in STM. Carotene is, finally, oxidized at fairly low potentials (+0.53 V, SCE) and therefore a case for two-step ET. Current-voltage relations were symmetric over more than one V, indicative of γ-values close to 0.5, but these correlations did not exhibit enough fine-structure to determine spectroscopic band shape parameters or for mechanistic discrimination. The opening of ET channels by low-lying redox levels in the in situ STM mode is most strikingly illustrated by Tao’s studies of iron protoporphyrin IX (FePP) coadsorbed with redox inactive H2PP on HOPG.203,217 In addition to molecular resolution for both molecules the redox active FePP showed a strong resonance in the current/oVerVoltage relation across the formal equilibrium potential. Structurally similar but electronically different adsorbates can thus be clearly distinguished at the single-molecule level. It also holds clues to the mechanism of the STM process. Resonance tunneling218 and coherent twostep ET183 are formally in keeping with the observed maximum but in both mechanisms the maximum is expected at a potential shifted from the equilibrium potential by the reorganization Gibbs free energy, ER, cf. Section 4.2.1. As noted, ER is small in the tunnel gap,183 and reservations as to the reference potential have been expressed.218 A maximum at the equilibrium potential is, however, fully in keeping with sequential two-step ET and γ ≈ 0.5.184 This illuminates the diagnostic value of the theoretical frames but also the need for considering more than a single tunneling mechanism. 4.3.3. STM of Functional Nanosize Molecular Assemblies. 4.3.3.1. Bridge-Assisted ET Systems Resembling Nanoscale DeVices. STM of large molecules with electron or hole states close to the Fermi levels of the enclosing electrodes offer perspectives in expanding areas of chemical or electrochemical nanoscale electronic systems with “device-like” functions.37-39 This notion applies when the electronic properties, ultimately

1144 J. Phys. Chem. B, Vol. 106, No. 6, 2002 of single molecules or supramolecular assemblies can be brought to display rectification, switch, amplification, single-electron tunneling, or other electronic function analogous to micro- and macroscopic electronics. Target molecules must be large in order to accommodate tunable electronic function. For use in singlemolecule technology, they must also be robust. Metalloproteins may thus have much to offer regarding fundamental structurefunction relationships in nanoscale electronics, but focus should be on smaller rigid molecular entities in actual devices. Such units could be phthalocyanins, porphyrins, aromatic ring systems, catenanes and rotaxanes, and other robust intermediatesize redox molecules. Attempts to construct single-molecule wires, rectifiers, switches, storage elements, photodiodes and -switches, amplifiers, etc. have rested on broad chemical system varieties: (i) selfassembled condensed porphyrins;219,220 (ii) organic donoracceptor molecules;190-193,221-223 (iii) carbon nanotubes;224 (iv) supramolecular ladders, grids, and other transition metal complex arrays;225-227 (v) polynuclear transition metal complexes. The metal ions can be structurally close to each other and interact strongly, creating a generic storage element,228,229 or spatially separate, with weak electronic interaction, constituting the basis for rectification and switching;37-39,230,231 (vi) singlemolecule conduction of molecules trapped in metallic nanogaps;34-36,39,70 (vii) differential resistance patterns in selfassembled heteropolytungstates210,211 and organic molecular arrays;232,233 (viii) molecular D-S-A photodiodes composed of porphyrin, ferrocene, and quinone units in LangmuirBlodgett films;230 and (ix) catenane- and rotaxane-based switches.194 These can be held “open” or “closed” by keeping the redox center in the oxidized and reduced state, respectively, by external potential control. 4.3.3.2. Two Case Studies. Monolayers of hexadecylquinolinium tricyanodimethanide between electrodes of gold, aluminum, or graphite and deposited aluminum or magnesium have been reported as the first case for single-molecule current rectification by intramolecular ET.192,193 The ground state is T-D+-S-A- T T-D-S-A, where T is the hexadecyl substituent required for Langmuir-Blodgett films, D+ is quinolinium, A- is cyanodimethanide, and S is an aromatic spacer. Electron flow on metallic substrates is from A- to D+. There is a low-lying charge transfer state 2.2 eV above the ground state,234 with a spectral bandwidth corresponding to a nuclear reorganization Gibbs free energy of 0.90 eV. The currentvoltage relation is strongly asymmetric, with much higher currents at positive bias with the A--end toward the negatively biased electrode. The threshold bias voltage for current onset is +1.5 V. This value is little affected in the temperature range 105-298 °K, but the transition from blocked to rectified current is sharper, and the current at a given bias voltage is smaller the lower the temperature. The view of rectification as intramolecular ET controlled by an electric field (section 4.2.3) accords roughly with the data.195 The ground state HOMO is below and the excited state LUMO above the Fermi levels of the electrodes at zero bias voltage. The threshold voltage accords roughly with the optical excitation energy if the LUMO energy variation follows the bias voltage. The reorganization free energy determined from the optical transition is notably smaller than the threshold bias voltage energy (cf. Figures 8 and 9). This could imply that rectification proceeds coherently, via a dynamically populated intermediate state where the donor is oxidized and the acceptor reduced, cf. section 4.2.3 and Figure 7. Reference 212 reported STM of a 6 nm gold nanoparticle linked to a gold substrate by a 20 methylene unit of R,ω-

polymethylenedithiol with a pyridinium group (bipy) in the center. The redox state of the latter determines the behavior of this junction. The views in section 4.2 accord with the STM pattern, but the system is more composite by the presence of the gold nanoparticle. Current-distance relations in aqueous solution under full electrochemical control could be determined for given substrate potentials at a fixed bias potential. The tunneling decay length, β-1, was much shorter at a high positive potential (+0.492 vs NHE), i.e., 0.6 Å, than at the low potential -0.21 V (NHE), with β-1 ) 1.5 Å. In the former case, bipy is fully oxidized. The potential in the latter case is close to the equilibrium potential of bipy2+/•+. The absolute energy of the redox orbital is 4.29 eV, and the absolute value of the gold substrate is 4.99 eV at +0.49 V (NHE) and 4.29 eV at -208 mV. The fast current-distance decay at high potential is in keeping with superexchange where the redox level is coupled to the electrodes purely electronically. Electrons tunnel through bipy in a barrier corresponding to the off-resonance vacant redox orbital, into the gold nanoparticle, and further to the tip. The redox level is thermally accessible at the low potential, and STM here is likely to involve two-step ET. If the two electronic coupling factors are approximately equal, this is the potential of maximum current. bipy•+ is oxidized by ET to the tip and then reduced by ET from the substrate, or bipy2+ is reduced by ET from the substrate and reoxidized by ET to the tip. In either case, the hopping mechanism reduces the tunneling distance significantly. The environmental reorganization Gibbs free energy from the main 290 nm optical absorption band of bipy2+ is about 0.31 eV. With a bias voltage of +0.2 V, this indicates that the transition involves sequential two-step ET rather than a dynamically populated bipy2+ state. The current-bias voltage could be recorded in a two-electrode configuration over a broad range where the substrate potential was varied from 0 to -2.0 V relative to the tip potential. A strong 0.2 V wide resonance at -1.75 V was apparent in the derivative current/bias voltage relation. This accords again with opening of an ET channel via bipy2+/•+. The discrepancy between the resonance voltage and the difference between the Fermi energy of the gold substrate and the redox level energy could be caused by a parallel but smaller upward energy shift of the redox level (γ ≈ 0.6) when the bias potential is scanned negative. This could be illuminated by current/voltage scans under full potentiostatic control. Several equidistant smaller resonances (0.3-0.35 V) between 0 and -1.5 V also appear. The spacing excludes coupling to local high-frequency modes, and the gold particles are probably too large for single-electron charging of the gold nanoparticle. These effects are presently elusive, but this system emerges as a potential target for multifarious properties of nanoscale systems approaching device behavior. 4.3.4. STM of Redox and Other Metalloproteins. Imaging of single biological macromolecules such as DNA and proteins early became a vision in STM. Phophorylase kinase and phosphorylase b,235 serum albumin,236 immunoglobin G,237 vicilin,238 cytochromes,239 hemoglobin,240 catalase,241,242 glucose oxidase,243 lysozyme,244 the multicenter metalloprotein nitrogenase,245 and photosynthetic reaction centers246 are cases for claimed molecular resolution. This was, however, for air ambient and often with high bias voltage or in aqueous solution (lysozyme) but with no potential control. Imaging of metalloproteins by in situ STM in aqueous buffer with full potentiostatic control dates from the mid-1990s. Imaging of functional proteins is fraught with recognized technological and fundamental difficulties. Immobilization is the most important among the

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Figure 10. Three-dimensional structure of P. aeruginosa azurin. The copper center with ligands and the disulfide group are highlighted. Coordinates from ref 250 and Brookhaven Data Bank. Graphics in Molscript.251

former. Fundamental limitations are, first, the materially “soft” nature of the biological macromolecules, for which resolution cannot be expected to compare with small rigid molecules. As the molecules are also large, high bias voltages are frequently needed. Both electric fields and pressure from the tip can therefore affect detrimentally the functional proteins. Large bias voltages mean, finally, that multiple charge-transfer patterns are competitive, with congestion of image contrasts. With the present state of in situ STM, questions to be addressed must show reverence for these properties. Focus has been on small redox proteins with interfacial behavior consolidated in protein electrochemistry. They have also been targets in theory of long-range ET. Imaging features to be looked for can therefore be identified. Horse heart cytochrome c on glassy carbon was the first case for in situ STM molecular resolution of a metalloprotein.157 Image resolution of cyt c covalently linked to anodically prepared surface functionalities on HOPG is molecular, with a submolecular feature.215 Molecular resolution was also achieved by covalent cyt c linking to platinum or gold.159 Voltammetric or other supporting data accompanied none of these studies. Other reports could not disclose cyt c on HOPG by in situ STM after noncovalent adsorption, but in situ AFM identified globular structures over a broad potential range.160 The adsorption process and parallel voltammetric time evolution could also be followed. STM of the redox metalloenzymes laccase (Polypherus Versicolor)247 and peroxidase (horseradish, HRP) on HOPG158 have been reported. Only the latter qualifies as in situ STM. HRP was covalently bound to surface oxide functionalities. Molecular resolution was achieved under conditions where the enzyme could be catalytically active in H2O2 reduction by electron exchange with the electrode. In situ STM of HRP under denaturing conditions disclosed other features distinct from those of native HRP. STM of metallothionein (rabbit liver),214 rubredoxin248 (Clostridium pasteurianum), and thiolated mutant cytochrome P-450249 on Au surfaces to molecular resolution in aqueous solution has been reported, but none of these cases were subject to potential control or supported by other data. The proteins have sulfur-containing surface residues (cysteine

or methionine) or large amounts of internal sulfide, potentially reactive to gold even at low electrochemical potentials. Potentiostatic control of proteins with these groups is therefore crucial. Proteins with given surface groups suitable for direct chemisorption such as thiolate and disulfide offer an attractive approach to gentle protein immobilization. Linking groups can be naturally present or introduced by chemical or microbiological modification. Well-defined orientations suitable for STM/ AFM and electrochemistry of functional oriented monolayers would be the desirable outcome. This concept accommodates localized surface charges or hydrophobic areas which attach the proteins to surfaces in narrow orientation distributions. A particular case, the blue copper protein azurin, will be addressed below. Azurin possesses two structural elements suitable for surface attachment, i.e., a surface disulfide group opposite the copper redox center and a hydrophobic surface area around this center (Figure 10). Both can be exploited, preparing the protein in two opposite, functional orientations. At the same time, azurin illuminates constraints in this strategy. The surface linking group may not be close to the redox center. This would hamper electrochemical ET but not necessarily in situ STM. The opposite limitation can also be envisaged; that is, the linking group is close to the redox center but remote from the probing STM tip. Inconveniently high bias voltages might then be needed. This scenario is represented by hydrophobically immobilized azurin on self-assembled variable-length alkanethiols. The chemisorbed atomic link, say sulfide could, finally, itself exhibit a strong STM contrast, impeding disclosure of details from the rest of the molecule. 4.4. Molecular Structure and Functional Control of a Redox Metalloprotein, Pseudomonas aeruginosa Azurin, on Pure and Modified Au(111) Surfaces. Recent studies of the blue single-copper protein Pseudomonas aeruginosa azurin (Figure 10) on single-crystal Au(111) surfaces by electrochemistry and in situ STM45-47,117,118,187 have disclosed a high degree of detail in structural mapping and functional control of the adsorbed protein. They have also pointed to the need for a comprehensive approach and illuminate both perspectives and

1146 J. Phys. Chem. B, Vol. 106, No. 6, 2002

Figure 11. Two molecular orientations of adsorbed azurin on Au(111). A. Direct adsorption on bare Au(111) via the disulfide group, with the copper center opposite to the Au(111) surface. B. Adsorption on an alkanethiol monolayer self-assembled on Au(111) by hydrophobic interactions with the surface region around the copper center which faces the Au(111) surface. From ref 118, with permission.

limitations of in situ STM approaches to electronic properties of redox proteins. 4.4.1. Adsorption and ET Function of P. aeruginosa Azurin at Bare Au(111) Electrodes. The molecule is immobilized in two opposite orientations, at bare and modified single-crystal Au(111)-surfaces (Figure 11). These studies are so far the only case for both orientational control and structural mapping of a functional redox protein under electrochemical potential control approaching the molecular level. Two features are crucial. One is the copper atom coordinated to two His, Cys, Met, and Gly close to one end of the molecule, and the other one is the surface disulfide group opposite the copper center. The location of the copper center enables facile electron exchange with solute reaction partners or electrode surfaces. The two functional units are linked by β strands facilitating electron relay also along this route. Prerequisites have been atomically planar electrode surfaces, a variety of electrochemical techniques combined with in situ STM, and reference molecules. Electrochemical and spectroscopic data support in situ STM as a case for mapping of a truly functional redox protein on the solid surface. Azurin voltammetry at edge-plane pyrolytic graphite is reversible and diffusion-controlled.252 Single-crystal Au(111)electrode surfaces are suitable for adsorption via the disulfide. ET function through the protein in the adsorbed state is retained, because of bonding of the sulfur atoms to the gold surface, as supported by several observations:45-47,117 (1) Reductive desorption in the potential range -0.8 to -1.1 V (SCE) is clearly apparent in differential pulse voltammetry. This feature is a fingerprint of Au-thiolate bond formation. The equivalent charge comprises 70-80% of a monolayer (7 × 10-12 mol cm-2), cf. point 3. (2) X-ray photoelectron spectroscopy discloses the same two S2p fingerprint peaks (163.3 and 162.2 eV) for adsorbed azurin as for adsorbed butanethiol and the amino acid cystine. A strong N1s signal (401 eV) is also seen. A sulfur peak at 164.1 eV reflects, interestingly, the two sulfur donor atoms at the copper center. (3) In situ STM shows about 70% monolayer coverage (Figure 12A), cf. point 1, that is stable for weeks. The molecular structures have the same lateral extension as azurin (3.7 ( 0.4 nm). These observations offer a coherent view of azurin adsorption via surface disulfide. The following other observations testify that ET function is retained in the adsorbed state:

Figure 12. A. In situ STM image of P. aeruginosa azurin on bare Au(111). 50 mM ammonium acetate, pH 4.6. Substrate potential -0.1 V (SCE), Vbias ) 0.20 V. Tunneling current 0.80 nA. Scan area: 110 × 110 nm. B. Ex situ STM image of P. aeruginosa azurin on decanethiol self-assembled on Au(111) prepared from 50 mM NH4Ac, pH 4.6. Bias voltage 0.81 V. Tunneling current 0.16 nA. Scan area: 100 × 100 nm.

(4) There are symmetric cathodic and anodic differential pulse signals in the Cu2+/+-potential region (0.10 V, SCE). The charge accords with that for reductive desorption. Such signals appear neither for cysteine and cystine nor for Zn-substituted azurin. (5) The interfacial ET rate constant at the equilibrium potential is 30 s-1 determined by impedance spectroscopy. This value is smaller by an order of magnitude than that for azurin adsorbed in the opposite orientation on alkanethiol-modified Au(111) surfaces. These points show that a stable functional azurin monolayer directly bound via surface disulfide has formed on the Au(111) surface and that single-molecule in situ STM of functional azurin has been achieved. 4.4.2. Adsorption and Interfacial ET of Azurin on SelfAssembled Alkanethiol Monolayers. Azurin adsorption on variable-length alkanethiol monolayers on Au(111) by hydrophobic interaction between the terminal alkanethiol methyl group and the hydrophobic protein surface around the copper center is an alternative adsorption mode.118 The molecular orientation is opposite to adsorption on bare Au(111), with the copper center facing the electrode. The most striking difference from adsorption on bare Au(111) is that stable, almost ideal azurin monolayer voltammetry emerges. The rate constants for electron tunneling between the electrode and the copper center, across the alkanethiols, are significantly higher than for tunneling through the protein in the direct adsorption mode, for similar tunneling distances. The following data disclose a coherent view of adsorption and ET of fully functional azurin: (I) Azurin adsorbs in stable submonolayers. Figure 12B shows a representative STM image of the azurin/decanethiol surface.

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Figure 14. A. Dependence of interfacial ET rate constants at zero overvoltage on alkane chain length for P. aeruginosa azurin adsorbed on variable-length alkanethiols self-assembled on Au(111). B. Dependence of interfacial ET rate constant on the overvoltage for P. aeruginosa azurin adsorbed on tetradecanethiol self-assembled on Au(111). 50 mM NH4Ac, pH 4.6.

Figure 13. A. Monolayer voltammograms of P. aeruginosa azurin on dodecanethiol monolayer self-assembled on Au(111). Different scan rates. B. Anodic (Ipa) and cathodic (Ipc) voltammetric current peak height dependence on the scan rate for the data in A. 50 mM NH4Ac, pH 4.6.

The high order of the alkanethiol and the uniform dimensions and distribution of the protein structures over the surface are notable. (II) Azurin monolayer voltammetry is reversible, with symmetric cathodic and anodic peaks and linear peak height dependence on the scan rate (Figure 13). The peaks separate at higher scan rates as kinetic control takes over. (III) Azurin monolayer coverage is stable and sensitive. The current decay on alkanethiols with more than nine carbon atoms is only a few percent after several hundred scans, and clear voltammetric peaks appear for less than 2% coverage.118 Saturation approaches 70% of a monolayer at µM concentrations for alkanethiols between 10 and 14 carbon atoms but is smaller for shorter chains. I-III show that azurin adsorbs in a fully functional state with narrow orientational distribution. A suitable combination of bare and pretreated Au(111) surfaces has thus enabled protein adsorption in two opposite orientations. Interfacial ET is much more facile in the alkanethiol than in the disulfide adsorption

mode. Alkanethiols thus provide a more efficient medium for long-range ET than adsorbed protein. Double layer capacitance and reductive desorption exhibit patterns which both depend characteristically on the alkylthiolate chain length and are affected in subtle ways by azurin adsorption. This is discussed in ref 118. The following other features show that adsorbed azurin monolayers are stable and uniform enough to accord with notions from ET theory: (IV) The interfacial standard ET rate constants over a broad range of thiol lengths can be obtained from voltammetric peak separation and electrochemical impedance spectroscopy.118 Figure 14A shows the dependence of the rate constants on the number of carbon atoms. As for capacitance data, a biphasic correlation is noted. The rate constants are independent of the thiol length for shorter thiols but follow an exponential dependence with the decay factor, β ≈ 1.0 per CH2 unit for longer thiols. This accords with patterns for covalently linked ferrocene on polycrystalline gold106 and electrostatically bound cyt c.123,126 The physical cause of the weak dependence at the shorter alkanethiols is elusive. Preorganization123 or penetration of azurin into the shorter-chain structures118 can be suggested, but such steps would still depend on the overpotential. They would be rate determining for shorter, electron tunneling for longer chains. (V) The overvoltage dependence of the rate constants is available from chronoamperometry.118 Figure 14B shows data for a 13 CH2 unit alkanethiol. The cathodic and anodic branches are symmetric and follow a quadratic dependence, with a limiting transfer coefficient R(η ) 0) ) 0.5 and the reorganization Gibbs free energy of 0.3 eV. There is no sign of electronic

1148 J. Phys. Chem. B, Vol. 106, No. 6, 2002 lability of the metallic electron density caused by varying excess electrode charges.101,102 These data propose azurin as a “prototype” redox protein in molecular protein assemblies of high order, stability, and sensitivity. The orientation of the assembled protein molecules can be controlled, ET function is retained, and overvoltage and distance dependence can be characterized in detail and framed by electrochemical ET theory. 4.4.3. In Situ STM Tunneling Contrast Variation of P. aeruginosa Azurin. In situ STM spectroscopy (sections 4.2 and 4.3) of redox metalloproteins would hold exciting perspectives for mapping single-biomolecule electronic properties, but technological and fundamental issues have posed barriers for a real spectroscopic dimension. A “technological” issue is the limited potential ranges for ordered layer stability. A broader issue is that high bias voltages, i.e., a significant fraction of a volt, are frequently needed. Tunneling through the proteins, assisted by the redox centers, can be identified but still involves tunneling steps over more than 10 Å through metal-free protein matter. Tunneling must therefore be assisted by high driving forces, i.e., bias or overvoltages. This raises, first, the risk of deformation or disassembly of the protein layers by mechanical contact or electric field effects in the tunnel gap. The bias voltages can, second, easily exceed the small solvent reorganization Gibbs free energy, taking STM spectroscopy into the range where the current variation is weak, cf. section 4.3.2. The following data for azurin substantiate the importance of these observations.187 Figure 15 shows in situ STM images in the substrate potential range from -0.35 to 0 V (SCE), at a fixed bias voltage of + 0.2 V. With the copper center close to the tip, this corresponds to -(0.25-0.30) to 0-0.1 V at the copper center, relative to the equilibrium potential, i.e., a broad potential range on the cathodic side of the equilibrium potential. A dense (≈70%) monolayer is clearly seen in most of the range, with molecular resolution and little contrast variation. The images become blurred, and the apparent surface coverage decreases at both the lower and upper limits of the stability potential range, but a switch back to potentials inside the range identifies the same kind of dense monolayer as before the potential excursions. This image pattern prompts two observations. The sequence in Figure 15 parts A and B resembles, first, the behavior expected if a redox process is involved. The upper potential limit of the stability range is close to the equilibrium potential of the copper center and would correspond to the energy level diagram in Figure 7. At the positive potential limit, the redox level is vacant and above the Fermi energy of the substrate. Thermal activation of the two-step ET process is then required, and the tunneling current is low. The redox level becomes occupied at substrate potentials across the equilibrium potential, but because of the relatively high bias voltage, the reduced level only relaxes to a position above or close to the Fermi level of the tip. An overvoltage range of about the bias voltage, with activationless high tunneling current weakly dependent on the overvoltage, therefore follows. The current only drops again when the overvoltage has assumed a higher negative value where the reduced level is trapped well below the Fermi level of the positively biased tip. This would correspond to the lower potential limit of monolayer stability in Figure 15. The broad potential range of robust high contrast and weaker contrasts on either side could reflect a redox spectroscopic feature convoluted with the bias voltage. However, although attractive, this is almost certainly not the only cause of the pattern in Figure 15, as indicated by the following second observation. Figure 15C shows a comparison between the area

Figure 15. Sequences of in situ STM images of P. aeruginosa azurin adsorbed on bare Au(111) for a range of Au(111) potentials at a fixed bias potential, Vbias ) 0.2 V. 50 mM NH4Ac, pH 4.6. A. The sequence a-f shows images for increasing negative potentials with the values (SCE) -0.15, -0.20, -0.20, -0.25, -0.25, -0.30, and -0.35 V. Azurin is in the reduced state throughout. Groups of azurin molecular structures are framed. The group of five molecules in the triangular frame in the upper part of e only contains four azurin molecules in f. B. The sequence g-j shows the in situ STM pattern of azurin for increasing positive potentials. The values (SCE) are -0.15, -0.10, -0.05, and 0.0 V. i and j are around the equilibrium potential of azurin and show lower apparent coverage and weaker contrasts than at the lower potentials. C. Comparison of the apparent coverage and contrast pattern at -0.15 V after potential excursion to the positive limit. k is the scan area under the tip, and l is a different area of the same sample.

scanned during the potential excursion beyond the lower potential limit of monolayer stability and a different area not scanned. A similar comparison applies to the positive stability potential limit. Both areas shown are imaged within the stability potential range but after the potential excursion. The adsorbate population is clearly lower in the area scanned during the whole potential sweep than in the area which was not scanned. The protein monolayer is thus robust to potential variations across the equilibrium potential and to changes in the redox state, but interference by tip-assisted desorption is apparent in the multiply scanned area. Such issues must be recognized but are not

Feature Article prohibitive for in situ imaging of metalloprotein adsorbate electronic structure changes. 5. Conclusions and Some Perspectives The discussion above has focused on mechanisms of the fundamental in situ STM process of adsorbate molecules with accessible redox levels, which can be brought to resonance with the Fermi levels of the substrate and tip by thermal fluctuations in the environmental nuclear configurations. The first observation is that different thermally assisted ET channels emerge, in addition to inherent superexchange via the low-lying fluctuating redox level. The most important ET channels accord with notions of coherent and sequential two-step ET and fully diabatic and partially and fully adiabatic two-step ET. Analogues of these are known in interfacial electrochemical ET, long-range solution chemical and biological ET, and optical processes such as resonance Raman scattering and hot fluorescence. The in situ STM processes accord with analytical theoretical formalism with features in common with that for these other processes. Pathways towards a “unified” theory to cover both the multifarious in situ STM mechanisms and analogous chemical and optical threelevel processes are, moreover, visible. The second observation is that the in situ STM configuration with two electrochemical interfaces is also an environment for new ET phenomena with no direct parallel in other ET processes. The most important novel observation is apparent in the fully adiabatic limit where the strong interactions at both metallic surfaces transmit a large number of electrons in a single in situ STM event. This could resolve issues related to the high observed current densities per molecule mostly observed in STM. The multielectron transfer nature of the in situ STM process in the fully adiabatic limit is rooted in the truly vibrationally coherent transmission of the electrons, whereas the intermediate occupied or vacant redox level relaxes toward vibrational equilibrium. The coherent transmission of a large number of electrons holds a perspective in the context of single-molecule device construction, where switching and other intended features would be strongly amplified. The observations regarding molecular in situ STM mechanisms of redox molecules offers novel single-molecule spectroscopic perspectives, with analogues in other recent singlemolecule spectroscopies. This is the third observation. The overvoltage and the bias voltage are the two external parameters, and the current-voltage band shapes offer detailed insight into the level energetics and electronic-vibrational coupling. The environmental reorganization (Gibbs free) energy in the tunneling gap is, however, small compared to this central quantity in electrochemical ET and ET processes in homogeneous solution. Convolution of the intrinsic spectral band shape with the bias voltage is therefore important. Comparison of in situ STM of redox molecules as a singlemolecule spectroscopy with other single-molecule spectroscopies is the fourth observation. In situ STM offers probably the highest structural resolution and in principle electronic or functional resolution of small and intermediate-size molecules. Structural resolution of biological macromolecules is competitive with other single-molecule spectroscopies and complementary by offering both electronic and topographic resolution. Functional resolution of single-molecule electronic properties for biological macromolecules must, however, be approached with adequate reverence for the molecular properties. Relatively high bias voltages are, for example, needed for electronic transmission through the electronically insulating molecular matter. This poses problems as to the behavior of the soft molecules under

J. Phys. Chem. B, Vol. 106, No. 6, 2002 1149 such drastic conditions and calls for appropriate conduct in the use of the theoretical frames. The most important perspective of in situ STM as a singlemolecule spectroscopy compared with other single-molecule spectroscopies is that this spectroscopy could offer visualization and control of the electronic properties of single molecules and biological macromolecules in action. Redox metalloenzymesubstrate complex formation must, for example, be accompanied by significant electronic structural changes compared to the free enzyme. This is apparent from the widely different interfacial electrochemical behavior of the enzyme and enzyme-substrate complex. Such changes could be reflected conspicuously in the in situ STM images. Such imaging and control would refer directly to the natural aqueous environment of the molecules but with reservations as to electrical field effects and spatial confinements. Mapping and control of single-molecule electronic reactivity would provide exciting insight into single-molecule functional individuality (stochastic features) and biomolecular electronic features in resting and active states. Biomolecules may not be suitable as components in working molecular and hybrid-molecular devices, but their multifarious behavior in single-molecule in situ STM is likely to represent all of the features needed for real device construction and offers guidance in these respects. The most direct device perspective of in situ STM of biological macromolecules is instead that structure and functional elements of enzyme electrodes, biosensors, multielectrode arrays, and other functional biotechnology toward the single-molecule level could be disclosed by this spectroscopy. This could again be important for multiple screening of biological liquids and in other contexts where such media are in contact with metallic surfaces. Reported cases of in situ STM spectroscopy are presently small in number. The final, and fifth observation, has been to overview the properties of some important cases and to show how their STM behavior can be interfaced with notions of in situ STM theory. Intermediate-size molecules such as FePP and R,ω-polymethylenedithiol-linked bipyridinium are robust enough to form stable monolayers and exhibit current-voltage spectroscopy. They are, further, large enough to undertake singlemolecule electronic function and still be targets for highresolution imaging. In situ STM of molecules such as these therefore promise to be a spectroscopic tool for single-molecule properties on metallic surfaces. Single-molecule behavior of redox metalloproteins is most accurately and comprehensively illustrated by P. aeruginosa azurin. The properties of this class of molecules have pointed to ways of controlled orientation of well-defined monolayers of fully functional protein molecules, comprehensive characterization of the electronic properties of the immobilized molecules, and single-molecule in situ STM, which approaches a spectroscopic dimension. Azurin on bare and modified Au(111)-surfaces, thus, at the same time, offers broader use of in situ STM of redox proteins and illuminates the prerequisites and limitations of in situ STM as a singlemolecule spectroscopy for biological macromolecules. Acknowledgment. We would like to thank Dr. Esben P. Friis for helpful discussions and for the preparation of Figure 1. Financial support from the Danish Technical Science Research Council and the EU Program INTAS (99-1093) is acknowledged. References and Notes (1) Janson, J.-C.; Ryde´n, L. Protein Purification, Principles, HighResolution Methods and Applications; Wiley VCH: Weinheim, Germany, 1998.

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