Direct Electrochemical and Spectroscopic Assessment of Heme

Even more importantly, cyt c is redox active through its Fe(II/III) heme couple, permitting direct electrochemical investigation of its integrity when...
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Anal. Chem. 2007, 79, 2303-2311

Direct Electrochemical and Spectroscopic Assessment of Heme Integrity in Multiphoton Photo-Cross-Linked Cytochrome c Structures Jennifer L. Lyon, Ryan T. Hill, Jason B. Shear, and Keith J. Stevenson*

Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station MC A5300, Austin, Texas 78712

Multiphoton excitation (MPE) lithography offers an effective, biocompatible technique by which three-dimensional architectures comprised of proteins, enzymes, and other relevant materials may be fabricated for use in biological studies involving cellular signal transduction and neuronal networking. We present a series of studies designed to investigate the integrity of cytochrome c (cyt c) photocross-linked via MPE. Specifically, we have used electrochemical methods and surface-enhanced Raman spectroscopy (SERS) to determine whether photo-cross-linked cyt c retains its well-characterized Fe(II/III) heme redox activity. Cyt c is observed to retain its native FeII/III electron-transfer properties, as the apparent electrontransfer rate constant, k0ET, for cyt c photo-cross-linked onto an indium-doped tin oxide (ITO) substrate was 8.4 ( 0.2 s-1, on the same order of magnitude as literature values though somewhat slower than other immobilized cyt c studies, most likely due to unoptimized entrapment in the photo-cross-linked matrix. SERS data reveals peaks corresponding to vibrational modes of an intact porphyrin ring with the Fe center intact. Cyt c has also been shown to demonstrate peroxidase-like activity, and we have evaluated the turnover rate of H2O2 at photo-cross-linked matrices relative to that at adsorbed monolayers of cyt c on glass substrates. The photo-cross-linked cyt c samples demonstrate apparent Michaelis-Menten parameters of Vm ) 0.34 fmol/s and kcat/Km on the order of 104 s-1 M-1, in agreement with previously published results for aqueous cyt c. Fluorescence data obtained for mediated H2O2 turnover also indicated enzymatic activity specifically at photo-cross-linked cyt c structures. The fabrication of three-dimensional (3D) biomolecular architectures has generated much interest for its potential applications in a number of biological schemes, including but not limited to neuronal networking, microfluidics, and cellular manipulation.1-7 * To whom correspondence should be addressed. Phone: (512) 232-9160. Fax: (512) 471-8696. E-mail: [email protected]. (1) Cunningham, L. P.; Bush, K. A.; Pins, G. D.; Campagnola, P. J. Polym. Mater. Sci. Eng. Prepr. 2006, 94, 46. (2) Pins, G. D.; Bush, K. A.; Cunningham, L. P.; Campagnola, P. J. J. Biomed. Mater. Res., Part A 2006, 78, 194-204. (3) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352-1355. 10.1021/ac0619377 CCC: $37.00 Published on Web 02/09/2007

© 2007 American Chemical Society

An attractive alternative to the cytotoxic conditions inherent to many hard lithography techniques is multiphoton excitation (MPE) lithography in physiological environments, which has been shown to effectively cross-link proteins in the presence of existing cellular environments without causing damage to them.5-7 MPE cross-linking typically occurs through nonlinear excitation of a photosensitizer (or in some cases, the excitation of the photocross-linking protein itself8) by a near-IR excitation source, which then induces irreversible cross-linking of residues associated with the protein of interest.9 Spatial control of MPE-cross-linked products is attainable by focusing a femtosecond-pulsed laser to submicrometer dimensions within an aqueous solution containing the molecule to be cross-linked (and photosensitizer, if necessary), allowing the fabrication of 3D pillars, freestanding cables, and other complex architectures.5-8 Some relatively simple structures prepared by MPE are shown in Figure 1. The actual cross-linking mechanism has been shown to involve both type I (free radical) and type II (singlet oxygen) reactions, depending on fabrication parameters such as photo-cross-linking solution composition and excitation wavelength.10-12 However, the exact functional groups or residues within individual proteins that undergo cross-linking to form the final 3D product are not well-characterized. Some experiments to this end have involved isolation of individual protein residues suspected to be responsible for cross-linking in attempts to elucidate reaction mechanisms,10,11,13 but a more general, qualitative approach involves confirming that residues involved in critical protein activities are not damaged to a prohibitive level as a result of cross-linking. For example, proteins with native affinities for certain molecules in their monomeric forms can be evaluated in (4) Cukierman, E.; Pankov, R.; Yamada, K. M. Curr. Opin. Cell Biol. 2002, 14, 633-640. (5) Kaehr, B.; Ertas, N.; Nielson, R.; Allen, R.; Hill, R. T.; Plenert, M.; Shear, J. B. Anal. Chem. 2006, 78, 3198-3202. (6) Allen, R.; Nielson, R.; Wise, D. D.; Shear, J. B. Anal. Chem. 2005, 77, 50895095. (7) Kaehr, B.; Allen, R.; Javier, D. J.; Currie, J.; Shear, J. B. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16104-16108. (8) Hill, R. T.; Lyon, J. L.; Allen, R.; Stevenson, K. J.; Shear, J. B. J. Am. Chem. Soc. 2005, 127, 10707-10711. (9) Shear, J. B. Anal. Chem. 1999, 71, 598A-605A. (10) Spikes, J. D.; Shen, H.-R.; Kopeckova, P.; Kopecek, J. Photochem. Photobiol. 1999, 70, 130-137. (11) Shen, H.-R.; Spikes, J. D.; Kopecek, P. J. Photochem. Photobiol., B 1996, 34, 203-210. (12) Verweij, H.; Van Steveninck, J. Photochem. Photobiol. 1982, 35, 265-267.

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direct electrochemistry of cyt c has been exhaustively investigated in the literature, allowing comparison between our experimental system and those previously reported.16-21 Herein we report evidence of a robust MPE cross-linking mechanism for cyt c, elucidated through not only direct electrochemistry but also Raman spectroscopy. We demonstrate that MPE-fabricated cyt c structures retain a significant Fe(II/III) redox response, as well as peroxidase-like enzymatic activity at the cyt c heme site. We are able to extensively probe and obtain quantitative data from photo-cross-linked matrixes with areas as small as 10 × 17 µm2 using these techniques. Using quantitative parameters obtained for electron transfer and enzymatic activity of photo-cross-linked cyt c, we can also directly compare the asfabricated structures’ properties to those of monomeric, aqueous cyt c. The methods presented here for cyt c analysis could easily be extended to the investigation of other MPE-cross-linked hemoproteins.

Figure 1. Differential interference contrast images of photo-crosslinked cyt c structures created via 2D raster scanning using a confocal scan box. (a) Parallel lanes created on ITO from a solution of 100 mg/mL cyt c and 4.5 mM FAD. The confocal scan box was used while translating the mechanical stage at a speed of 3 µm/s. Inset: A single structure created on a glass substrate by scanning four times over an area 10 × 17 µm2, created from 200 mg/mL cyt c solution. (b) Diagonal lines created by combining raster scanning with horizontal translation of the mechanical stage at 7 µm/s through a solution of 100 mg/mL cyt c and. 4.5 mM FAD.

terms of these affinities after post-cross-linking. Basu and Campagnola have used this approach to investigate the enzymatic activity of MPE-fabricated alkaline phosphatase structures via fluorescence assays of a phosphatase-catalyzed fluorescein,14 and the Shear group has demonstrated retention of avidin-biotin affinity through both avidin coupling to cross-linked matrices of biotinylated proteins6 and biotin affinity that scales directly with avidin content within 3D microstructures.7 We sought to continue the investigation of MPE cross-linking mechanisms by assessing the retention of Fe(II/III) heme activity in photo-cross-linked cytochrome c (cyt c), fabricated in aqueous physiological medium both with and without a flavin adenine dinucleotide (FAD) photosensitizer. Cyt c is unique among the hemoproteins in that its heme is covalently bound to surrounding cysteine residues rather than being electrostatically retained within a hydrophobic cleft of amino acids.15 This covalent linkage of the porphyrin ring within cyt c stabilizes the protein’s native conformation; therefore, the presence of an intact heme postfabrication could confirm a nondenaturing photo-cross-linking mechanism. Even more importantly, cyt c is redox active through its Fe(II/ III) heme couple, permitting direct electrochemical investigation of its integrity when fabricated onto an electrode support. The (13) Shen, H. R.; Spikes, J. D.; Smith, C. J.; Kopecek, J. J. Photochem. Photobiol., A 2000, 133, 115-122. (14) Basu, S.; Campagnola, P. J. Biomacromolecules 2004, 5, 572-579. (15) Turano, P.; Lu, Y. In Handbook on Metalloproteins; Bertini, I., Sigel, A., Sigel, H., Eds.; Marcel Dekker: New York, 2001; pp 269-286.

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EXPERIMENTAL SECTION Reagents. Cytochrome c (cyt c) from bovine heart was purchased from Sigma-Aldrich (St. Louis, MO, C3131) and was stored in a desiccator at -20 °C until immediately before use. Gel permeation chromatography using an Amersham Biosciences HiPrep 16/60 column indicated that >98% of cyt c was in its Fe(III) oxidation state as received and less than 2% was denatured (calculated by integration of peaks corresponding to both free heme and oligomeric cyt c); therefore, no further purification of cyt c was performed prior to use. Flavin adenine dinucleotide (FAD; Sigma-Aldrich, F6625) was also stored in a desiccator at -20 °C. Bovine serum albumin (BSA; Equitech-Bio, Kerrville, TX, BAH64-0100) and methylene blue (Sigma-Aldrich, M4159) were stored at 4 °C and room temperature, respectively. Amplex red was purchased from Invitrogen, diluted to 0.1 M in DMSO immediately upon arrival, divided into 5 µL aliquots, and stored at -20 °C until immediately before use. Hydrogen peroxide (H2O2) (30% w/v) was purchased from Fisher Scientific (Fairlawn, NJ, H325-100) and stored at 4 °C at all times. H2O2 standards were prepared by serial dilution of 30% w/v H2O2 in 0.1 M K2HPO4/ 0.05 M citric acid buffer, pH ) 5.0. All other reagents were obtained from Sigma-Aldrich and used as received. All solutions were prepared using ultrapure (18 MΩ cm) H2O. MPE Fabrication. Cross-linked protein structures were fabricated on a Zeiss Axiovert (inverted) microscope using a femtosecond titanium: sapphire (Ti:S) laser (Spectra Physics, Mountain View, CA) typically tuned to 740 nm. The laser output was adjusted to approximately fill the back aperture of a highpower objective (Zeiss Fluar, 100×/1.3 numerical aperture, oil immersion). Average laser powers entering the microscope were 20-60 mW. Photo-cross-linked protein structures were created by raster scanning the focused laser beam within the focal plane (16) Burgess, J. D.; Hawkridge, F. M. In Electroanalytical Methods for Biological Materials; Brajter-Toth, A., Chambers, J. Q., Eds.; Marcel Dekker: New York, 2002; pp 109-142. (17) Runge, A. F.; Saavedra, S. S. Langmuir 2003, 19, 9418-9424. (18) El Kasmi, A.; Leopold, M. C.; Galligan, R.; Robertson, R. T.; Saavedra, S. S.; El Kacemi, K.; Bowden, E. F. Electrochem. Commun. 2002, 4, 177-181. (19) Willit, J. L.; Bowden, E. F. J. Phys. Chem. 1990, 94, 8241-8246. (20) Koller, K. B.; Hawkridge, F. M. J. Am. Chem. Soc. 1985, 107, 7412-7417. (21) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J. Electroanal. Chem. Interfacial Electrochem. 1984, 161, 355-376.

using galvanometer-driven mirrors (BioRad MRC600 confocal scanner). Although some day-to-day optimization of laser power was required due to possible changes in beam alignment and laser pulse properties, once appropriate conditions were determined, structures generally could be fabricated reproducibly with errors of ∼0.5 to 1 µm. Geometric Estimates of Photo-Cross-Linked Cyt c Matrixes. To determine the volume of photo-cross-linked cyt c structures, in situ atomic force microscopy (AFM) was performed using a Digital Instruments Bioscope microscope in combination with a Nanoscope IV controller (Veeco Metrology, Santa Barbara, CA). Samples were probed in a buffered aqueous environment consisting of 18 mM Na2HPO4/0.1 M NaClO4 (pH 7.40 ( 0.03) using triangular silicon nitride cantilever tips (cantilever length, 100 µm; resonant frequency, 11 kHz; spring constant, 0.02 N/m; Olympus, OMCL-TR400PSA). Direct Electrochemistry. Direct electrochemical analysis of cyt c was performed using a CH Instruments 440 potentiostat (Austin, TX) interfaced with CH Instruments 440 software. The working electrodes were composed of indium tin oxide (ITO, 1520 Ω/square; Metavac, Inc., Holtsville, NY)-coated glass coverslips of no. 1 thickness (Erie Scientific, Portsmouth, NH). Electrical contact was made with ITO using indium foil (Alfa Aesar, Ward Hill, MA). A Pt wire counter electrode and Ag/AgCl reference electrode (saturated KCl, World Precision Instruments) were also used. All reported potentials are versus Ag/AgCl (saturated KCl). For direct electrochemical analysis, cyt c microstructures were constructed within a Teflon chamber fitted with a viton O-ring to expose 0.242 cm2 electrode area using solutions of 100 mg/mL cyt c and 4.5 mM FAD in 18 mM Na2HPO4/0.1 M NaClO4 buffer (pH 7.40 ( 0.03). The chamber was left intact, and the crosslinked structure was rinsed 30 times with Na2HPO4/NaClO4 buffer prior to its use as a single-compartment, 2 mL cell for electrochemical analysis. Raman Spectroscopy. Raman spectroscopy was performed with a Renishaw InVia system using 514.5 nm incident radiation operating at 10% of its maximum power. A 50× objective was used, resulting in an approximately 2 µm spot size. Prior to acquiring each spectrum, samples were photobleached for 20 s to reduce fluorescence background. Raman spectra shown here are averaged over nine 10 s acquisitions. For Raman analysis, cyt c microstructures were constructed from a photo-cross-linking solution of the same composition as that used for direct electrochemical analysis. Additionally, the ITO substrate was pretreated prior to photo-cross-linking by soaking in 200 mg/mL BSA in phosphate/perchlorate buffer to promote highly specific adsorption of Ag particles for SERS enhancement, following a previously described protocol.8 For SERS enhancement, the structures were modified with electrostatically bound Ag nanoparticles using a procedure that has been described in our previous publications.8,22 Briefly, structures were incubated with protein-Au conjugates (EY Laboratories, San Mateo, CA) for 10 min in 2 mM borate buffer (pH ) 8-9) followed by 20 rinses with 10 µL of H2O. A silver enhancement solution kit (EY Labs) was applied to the structures for ∼13 min, during which time Ag ions were catalytically reduced at the nanoparticle-protein sites. The structures were then subjected to one final cycle of 30 rinses with H2O. (22) Hill, R. T.; Shear, J. B. Anal. Chem. 2006, 78, 7022-7026.

Enzymatic Activity. Electrochemical experiments for assessment of cyt c enzymatic activity were performed at room temperature (23 ( 2 °C) using an Autolab PGSTAT30 potentiostat interfaced with Autolab GPES version 4.9 software. For enzymatic analysis, 200 mg/mL cyt c was photo-cross-linked directly onto a glass coverslip without the use of FAD, within an in-housefabricated poly(dimethylsiloxane) (PDMS) well of area 8.55 mm2, pretreated by soaking in BSA/methylene blue to prevent nonspecific protein adsorption.22 H2O2 standards were analyzed in a standard single-compartment, glass three-electrode cell of 5 mL volume, using a Ag/AgCl reference electrode and a Au wire counter electrode (Strem). The working electrode was a 10 µm diameter glassy carbon (GC) disk-in-glass microelectrode (Cypress Systems). It was polished successively with 0.3 and 0.05 µm alumina slurries on a polishing cloth (Buehler) to a mirror finish, ultrasonically cleaned for 20 min in ultrapure H2O, and used immediately in experiments. A Faraday cage was used to shield the three-electrode cell from external noise. All reported potentials are versus Ag/AgCl (saturated KCl). Wide-field fluorescence measurements were made on the Axiovert microscope using a mercury lamp excitation source and a 10× Fluar 0.5 numerical aperture objective. Fluorescence emission was collected using standard red filter sets (Chroma, Rockingham, VT). Fluorescence was detected using a 12-bit 1392 × 1040 element CCD (CoolSnap HQ; Photometrics, Tucson, AZ). Data were processed using Image J and Metamorph (Universal Imaging, Sunnyvale, CA) image analysis software. RESULTS AND DISCUSSION Geometric Estimates of Photo-Cross-Linked Cyt c Matrixes. As we have demonstrated previously,8,22 cyt c structures may be cross-linked into a variety of architectures on both ITO and glass substrates. In the present experiments, cyt c structures are fabricated at a rate of ∼0.005 mm2 area/min, though our substrates are of comparatively larger areas (0.242 cm2 and 8.55 mm2 for ITO and glass, respectively). Thus, while at first glance the images in Figure 1 may imply that the matrixes occupy a significant portion of the substrates, in reality they cover only ∼0.1% of the area contained in our experimental cells, depending on the length of photo-cross-linking time chosen. However, the amount of cyt c contained within these dense “islands” on the substrate is enough to impart significant changes in heme-related activity relative to that of a substrate containing only nonspecifically adsorbed cyt c (vide infra). In order to quantitatively assess the extent of activity at these structures, it is necessary to estimate the number of cyt c molecules present. We achieved this by determining the volume of cyt c matrixes using in situ atomic force microscopy (AFM) in the same buffer used for their fabrication. Through this method, the average height of the photo-cross-linked matrixes was determined to be 200 nm (see the Supporting Information). With the use of this information in conjunction with photo-cross-linked area calculations and the volume of a single cyt c molecule,17 the approximate number of cyt c molecules contained within a photocross-linked matrix of 0.095 mm2 area (the area used for direct electrochemical studies described below) was determined to be 8.22 × 1011. We note that this calculation assumes a solid matrix of cyt c, though the structures appear somewhat porous in images shown in our previous work.8 However, it should be noted that Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

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Figure 2. Cathodic peak current response as a function of scan rate for 0.095 mm2 photo-cross-linked cyt c (closed squares) and adsorbed cyt c (open squares) obtained via cyclic voltammetry. Inset: peak current data obtained at scan rates e250 mV/s, shown for clarity. CV parameters: ITO working electrode (area: 0.242 cm2), Ag/AgCl reference electrode, Pt wire counter electrode. Photo-crosslinking solutions consisted of 100 mg/mL cyt c/4.5 mM FAD in 18 mM Na2HPO4/0.1 M NaClO4 supporting electrolyte, pH ) 7.40.

these images were collected ex situ (i.e., without buffer present), and as such the dehydrated protein structures exhibit a more porous appearance. When fully hydrated, as permitted by the present in situ AFM conditions, the structures appear much less porous, and their average dimensions in this case support our calculation of the number of cyt c molecules contained within this geometric structure. To make this clearer we have added AFM images displaying the variation of structure height (Figure S1), along with histograms indicating that the average thickness is ca. 200 nm. Direct Electrochemistry of Photo-Cross-Linked Cyt c Matrixes. As stated previously, cyt c was chosen from the many proteins amenable to MPE cross-linking due to the inherent, welldocumented redox activity of its Fe(II/III) heme.16-21 To evaluate whether cyt c retains its redox activity upon photo-cross-linking, 3D matrixes were cross-linked directly onto an indium-doped tin oxide (ITO)-coated cover glass, which was then used as a working electrode to investigate the cyt c Fe(II/III) redox couple. Cyt c has demonstrated robust redox activity when adsorbed at ITO; thus, control experiments were performed in which an ITO-coated cover glass was exposed to the photo-cross-linking solution for the same amount of time as it would be during MPE cross-linking and also evaluated via cyclic voltammetry. Cyclic voltammograms obtained from both adsorbed cyt c controls and photo-cross-linked cyt c samples (Supporting Information, Figure S2) demonstrate both cathodic and anodic peaks centered about E1/2 ) -0.01 V versus Ag/AgCl, in good agreement with previously published data for cyt c adsorbed at ITO.17-21 This voltammetry was observed to be reproducible for upward of 25 consecutive scans, slightly decreasing in intensity following the initial scan and then remaining consistent for subsequent scans. The ratios of the integrated charge, Qc/Qa, calculated from CVs obtained at 50 mV/s indicated that the Fe(II)/Fe(III) conversion is quasireversible in both photo-cross-linked and adsorbed cyt c samples, with Qc/Qa ) 0.84 and 1.12 for photocross-linked and adsorbed cyt c, respectively. On the basis of our calculated charge ratios, there appears to be a greater number of redox-active Fe(II) centers present in photo-cross-linked cyt c structures relative to adsorbed cyt c. Figure 2 shows the cathodic peak current obtained as a function of scan rate for both an 2306 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

adsorbed cyt c control and an ITO cover glass modified with 0.095 mm2 photo-cross-linked cyt c. An increase in peak current upon addition of photo-cross-linked cyt c is observed at all scan rates (data taken at scan rates e250 mV/s is shown in the inset for clarity). An increase in anodic peak currents is also observed (data not shown). Figure 2 shows that a significant amount of redoxactive cyt c is adsorbed at ITO when subjected to an incubation time matching that of the photo-cross-linking solution preparation conditions; we therefore assume that this amount of adsorbed cyt c is also present in all of our photo-cross-linked samples, in addition to the cyt c contained within the photo-cross-linked matrix itself. With the use of the equation Γ ) Q/nFA, where Γ is the surface coverage of an adsorbed redox species in mol/cm2, Q is the charge (in C) determined by integrating the cyclic voltammetry peaks, n is the number of electrons transferred in the redox reaction, F is Faraday’s constant (96 485 C/mol), and A is the area of the working electrode, the number of electroactive cyt c molecules adsorbed on the ITO electrode for a control experiment was calculated at 9.99 × 1011. (This value corresponds to 6.84 pmol/cm2 surface coverage, which is somewhat lower than a complete monolayer surface coverage and is reflective of the high ionic strength of the buffer solution.) Assuming that the same amount of cyt c is nonspecifically adsorbed at ITO in addition to the presence of the photo-cross-linked matrixes, the total amount of cyt c contained in a photo-cross-linked sample is the sum of the adsorbed cyt c and the amount of cyt c determined to be present in a 0.095 mm2 matrix, or 1.82 × 1012 molecules. Under this assumption, the number of cyt c molecules in a photo-crosslinked sample is approximately double relative to that of the control. The average percent increase of the cathodic peak current across all scan rates is 84.9% for the data shown in Figure 2, suggesting that nearly all of the cyt c molecules contained within the photo-cross-linked matrix are redox active and able to participate in electron transfer at ITO. It is important to note that the reproducibility between photocross-linked samples is rather poor due to fluctuating conditions in the photo-cross-linking solution and exposure time of this solution to ITO. Additionally, the high concentration of 100-200 mg/mL cyt c used in the photo-cross-linking solution is subject to protein aggregation, potentially inducing denaturation of aggregated cyt c molecules.16 Furthermore, the amount of time required to fabricate 0.095 mm2 cyt c via photo-cross-linking is approximately 20 min, during which cyt c can aggregate, presumably with varying degrees of redox activity, and randomly deposit on ITO (i.e., with no preferred orientation). To demonstrate the effects of the adsorption of cyt c from solutions containing different concentrations of cyt c on observed voltammetric response, a CV of cyt c adsorbed at ITO from a 30 µM solution is also shown in Figure S2b. While the well-behaved voltammetry observed in this case closely resembles previous findings of cyt c adsorbed from micromolar solutions,17-19 the CVs containing cyt c adsorbed from the 100 mg/mL (∼8 mM) cross-linking solution deviate in peakto-peak separation and peak shape, suggesting that the higher concentration of cyt c leads to aggregation effects which a produce a less ideal voltammtric response, suggestive of slower electrontransfer kinetics and diminished redox reversibility. While care was taken to use freshly made photo-cross-linking solutions for each experimental trial to minimize formation of aggregates, scan-

rate-dependent peak current data obtained from five separate sets of photo-cross-linked cyt c matrixes nevertheless demonstrate widely fluctuating changes (increases) in cathodic peak current relative to that of their respective adsorbed cyt c controls. Therefore, we did not attempt to quantify how the amount of photo-cross-linked cyt c scales with the overall average percent increase in cathodic peak current, though we do observe that these currents are indeed larger when photo-cross-linked cyt c is present. In an effort to determine a more consistent electrochemical parameter by which we could compare photo-cross-linked cyt c activity to that of control studies involving nonspecifically adsorbed cyt c, we hypothesized that electron transfer through the photocross-linked cyt c matrix would be sluggish compared to that of the nonspecifically adsorbed monolayer of cyt c, due to the random heme orientation and varying electron-transfer distances (0 and 200 nm based on our AFM measurements) between matrixentrapped heme units and the ITO surface. Under this assumption, the redox activity of photo-cross-linked cyt c can be elucidated through comparison of the apparent electron-transfer rate constant (k0ET) between the photo-cross-linked matrixes relative to that of an adsorbed cyt c control. This kinetic parameter may be extracted from the peak-to-peak separation, ∆Ep, as a function of scan rate for an adsorbed species using the method of Laviron.23 Figure 3a shows ∆Ep as a function of scan rate averaged over a series of three trials of 0.095 mm2 photo-cross-linked cyt c, while Figure 3b shows k0ET as a function of scan rate, obtained from the ∆Ep data plotted in Figure 3a, for photo-cross-linked cyt c. As previously observed by El Kasmi et al.,18 k0ET increases linearly before reaching a scan-rate-independent region around 2V/s. By extrapolating from this plateau, we find the intrinsic k0ET for photocross-linked cyt c to be 8.4 ( 0.2 s-1. As expected, this value is somewhat lower than those previously reported for adsorbed monolayers of cyt c at ITO (between 10 and 25 s-1),18 and we attribute this apparent decrease to the ∼100-fold increase in thickness of our photo-cross-linked matrixes relative to the average thickness of a monolayer of adsorbed cyt c.17 The ∆Ep values shown in Figure 3a are also somewhat wider than that expected for an adsorbed redox couple; in fact, 0 mV peak splitting is never observed, even at the slowest scan rates. This observation has been previously reported18 and attributed to the many random conformations of redox-active proteins immobilized at an electrode surface: The varying distances between the redox centers and the electrode inherent in such random conformations result in broader voltammetric peak-topeak separation. Evidence of the heme-to-electrode distance affecting k0ET has been reported in the literature through kinetic analysis of cyt c adsorbed to self-assembled monolayers of varying chain lengths.24-26 This explanation can also be applied to the layer (23) Laviron, E. J. Electroanal. Chem. Interfacial Electrochem. 1979, 101, 1928. (24) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759-2766. (25) Fedurco, M. Coord. Chem. Rev. 2000, 209, 263-331. (26) Wei, J.; Liu, H.; Dick, A. R.; Yamamoto, H.; He, Y.; Waldeck, D. H. J. Am. Chem. Soc. 2002, 124, 9591-9599.

Figure 3. (a) Peak-to-peak potential separation, ∆Ep as a function of scan rate obtained from CVs collected from three 0.095 mm2 photocross-linked cyt c samples. (b) log k0ET as a function of scan rate, extracted from ∆Ep data in (a) using the method of Laviron (ref 23). For both (a) and (b), CV parameters and experimental conditions are the same as those reported in Figure 2.

of our photo-cross-linked cyt c in immediate contact with the ITO electrode, assuming the MPE mechanism promotes cross-linking through a number of different cyt c amino acid residues, resulting in a randomly assembled cyt c matrix and thus varying distances of photo-cross-linked cyt c hemes in this first protein layer relative to the ITO surface. Additionally, hemes located sufficiently far away from the electrode (more than a few nanometers) most likely relay electrons to and from ITO through heme-heme exchange, as previously demonstrated for a multilayer cyt c electrode assembly,27 as well as between cyt c and other heme-containing proteins.25 Spikes and co-workers have previously suggested that tyrosine, histidine, and lysine residues participate in photo-crosslinking;10,11,13 since cyt c contains these residues at multiple sites, it is conceivable that cross-linking can occur at several different locations on the protein surface, promoting random assembly within the photo-cross-linked matrix and thus varying efficiencies in heme-heme electron transfer. Additionally, El Kasmi et al. have attributed large ∆Ep values to conformational changes within the protein to enable facile electron transfer between the Fe(II) and Fe(III) states, proposing a “square” redox mechanism to explain the wide ∆Ep values and subsequently sluggish electron-transfer kinetics of cyt c.18 In such a mechanism, the oxidized and reduced forms of the protein are associated with different preferred conformations, and relaxation (27) Beissenhirtz, M. K.; Scheller, F. W.; Stocklein, W. F. M.; Kurth, D. G.; Mohwald, H.; Lisdat, F. Angew. Chem., Int. Ed. 2004, 43, 4357-4360.

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into each of these preferred conformations manifests itself in the overall k0ET value. (It is important to note that in this portion of the discussion, the term “preferred conformation” refers to the relative position of non-redox-active protein residues surrounding the heme, and not a physical translation of the heme itself relative to the electrode surface.) The two apparent rate constants associated with relaxation to the preferred conformations for oxidized and reduced cyt c, respectively, along with the two electron-transfer rate constants associated with the oxidation and reduction of cyt c, form a “square” scheme illustrating the overall redox mechanism.18,24 This concept of adsorbed cyt c conformational changes is also supported by SERS studies by Hildebrandt and Stockburger.28 At sufficiently fast scan ratessabove 2 V/s in our case, as evident from Figure 3bsinadequate time is allotted for relaxation into the preferred conformations to occur, and k0ET becomes independent of this process, as demonstrated by the plateau region in Figure 3b. At sufficiently slow scan rates, enough time is allotted for preferred conformational changes to take place, and the molecular reorientation of Fe(II) and Fe(III) prior to their oxidation or reduction, respectively, influences k0ET and results in a limiting ∆Ep value at v ) 0 V/s, accounting for the >0 mV peak splitting we observe. The ∆Ep values as a function of scan rate for adsorbed cyt c controls followed those of Figure 3a very closely (see the Supporting Information), and k0ET for adsorbed cyt c was 9.0 ( 0.2 s-1. As expected, the dependence of k0ET on the scan rate again followed the square mechanism for adsorbed cyt c described above. While the addition of photo-cross-linked cyt c does indeed appear to result in slightly less facile electron transfer, it remained difficult to determine from the direct electrochemistry of cyt c whether the addition of photo-cross-linked matrixes occupying only 0.1% of the total ITO working electrode area substantially altered the electrochemistry observed from the adsorbed cyt c monolayer ubiquitous to all samples. For this reason, we elected to utilize additional analytical tools that would enable us to specifically evaluate the redox properties of photo-cross-linked cyt c structures. Raman Spectroscopy. Raman spectroscopy was chosen to elucidate the integrity of the cyt c heme after photo-cross-linking due to the precise control of sampling area afforded by this technique. By focusing the excitation laser beam to a spot size of ∼2 µm, the beam may be contained entirely within the area of a photo-cross-linked matrix, allowing unambiguous analysis of the matrix without contribution from adsorbed cyt c on the surrounding support. Additionally, Raman analysis of cyt c’s porphyrin vibrational modes is abundant in the literature.28-35 Figure 4 shows Raman spectra for a 2 µm spot of both a photocross-linked cyt c matrix and adsorbed cyt c on a glass coverslip, (28) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710-6721. (29) Zhou, J.; Zheng, J.; Jiang, S. J. Phys. Chem. B 2004, 108, 17418-17424. (30) Bonifacio, A.; Van der Sneppen, L.; Gooijer, C.; Van der Zwan, G. Langmuir 2004, 20, 5858-5864. (31) Keating, C. D.; Kovaleski, K. K.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9414-9425. (32) Hu, S.; Morris, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J. Am. Chem. Soc. 1993, 115, 12446-12458. (33) Cartling, B. In Biological Applications of Raman Spectroscopy; Spiro, T. G., Ed.; John Wiley and Sons: New York, 1988; Vol. 3, pp 217-249. (34) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1986, 90, 6017-6024. (35) Cotton, T. M.; Schultz, S. G.; Van Duyne, R. P. J. Am. Chem. Soc. 1980, 102, 7960-7962.

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Figure 4. Surface-enhanced Raman spectroscopic data for photocross-linked (top) and adsorbed (bottom) cyt c, indicating retention of an intact heme in both cases.

both excited with a 514.5 nm Ar laser. The diminished signal-tonoise ratio (S/N) in the individual vibrational stretches for the photo-cross-linked cyt c structures relative to that for adsorbed cyt c is most likely due to SERS effects produced by differences in local concentrations of cyt c. Larger signal enhancements are seen for adsorbed cyt c because the concentration is lower in the excitation volume, consistent with concentration based SERS effects as detailed previously.36 Regardless, the Raman data presented were reproducibly collected from several photo-crosslinked cyt c samples and adsorbed controls and also demonstrate the well-characterized cyt c signature; thus, we feel confident in annotating the spectrum shown in Figure 4. The adsorbed cyt c spectrum correlates very well to that of an Fe(III) heme, with all the annotated peaks in Figure 4 (bottom) corresponding to vibrational stretches reported in the literature for low-spin, six-coordinate Fe(III) heme cyt c.33-35 Generally speaking, the frequencies of porphyrin skeletal modes above 1350 cm-1 correlate to the oxidation state of the heme iron,33 and the peaks occurring at 1371, 1504, 1563, 1584, and 1636 cm-1 are known to correspond to vibrational stretches within the porphyrin ring about the ν4, ν3, ν11, ν19, and ν10 modes, respectively, while the Fe center is in its low-spin, six-coordinate state.34,35 By contrast, in the spectrum of photo-cross-linked cyt c (Figure 4, top) the stretch in the v10 mode has shifted by 10 to 1626 cm-1, characteristic of a high-spin, six-coordinate Fe(III) heme. The v2 mode stretch is also observed to shift to a high-spin Fe(III) value, appearing at 1570 cm-1.34 Interestingly, the shifts appearing in the photo-cross-linked cyt c spectrum at 1092, 1399, 1463, and 1540 cm-1 are reportedly indicative of a low-spin Fe(II) contained in the porphyrin ring.33 When viewed in conjunction with the two shifts assigned to a highspin Fe(II) heme, it appears that photo-cross-linked cyt c exists in a mixed oxidation state. Perhaps a partial chemical reduction within the heme may occur as a result of photo-cross-linking. Regardless, the presence of these peaks corresponding to either or both heme oxidation states indicates that the porphyrin ring and iron center have remained intact throughout the photo-crosslinking process. Nevertheless, the SERS data presented in Figure (36) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 99, 2957-2975.

Figure 5. Scheme depicting amplex red-mediated electrochemical H2O2 detection catalyzed by photo-cross-linked cyt c.

4 only suggests the presence of an intact heme and cannot be used to directly evaluate the heme’s redox activity. Evaluation of Cyt c Peroxidase Activity. In order to assess whether redox activity of the cyt c heme was maintained, we utilized photo-cross-linked cyt c structures in an enzymatic sensing scheme for mediated electrochemical detection of H2O2. In addition to its intrinsic redox activity at the heme site, cyt c has demonstrated peroxidase-like activity in many instances reported in the literature.37-41 The proposed mechanism for this enzymatic catalysis involves excitation of the porphyrin ring to an oxoiron(IV)porphyrin radical cation by one molar equivalent of H2O2, similar to the catalytic mechanism of other peroxidases.42,43 The H2O2 sensing scheme we used to evaluate the peroxidase-like activity of photo-cross-linked cyt c is outlined in Figure 5. In this scheme, H2O2 reduction to H2O is catalyzed by the cyt c (Fe(III)) heme, inducing the FeIV)O porphyrin state. The heme is reduced back to Fe(III) through 2en donation by a solvated mediator, 10acetyl-3,7-dihydroxyphenoxazine (available commercially as amplex red). Oxidized amplex red, known as resorufin, is both redox active44 and fluorescent (excitation 563 nm, emission 587 nm45) and has been used extensively in biological fluorescence assays.45-47 Figure 6a shows a 2 × 2 matrix of photo-cross-linked cyt c (total area ) 680 µm2, glass substrate) subjected to a flow of 200 µM H2O2 and 20 µM amplex red.22 It can be clearly seen that significant fluorescence is observed at the cyt c structures relative (37) Vazquez-Duhalt, R. J. Mol. Catal. B: Enzym. 1999, 7, 241-249. (38) Zhao, G.-C.; Yin, Z.-Z.; Zhang, L.; Wei, X.-W. Electrochem. Commun. 2005, 7, 256-260. (39) Busi, E.; Howes, B. D.; Pogni, R.; Basosi, R.; Tinoco, R.; Vazquez-Duhalt, R. J. Mol. Catal. B: Enzym. 2000, 9, 39-48. (40) Kim, N. H.; Jeong, M. S.; Choi, S. Y.; Kang, J. H. Bull. Korean Chem. Soc. 2004, 25, 1889-1892. (41) Orii, Y. J. Biol. Chem. 1982, 257, 9246-9248. (42) Vazquez-Duhalt, R. In Advances in Bioprocess Engineering II; Galindo, E., Ramirez, O. T., Eds.; Kluwer Academic Publishers: The Netherlands, 1998; p 183. (43) Fukuzumi, S. In The Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: San Diego, CA, 2000; Vol. 8, pp 123128. (44) Gajovic-Eichelmann, N.; Bier, F. F. Electroanalysis 2005, 17, 1043-1050. (45) Zhou, M.; Diwu, Z.; Panchuk-Voloshina, N.; Haugland, R. P. Anal. Biochem. 1997, 253, 162-168. (46) Mohanty, J. G.; Jaffe, J. S.; Schulman, E. S.; Raible, D. G. J. Immunol. Methods 1997, 202, 133-141. (47) Reszka, K. J.; Wagner, B. A.; Burns, C. P.; Britigan, B. E. Anal. Biochem. 2005, 342, 327-337.

Figure 6. (a) Fluorescence image of resorufin response at a 2 × 2 matrix of photo-cross-linked cyt c (area of each cyt c structure is approximately 10 × 17 µm2), generated via flow of 20 µM amplex red/200 µM H2O2. Data was averaged from 11 acquired images using 1 × 1 binning and was subjected to flat field correction. Experimental conditions: 20 mM HEPES/0.1 M NaCl buffer, pH 7.40; 150 µL/min flow rate. (b) Peak current response for amplex red-mediated H2O2 detection at 170 µm2 photo-cross-linked cyt c as a function of H2O2 concentration using square wave voltammetry. SWV parameters: amplitude ) 25 mV, step height ) 5 mV, frequency ) 25 Hz. Experimental conditions: 10 µm diameter GC working electrode, Ag/ AgCl (saturated KCl) reference electrode, Au wire counter electrode, 0.1 M K2HPO4/0.05 M citric acid buffer, pH ) 5.0, 10 µM amplex red. Photo-cross-linking solutions consisted of 200 mg/mL cyt c in 18 mM Na2HPO4/0.1 M NaClO4 supporting electrolyte, pH ) 7.40. Inset: Lineweaver-Burke analysis of the plateau region in (b).

to the surrounding glass substrate, indicating an extremely high density of resorufin formation at photo-cross-linked cyt c and thus indicative of robust heme activity. Though fluorescence-based flow studies aid in the ability to qualitatively assess local activity of microstructures, they are not optimal when trying to elucidate quantitative enzyme kinetics data. For example, the experimental setup in Figure 6a would require that assumptions be made to describe the diffusion of fluorescing dye molecules under flow conditions and the number of active molecules present in a given matrix. Also, accurate characterizations of the enzyme activity at steady-state conditions would be essential. Such characterization via fluorescence is difficult for protein structures of micrometer dimensions. Additionally, sufficiently high H2O2 concentrations (such as the 200 µM H2O2 flow used in acquiring Figure 6a) have been reported by Andrieux et al. to convolute the apparent catalytic activity of multilayered enzyme films due to substrate-induced inhibition that scales with increasing film thickness.48 Given that the photo-cross-linked cyt c structure mimics a multilayered enzyme assembly, such inhibition could also occur in this case, making straightforward enzymatic evaluation difficult. Fortunately, the electrochemical reduction of resorufin to dihydroresorufin at a GC working electrode (Figure 5) may be quantitatively and sensitively analyzed through electrochemical reduction and the observed current response. Because H2O2 consumption and amplex red conversion to resorufin occur in a 1:1 ratio through the cyt c heme, H2O2 consumptions and thus cyt c enzymatic activitysmay be directly extracted from the current response generated by resorufin reduction. We have recently developed an enzymatic H2O2 electrochemical sensing (48) Andrieux, C. P.; Liomges, B.; Saveant, J.-M.; Yazidi, D. Langmuir 2006, 22, 10807-10815.

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scheme utilizing amplex red in our labs,49 and we incorporated cyt c matrixes photo-cross-linked onto glass supports into this sensing scheme to evaluate the peroxidase activity of photo-crosslinked cyt c and hence the redox activity of its heme. We again used control experiments of cyt c adsorbed at glass from the photo-cross-linking solution to compare our results. Figure 6b shows the peak current response as a function of H2O2 concentrations varying between 300 pM and 4 µM for a 8.55 mm2 area of a glass slide containing 170 µm2 photo-cross-linked cyt c (again, we assume that cyt c nonspecifically adsorbed to the glass substrate during the photo-cross-linking process is also present). By working in this low concentration range, substrate inhibition effects can be significantly reduced,48 allowing a more straightforward comparison of photo-cross-linked cyt c’s enzymatic activity to that of adsorbed cyt c. In Figure 6b, classic MichaelisMenten (M-M) behavior is exhibited as the peak current response scales linearly with increasing H2O2 concentration, eventually becoming concentration-independent as the maximum enzymatic turnover rate has been achieved.50 This response is similar to that observed in other studies in our laboratory involving HRP using our mediated sensing scheme.49 The maximum velocity of H2O2 consumption by cyt c, Vm, can be determined by dividing the current obtained through extrapolation of the plateau region in Figure 6b by the quantity nF, where n ) 2 electrons transferred in the reduction of one resorufin molecule and F is Faraday’s constant. For the sample analyzed in Figure 6b, Vm ) 0.34 fmol/ s. This value is in close agreement with those reported by our laboratory for horseradish peroxidase activity using the same detection scheme.49 The turnover number, kcat, may be determined by dividing Vm by the number of moles of cyt c present. For 170 µm2 photo-cross-linked cyt c, and assuming an adsorbed cyt c surface coverage on glass to be as extensive as that determined earlier using direct cyt c electrochemistry at ITO (6.84 pmol/cm2, noted above), kcat ) 5.8 × 10-4 s-1. For comparison, Vm for an adsorbed cyt c control on glass using the same solution conditions from Figure 6b was 0.41 fmol/s, and kcat ) 7.0 × 10-4 s-1 (see the Supporting Information). These values are somewhat low compared to kcat values previously reported for cyt c-catalyzed H2O2 reactions,37,38 which we again attribute to the nonuniform distribution of cyt c on the glass substrate due to aggregates formed in the highly concentrated cyt c solutions necessary for photo-cross-linking. From these Vm and kcat values, it appears that photo-cross-linked cyt c again exhibits sluggish behavior relative to that of adsorbed cyt c, which is probably a consequence of the dense, heme-entrapped composition of the photo-cross-linked matrix. Nevertheless, it is remarkable that this decrease in Vm and kcat can be observed consistently with the addition of only 170 µm2 photo-cross-linked cyt c, which effectively increases the number of cyt c molecules by only 0.4% relative to those assumed to be adsorbed to the glass. Vm and kcat values for photo-cross-linked samples were always lower than those of cyt c adsorbed from the same solution conditions in a series of five trials. Perhaps the most important parameter that can be extracted from M-M enzymatic analysis is Km, the Michaelis-Menten constant, which is equal to the concentration of substrate at which

half the active enzyme sites in the sample are occupied by substrate, forming the “enzyme-substrate complex” that serves as an intermediate to product formation in M-M theory. Km is acquired through Lineweaver-Burke analysis, i.e., a doublereciprocal plot of 1/ip versus 1/[substrate]. The line-of-best-fit obtained through linear regression analysis adheres to the following equation:51

(49) Lyon, J. L.; Stevenson, K. J. Anal. Chem. 2006, 78, 8518-8525. (50) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman & Co.: New York, 1988.

(51) Nakaminami, T.; Ito, S.-i.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1999, 71, 1068-1076.

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ip-1 ) imax-1 + (Kmimax-1)[H2O2]-1

(1)

where Km can be extracted by dividing the slope of the line by its y-intercept (imax-1). The Lineweaver-Burke plot shown in the inset of Figure 6b yields Km ) 54 ( 2 nM, while Lineweaver-Burke analysis of the adsorbed cyt c control gives Km ) 4.8 ( 0.3 nM. The increase in Km by as much as an order of magnitude upon addition of a photo-cross-linked structure was also observed throughout the five experimental trials. A larger Km suggests that more active hemes are present in the photo-cross-linked cyt c sample, thus requiring a higher H2O2 concentration to form heme-substrate complexes with half of them. Alternatively, Km can be considered as a dissociation constant for the enzyme-substrate complex in cases where the rate of complex dissociation into enzyme and substrate is much greater than the rate of product formation.50 Under this assumption, the larger Km value observed for photo-cross-linked cyt c suggests a weaker affinity for product formation relative to that of adsorbed cyt c. However, the fluorescence image shown in Figure 6a contradicts this argument, as a clear increase in resorufin fluorescence is localized at the photo-cross-linked cyt c structures. When analyzed in conjunction with one another, Figure 6, parts a and b, supports the argument that the larger apparent Km is reflective of a increased H2O2 concentration necessary to fill the increasing number of active heme sites due to the presence of photo-cross-linked cyt c. For further comparison of our enzyme kinetic parameters to those previously published for cyt c, the constant kcat/Km was calculated. This constant was determined to be on the order of 104 s-1/M-1, which is on the same order of magnitude as some previously published cyt c peroxidase-like H2O2 sensors.39-41 Additionally, Basu and Campagnola have reported kcat/Km values of 105-106 s-1/M-1 for photo-cross-linked alkaline phosphatase activity deduced via fluorescence.14 CONCLUSION This study presents a unique approach to the assessment of cyt c protein integrity that is maintained upon multiphoton photocross-linking. When considered in conjunction with one another, the direct electrochemical data, SERS analysis, and enzymatic parameters indicate that cyt c retains its heme redox activity when subjected to MPE photo-cross-linking conditions. These results give insight into the reaction mechanisms governing MPE: from our results, we can deduce that cross-linking does not significantly degrade the porphyrin ring, nor does the iron center undergo irreversible oxidation state changes, though Fe does appear to undergo a spin state change and/or partial chemical reduction relative to cyt c as adsorbed directly from solution. Direct

electrochemical studies indicate that photo-cross-linked cyt c matrixes exhibit intrinsic electron-transfer behavior that differs only subtly from adsorbed monolayers of cyt c, while enzymatic analysis of a single 10 × 17 um2 cyt c matrix produces results that differ more widely (ca. a factor of 10) from those measured from adsorbed cyt c. Future studies will focus on development of photo-cross-linking strategies that promote more facile electron transfer between heme centers and electrode supports. The electrochemical and spectroscopic methodologies presented herein for characterizing electron-transfer reactivity in immobilized proteins will assist in the elucidation of other cross-linkable hemoproteins, such as hemoglobin and myoglobin. Moreover, the enzymatic electrochemical sensing scheme is capable of modeling local activity of a wide variety of peroxidase-based protein matrixes, thus providing a novel method with which to assess the functionalities of enzymes (or enzyme-mimicking proteins) post-cross-linking. Quantitative characterization of these matrixes’ enzymatic parameters promotes their application as enzymatic sensors that may be fabricated among existing cellular microenvironments.

ACKNOWLEDGMENT Financial support of this work was provided in part by the R. A. Welch Foundation (Grants F-1331 and F-1529) and the NSF (Grant CHE-0134884). J.L.L. acknowledges the Welch Foundation for Summer Fellowship support. We thank Dr. David E. Graham for use of chromatography equipment and Ms. Lauren Tolles for assistance in collecting the AFM image shown in Figure S1. SUPPORTING INFORMATION AVAILABLE In situ AFM images and height profiles and distributions of photo-cross-linked cyt c, representative cyclic voltammograms of photo-cross-linked and adsorbed cyt c, plots of log k0ET versus scan rate and M-M peroxidase activity for adsorbed cyt c. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review October 12, 2006. Accepted January 2, 2007. AC0619377

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