Accurately measuring respiratory activity of single living cells by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is a powerful tool to examine the respiratory activity of living cells. However, in SECM measurements of cell respiratory activity, the signal recorded usually also includes the signal corresponding to the cell topography. Therefore, measurements of cell respiratory activity using conventional SECM techniques are not accurate. In the present work, we develop a method for accurate measurement of the respiratory activity of single living cells using SECM. First, cells are immobilized on a glass substrate modified with collagen. Then, a Pt ultramicroelectrode tip of SECM held at −0.50 V is scanned along the central line across a living cell and a SECM scan curve, i.e., the relationship of the tip current versus the displacement (the first scan curve) is recorded with a negative peak. The peak current i_p on this first scan curve is composed of i_p1, which corresponds to the cell respiratory activity and i_p2, which corresponds to the cell topography. In order to isolate the i_p2 component, the cell is killed by exposing it to 1.0 × 10⁻³ mol/L KCN for 10 min. The tip is then scanned again with the same trace over the dead cell, and a second SECM scan curve is recorded. Noting that the topography of the dead cell is the same as that of the living cell, this second scan curve with a negative peak corresponds now only to the cell topography. Thus, i_p2 is obtained from the second SECM scan curve. Finally, i_p1 corresponding to the respiratory activity of the living cell can be accurately calculated using i_p1 = i_p − i_p2. This method can be used to monitor real-time change in the respiratory activity of single cells after exposing them to KBr, NaN₃ and KCN.     

Localization of proteins in paint cross-sections by scanning electrochemical microscopy as an alternative immunochemical detection technique

The qualitative identification of proteinaceous substances, as well as their location within a complex paint stratigraphy, is one of the most challenging issues in the characterization of painting materials. Nevertheless, information on paint components represent a crucial task for studies concerning both the ancient painting techniques adopted and the state of conservation, being fundamental investigations for the selection of appropriate conservation actions. The present research was aimed at developing a new detection approach for the immunochemical localization of ovalbumin in paint cross-sections based on the use of scanning electrochemical microscopy (SECM). The immunochemical analyses were performed using an anti-ovalbumin primary antibody and a secondary antibody labelled with horseradish peroxidase (HRP). SECM measurements were performed in feedback mode using benzoquinone (BQ)/hydroquinone (H₂Q) redox couple. In presence of hydrogen peroxide (H₂O₂), HRP catalyzes the re-oxidation of H₂Q to BQ and the increment of BQ concentration in correspondence of the target protein was detected by SECM through the electrochemical reduction of the regenerated BQ at the microelectrode. Indeed, the localization of ovalbumin was possible thanks to a clear discrimination of SECM currents, achieved by the comparison of the measurements recorded before and after H₂O₂ administration, based on the HRP on/off approach. The method was evaluated both on samples from standard mocks-up and on a historical sample, collected from a Renaissance wood painting. The obtained results were promising, foreseeing a wider application of SECM on cultural heritage researches.     

Fabrication of Active Horseradish Peroxidase Micropatterns with a High Resolution by Scanning Electrochemical Microscopy

We used a new reactive species OH^? to fabricate active horseradish peroxidase (HRP) micropatterns with a high resolution by scanning electrochemical microscopy (SECM) coupled with a carbon fiber disk electrode as the SECM tip. In this method, except for active HRP micropatterns predesigned other regions on a HRP‐immobilized substrate were deactivated by OH^? generated at the tip held at ?1.7 V in 1.0 mol/L KCl containing 2.0×10^?3 mol/L benzoquinone (BQ) (pH 8.0). The feedback mode of SECM with a tip potential of ?0.2 V was used to characterize the active HRP micropatterns in 1.0 mol/L KCl containing 2.0×10^?3 mol/L BQ and 2.0×10^?3 mol/L H₂O₂.     

Scanning electrochemical microscopy for biosurface imaging

Scanning electrochemical microscopy (SECM) is a powerful technique that can provide chemical identity, quantification, and spatiotemporal information on biosurfaces. The ability of SECM for noninvasive and high-resolution electrochemical imaging has made it valuable for the study of cell phenotypes and functions. This review focuses on the latest advances of SECM technique for the biosurface imaging. The SECM measurements of different biomarkers, including oxygen consumption rate and enzyme activity of cell aggregates, redox state of cardiomyocytes, and bacterial metabolic activity, are introduced. The applicability of SECM on membrane permeability measurements, neurotransmitter measurements, and intracellular measurements is discussed.     

Construction and interrogation of enzyme microarrays using scanning electrochemical microscopy - optimisation of adsorption and determination of enzymatic activity

Scanning electrochemical microscopy (SECM) has been used to image and study the catalytic activity of horseradish peroxidase (HRP) immobilised in a patterned fashion onto glass slides. Microarrays of HRP islands could be deposited on amino-modified glass slides using glutaraldehyde crosslinking combined with the SECM being used as a micro-deposition device. The enzymatic activity of the immobilised enzyme on the surface was in the presence of its substrate observed to give rise to substantial positive feedback between the slide and the SECM microelectrode tip. Conversely when either blank slides - or slides coated with HRP which had been subsequently thermally denatured were utilised, these showed negative feedback effects. Various conditions such as enzyme concentration, incubation time and substrate concentration were systematically varied to optimise sensitivity. Regular arrays of HRP could be assembled and when imaged, displayed lower limits of detection of 1.2 × 10(-12) mol ml(-1) of benzoquinone.     

Electrochemical detection of receptor-mediated endocytosis by scanning electrochemical microscopy

We report a scanning electrochemical microscopy (SECM)-based receptor-mediated endocytosis detection method. Epidermal growth factor receptor (EGFR), which is one of the key membrane proteins associated with cancer, was used as a model for receptor-mediated endocytosis. EGFR molecules on the outer cell membrane were detected by SECM by using alkaline phosphatase (ALP) as a labeling enzyme. Since SECM detected the ALP activity on the outer membrane, the procedure helped discriminate the EGFR on the outer membrane from the intracellular EGFR involved in endocytosis. SECM showed a marked decrease in the current responses generated due to ALP activity by 93% on addition of the epidermal growth factor, indicating clearly that EGF triggered the endocytosis, which led to the withdrawal of most EGFRs from the outer membrane.     

Fabricating and imaging carbon-fiber immobilized enzyme ultramicroelectrodes with scanning electrochemical microscopy.

The scanning electrochemical microscope (SECM) is used to image the activity of enzymes immobilized on the surfaces of disk-shaped carbon-fiber electrodes. SECM was used to map the concentration of enzymatically produced hydroquinone or hydrogen peroxide at the surface of a 33-microm diameter disk-shaped carbon-fiber electrode modified by an immobilized glucose-oxidase layer. Sub-monolayer coverage of the enzyme at the electrode surface could be detected with micrometer resolution. The SECM was also employed as a surface modification tool to produce microscopic regions of enzyme activity by using a variety of methods. One method is a gold-masking process in which microscopic gold patterns act as mask for producing patterns of chemical modification. The gold masks allow operation in both a positive or negative process for patterning enzyme activity. A second method uses the direct mode of the SECM to produce covalently attached amine groups on the carbon surface. The amine groups are anchors for attachment of glucose oxidase by use of a biotin/avidin process. The effect of non-uniform enzyme activity was investigated by using the SECM tip to temporarily damage an immobilized enzyme surface. SECM imaging can observe the spatial extent and time-course of the enzyme recovery process.     

Detection and Quantification of Sulfonamide Antibiotic Residues in Milk Using Scanning Electrochemical Microscopy

The use of scanning electrochemical microscopy (SECM) for the qualitative and quantitative determination of sulfapyridine (SPY) in milk is described. A direct competitive immunoassay was performed involving an antibiotic horseradish peroxidase (HRP)‐labeled analog and using selective capture antibodies immobilized on the surface of Protein G‐modified glassy carbon plates. SECM detection was accomplished by means of the sample generator/tip collector (GC) mode involving the reduction of benzoquinone (BQ) generated upon the HRP‐catalyzed oxidation of hydroquinone (HQ) at the modified substrate surface in the presence of H₂O₂. The detection limit for SPY in milk samples was as low as 0.13 ng mL^?1.     

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.     

On the diffusion of ferrocenemethanol in room-temperature ionic liquids: an electrochemical study

The electrochemical behaviour of ferrocenemethanol (FcMeOH) has been studied in a range of room-temperature ionic liquids (RTILs) using cyclic voltammetry, chronoamperomery and scanning electrochemical microscopy (SECM). The diffusion coefficient of FcMeOH, measured using chronoamperometry, decreased with increasing RTIL viscosity. Analysis of the mass transport properties of the RTILs revealed that the Stokes-Einstein equation did not apply to our data. The "correlation length" was estimated from diffusion coefficient data and corresponded well to the average size of holes (voids) in the liquid, suggesting that a model in which the diffusing species jumps between holes in the liquid is appropriate in these liquids. Cyclic voltammetry at ultramicroelectrodes demonstrated that the ability to record steady-state voltammograms during ferrocenemethanol oxidation depended on the voltammetric scan rate, the electrode dimensions and the RTIL viscosity. Similarly, the ability to record steady-state SECM feedback approach curves depended on the RTIL viscosity, the SECM tip radius and the tip approach speed. Using 1.3 μm Pt SECM tips, steady-state SECM feedback approach curves were obtained in RTILs, provided that the tip approach speed was low enough to maintain steady-state diffusion at the SECM tip. In the case where tip-induced convection contributed significantly to the SECM tip current, this effect could be accounted for theoretically using mass transport equations that include diffusive and convective terms. Finally, the rate of heterogeneous electron transfer across the electrode/RTIL interface during ferrocenemethanol oxidation was estimated using SECM, and k(0) was at least 0.1 cm s(-1) in one of the least viscous RTILs studied.     

Electrochemical evidence for asialoglycoprotein receptor – mediated hepatocyte adhesion and proliferation in three dimensional tissue engineering scaffolds

Asialoglycoprotein receptor (ASGPR) is one of the recognition motifs on the surface of hepatocytes, which promote their adhesion to extracellular matrix in liver tissue and appropriate artificial surfaces. ASGPR-mediated adhesion is expected to minimize trans-differentiation of hepatocytes in vitro that is generally observed in integrin-mediated adhesion. The aim of the present study is to verify the role of ASGPR in hepatocyte adhesion and proliferation in scaffolds for hepatic tissue engineering. Scanning Electrochemical Microscopy (SECM) is emerging as a suitable non-invasive analytical tool due to its high sensitivity and capability to correlate the morphology and activity of live cells. HepG2 cells and rat primary hepatocytes cultured in Polyvinyl alcohol (PVA)/Gelatin hydrogel scaffolds with and without galactose (a ligand for ASGPR) modification are studied using SECM. Systematic investigation of live cells cultured for different durations in scaffolds of different compositions (9:1 and 8:2 PVA:Gelatin with and without galactose) reveals significant improvement in cell–cell communication and proliferation on galactose incorporated scaffolds, thereby demonstrating the positive influence of ASGPR-mediated adhesion. In this work, we have also developed a methodology to quantify the respiratory activity and intracellular redox activity of live cells cultured in porous tissue engineering scaffolds. Using this methodology, SECM results are compared with routine cell culture assays viz., MTS ((1-Oxyl-2,2,5,5,-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate) and Albumin assays to demonstrate the better sensitivity of SECM. In addition, the present study demonstrates SECM as a reliable and sensitive tool to monitor the activity of live cells cultured in scaffolds for tissue engineering, which could be used on a routine basis.     

Study on Electrocatalytic Activity of Platinum for Hydrogen Oxidation in Acidic Solution by Scanning Electrochemical Microscopy

The electrocatalytic activity of platinum for hydrogen oxidation in 0.01 M H₂SO₄ + 0.1 MNa₂SO₄ solution has been investigated by scanning electrochemical microscopy (SECM) technique. The cyclic voltammogram (CV), approach curve, area scan imaging and chronoamperometric methods have been used. The results indicate that the imaging capability of the SECM feedback mode can be used more efficiently to visually identify materials' electrocatalytic activity, compared with the approach curve method for identification of the conductive or insulating nature of a surface. The SECM imaging method has demonstrated the effects of Pt substrate potential on the electrocatalytic oxidation of hydrogen under a constant tip potential. It is found that the more positive the Pt substrate potential, the lower the electrocatalytic activity of the Pt. Furthermore, the chronoamperometric results support the variation of the electrocatalytic activity with the Pt substrate potential as well.     

Scanning electrochemical microscopic study of hydrogen oxidation and evolution at electrochemically deposited pt nanoparticulate electrode incorporated in polyaniline.

Scanning Electrochemical Microscopy (SECM) feedback mode and substrate generation-tip collection (SG-TC) mode coupled with chronoamperometric approach were used to investigate H2 oxidation and hydrogen evolution reaction (HER) at a polyaniline (PAni) coated highly oriented pyrolytic graphite (HOPG) electrode. Using the former mode, the heterogeneous electron transfer (ET) kinetics for H2 oxidation was studied, while the latter mode allowed mapping of the distribution of local [H2] at the nanoparticulate/aqueous interface, followed by monitoring the transients at the tip. These preliminary studies demonstrate that SECM is useful in evaluating the activity of nanophase electrocatalysts. Particularly, if one employs nanometer-sized tips or hydrodynamic microjet electrodes where the mass transfer rate is significantly high, it should be possible to investigate the ET kinetics more accurately.     

In situ scanning electrochemical microscopy (SECM) detection of metal dissolution during zinc corrosion by means of mercury sphere-cap microelectrode tips

This work presents a scanning electrochemical microscopy (SECM)-based in situ corrosion probing methodology that is capable of monitoring the release of zinc species in corrosion processes. It is based on the use of Hg-coated Pt microelectrodes as SECM tips, which offer a wider negative potential range than bare platinum or other noble-metal tips. This allows for the reduction of zinc ions at the tip to be investigated with low interference from hydrogen evolution and oxygen reduction from aqueous solutions. The processes involved in the corrosion of zinc during its immersion in chloride-containing solutions were successfully monitored by scanning the SECM tip, set at an adequate potential, across the sample either in one direction or in the X-Y plane parallel to its surface. In this way, it was possible to detect the anodic and cathodic sites at which the dissolution of zinc and the reduction of oxygen occurred, respectively. Additionally, cyclic voltammetry (CV) or constant potential measurements were used to monitor the release of zinc species collected at the tip during an SECM scan.     

Investigation of the interactions between silver nanoparticles and Hela cells by scanning electrochemical microscopy

The interactions between Hela cells and silver nanoparticles (AgNPs) have been studied by scanning electrochemical microscopy (SECM) with both IrCl(6)(2-/3-) and Fe(CN)(6)(3-/4-) as the dual mediators. IrCl(6)(2-), which can be produced in situ and react with AgNPs, is used as the mediator between the AgNPs on the cells and the SECM tip. Another redox couple, Fe(CN)(6)(3-/4-), which has a similar hydrophilicity to IrCl(6)(2-/3-), but cannot react with AgNPs, is also employed for the contrast experiments. The cell array is cultured successfully onto a Petri dish by microcontact printing (muCP) technique, which can provide a basic platform for studying of single cells. The approach curve and line scan are the two methods of SECM employed here to study the Hela cells. The former can provide the information about the interaction between Hela cells and AgNPs whereas the later gives the cell imaging. The permeability of cell membranes and morphology are two main factors which have effects on the feedback mode signals when K(3)Fe(CN)(6) is used as the mediator. The permeability of the cell membranes can be ignored after interaction with high concentration of AgNP solution and the height of the Hela cells is slightly decreased in this process. The kinetic rate constants (k(0)) between IrCl(6)(2-) and Ag on the Hela cell can be evaluated using K(3)IrCl(6) as the mediator, and they are increased with the higher concentrations of the AgNP solutions. The k(0) is changed about 10 times from 0.43 +/- 0.04 x 10(-4) to 1.25 +/- 0.07 x 10(-4) and to 3.93 +/- 1.9 x 10(-4) cm s(-1) corresponding to 0, 1 and 5 mM of AgNO(3) solution. The experimental results demonstrate that the AgNPs can be adsorbed on the cell surface and detected by SECM. Thus, the amount of AgNPs adsorbed on cell membranes and the permeability or morphology changes can be investigated simultaneously using this approach. The dual mediator system and cell array fabricated by muCP technique can provide better reproducibility because they can simplify experiments, and provide a platform for further single cell detection.     

Competitive decay pathways of the radical ions formed by photoinduced electron transfer between quinones and 4,4'-dimethoxydiphenylmethane in acetonitrile

The reactivity of the cation radical of (4-MeOC6H4)2CH2 photosensitized by 1,4-benzoquinone (BQ), 2,5-dichloro-1,4-benzoquinone (Cl2BQ), and tetrachloro-1,4-benzoquinone (chloranil, CA) was investigated in acetonitrile. The main photoreaction products obtained by steady-state irradiation were identified to be: (4-MeOC6H4)2-CHOC6H4OH, sensitized by BQ; (4-MeOC6H4)2CHCl, sensitized by Cl2BQ; (4-MeOC6H4)2CHOH, sensitized by CA. The mechanism of their formation was investigated by nanosecond laser flash photolysis that allowed transient species (radical ions, neutral radicals, and ions) to be detected and characterized in terms of absorption spectra, formation quantum yields, and decay rate constants. For all systems, the interaction between the triplet quinone (Q) and (4-MeOC6H4)2CH2 produced the corresponding radical ions (quantum yield phi > or = 0.72) which mainly decay by back electron transfer processes. Less efficient reaction routes for the radical ions Q*- and (4-MeOC6H4)2CH2*+ were also: i) the proton-transfer process with the formation of the radical (4-MeOC6H4)2CH* by use of Cl2BQ; ii) the hydrogen-transfer process with the formation of the cation (4-MeOC6H4)2CH+ in the case of CA. Instead. BQ sensitized a much higher yield of BOH* and (4-MeOC6H4)2CH*, mainly by the direct interaction of triplet BQ with (4-MeOC6H4)2CH2. It was also shown that the presence of salts decreases significantly the rate of the back electron transfer process and enhances the quantum yields of formation of the neutral radicals and ions when Cl2BQ and CA are used, respectively. The behavior of BQ*-, Cl2BQ*-, and CA*- appears to be mainly determined by the Mulliken charges on the oxygen atom obtained from quantum mechanical calculations with the model B3LYP/6-311G(d,p). Spin densities seem to be much less important.     

Fabrication and Use of Dual‐function Iridium Oxide Coated Gold SECM Tips. An Application to pH Monitoring above a Copper Electrode Surface during Nitrate Reduction.

The fabrication of a gold microelectrode modified with iridium oxide film (IrO_x) and its use as tip with a dual function in SECM experiments is reported. The defective structure of the coating onto the microelectrode surface was used as strategy to combine the advantages of both amperometric (for current‐distance determination) and potentiometric (for pH sensing) SECM operation modes. Approach curves, using oxygen and hexaammineruthenium(III) as redox mediators, were obtained without significant loss of the performance and reproducibility of the potentiometric pH response. This allowed the precise positioning of the proposed tip above a substrate in SECM experiments and, subsequently, to monitor pH at the substrate surface. The IrO_x modified microelectrode was applied successfully in SECM experiments involving the local proton consumption during the nitrate reduction at a copper cathode surface.     

Photoelectrochemistry on Ru(II)-2,2'-bipyridine-phosphonate-derivatized TiO2 with the I3-/I- and quinone/hydroquinone relays. Design of photoelectrochemical synthesis cells

Photocurrent measurements have been made on nanocrystalline TiO2 surfaces derivatized by adsorption of a catalyst precursor, [Ru(tpy)(bpy(PO3H2)2)(OH2)]2+, or chromophore, [Ru(bpy)2 (bpy(PO3H2)2)]2+ (tpy is 2,2':6',2' '-terpyridine, bpy is 2,2'-bipyridine, and bpy(PO3H2)2 is 2,2'-bipyridyl-4,4'-diphosphonic acid), and on surfaces containing both complexes. This is an extension of earlier work on an adsorbed assembly containing both catalyst and chromophore. The experiments were carried out with the I3-/I- or quinone/hydroquinone (Q/H2Q) relays in propylene carbonate, propylene carbonate-water mixtures, and acetonitrile-water mixtures. Electrochemical measurements show that oxidation of surface-bound Ru(III)-OH2(3+) to Ru(IV)=O(2+) is catalyzed by the bpy complex. Addition of aqueous 0.1 M HClO4 greatly decreases photocurrent efficiencies for adsorbed [Ru(tpy)(bpy(PO3H2)2)(OH2)]2+ with the I3-/I- relay, but efficiencies are enhanced for the Q/H2Q relay in both propylene carbonate-HClO4 and acetonitrile-HClO4 mixtures. The dependence of the incident photon-to-current efficiency (IPCE) on added H2Q in 95% propylene carbonate and 5% 0.1 M HClO4 is complex and can be interpreted as changing from rate-limiting diffusion to the film at low H2Q to rate-limiting diffusion within the film at high H2Q. There is no evidence for photoelectrochemical cooperativity on mixed surfaces containing both complexes with the IPCE response reflecting the relative surface compositions of the two complexes. These results provide insight into the possible design of photoelectrochemical synthesis cells for the oxidation of organic substrates.     

Simultaneous detection of uric acid and glucose on a dual-enzyme chip using scanning electrochemical microscopy/scanning chemiluminescence microscopy

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1∼2 μm) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s⁻¹ while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable.     

Quantification of the surface diffusion of tripodal binding motifs on graphene using scanning electrochemical microscopy

The surface diffusion of a cobalt bis-terpyridine, Co(tpy)(2)-containing tripodal compound (1·2PF(6)), designed to noncovalently adsorb to graphene through three pyrene moieties, has been studied by scanning electrochemical microscopy (SECM) on single-layer graphene (SLG). An initial boundary approach was designed in which picoliter droplets (radii ~15-50 μm) of the tripodal compound were deposited on an SLG electrode, yielding microspots in which a monolayer of the tripodal molecules is initially confined. The time evolution of the electrochemical activity of these spots was detected at the aqueous phosphate buffer/SLG interface by SECM, in both generation/collection (G/C) and feedback modes. The tripodal compound microspots exhibit differential reactivity with respect to the underlying graphene substrate in two different electrochemical processes. For example, during the oxygen reduction reaction, adsorbed 1·2PF(6) tripodal molecules generate more H(2)O(2) than the bare graphene surface. This product was detected with spatial and temporal resolution using the SECM tip. The tripodal compound also mediates the oxidation of a Fe(II) species, generated at the SECM tip, under conditions in which SLG shows slow interfacial charge transfer. In each case, SECM images, obtained at increasing times, show a gradual decrease in the electrochemical response due to radial diffusion of the adsorbed molecules outward from the microspots onto the unfunctionalized areas of the SLG surface. This response was fit to a simple surface diffusion model, which yielded excellent agreement between the two experiments for the effective diffusion coefficients: D(eff) = 1.6 (±0.9) × 10(-9) cm(2)/s and D(eff) = 1.5 (±0.6) × 10(-9) cm(2)/s for G/C and feedback modes, respectively. Control experiments ruled out alternative explanations for the observed behavior, such as deactivation of the Co(II/III) species or of the SLG, and verified that the molecules do not diffuse when confined to obstructed areas. The noncovalent nature of the surface functionalization, together with the surface reactivity and mobility of these molecules, provides a means to couple the superior electronic properties of graphene to compounds with enhanced electrochemical performance, a key step toward developing dynamic electrode surfaces for sensing, electrocatalysis, and electronic applications.