Films of Manganese Oxide Nanoparticles with Polycations or

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Films of Manganese Oxide Nanoparticles with Polycations or Myoglobin from Alternate-Layer Adsorption† Yuri Lvov,*,‡ Bernard Munge,§ Oscar Giraldo,§ Izumi Ichinose,| Steven L. Suib,*,§ and James F. Rusling*,§ Institute for Micromanufacturing, Louisiana Tech University, Ruston, Louisiana 71272, and Department of Chemistry, Box U-60, University of Connecticut, Storrs, Connecticut 06269-3060, and Faculty of Engineering, Kyushu University, Fukuoka 812, Japan Received January 28, 2000. In Final Form: April 17, 2000 Alternate adsorption of manganese oxide nanoparticles with polycations poly(dimethyldiallylammonium) (PDDA) or myoglobin (Mb) onto silver, quartz, and rough pyrolytic graphite gave stable, porous, ultrathin films. Quartz crystal microbalance (QCM) and UV-vis absorbance revealed regular film growth at each adsorption step for MnO2 and PDDA and for SiO2 nanoparticles and Mb. Scanning electron microscopy of MnO2/PDDA films showed smooth surfaces on the 20 nm scale and cross sections consistent with individual nanoparticles. QCM during growth of films of Mb and MnO2 reflected a competition for adsorption of the protein by the film surface and dispersed MnO2 nanoparticles. Nevertheless, films of Mb and MnO2 up to 30 nm thick on rough pyrolytic graphite electrodes could be constructed. These novel films featured reversible interconversion of the protein’s heme FeIII/FeII redox couple with 10 electroactive layers of protein, considerably more than for polyion-Mb films on smooth gold (ca. 1.3 electroactive layers), and coiled PSS/Mb films on rough graphite (7 electroactive layers). Shifts in redox potential caused by CO complexation of the heme FeII, BET specific areas, and electrochemically driven catalytic reduction of oxygen suggest that the Mb/MnO2 films are highly porous to gas molecules. To our knowledge, these films represent the first nanofabrication of inorganic particles with functional proteins by the layer-by-layer method.

Introduction A novel technique for ultrathin film assembly was recently developed which employs alternate adsorption of oppositely charged polyions.1 Film growth proceeds as in the following example: A solid with a negatively charged surface is immersed in a solution containing a cationic polyelectrolyte, and a layer of polycation is adsorbed. When adsorption is done at a relatively high concentration of polyelectrolyte, a number of ionic groups remain exposed to the interface with the solution. Thus, surface charge is effectively reversed, preventing further polycation adsorption. After being rinsed in pure water, the substrate is immersed in a solution of polyanions. A new layer is adsorbed, and the original negative surface charge is restored. By repeating both steps, alternating multilayer assemblies are obtained with reproducible layer thicknesses. This approach is general, and in principle there is no restriction on the choice of polyelectrolytes. The procedure has been successful with linear polyions,1 proteins,2 ceramics,3 and charged nanoparticles.4 Films containing electroactive metalloproteins have been constructed on † Part of the Special Issue “Colloid Science Matured, Four Colloid Scientists Turn 60 at the Millennium”. ‡ Louisiana Tech University. § University of Connecticut. | Kyushu University.

(1) For examples, see: (a) Decher, G. Science 1997, 227, 1232. (b) Lvov, Y.; Decher G.; Mo¨hwald, H. Langmuir 1993, 9, 481. (c) Ferreira M.; Rubner, M. Macromolecules 1995, 28, 7107. (2) (a) Lvov, Y.; Ariga, K.; Ichinose I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (b) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald H., Eds.; Marcel Dekker: New York, 2000; pp 125-167. (3) Kleinfeld E.; Ferguson, G. Science 1994, 265, 370.

electrodes by this method.5 For successful assembly of nanoparticle films, alternation with “soft” linear or branched polyion layers is important. Flexible polyions penetrate between particles and act as an electrostatic glue. Layer-by-layer film assembly by alternate adsorption of charged nanoparticles and macromolecules provides a route for extending three-dimensional molecular architecture in a direction perpendicular to the solid support. Clusters of manganese dioxide were recently made in a phase transfer synthesis by preparing sols of tetraalkylammonium permanganate salts dispersed in mixed aqueous/nonaqueous solvent.6 Sizes of particles are regulated by controlling pH, aging time, temperature, and the nature of the tetraalkylammonium ion. Small-angle neutron scattering of these colloidal sols showed that the particulates are disk shaped and that diameters can be controlled starting at around 20 Å. Such stable precursor sols are quite unusual for manganese oxide systems. Coordination environments of this system have also been studied by X-ray absorption fine structure.7 (4) (a) Kotov, N.; Dekany I.; Fendler, J. H. J. Phys. Chem. 1995, 99, 13065. (b) Fendler, J. H.; Meldrum, F. Adv. Mater. 1995, 7, 607. (c) Schmitt, J.; Decher, G.; Dressik, W.; Brandow, S.; Geer, R.; Shashidhar R.; Calvert, J. Adv. Mater. 1997, 9, 61. (d) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (e) Lvov, Y.; Rusling, J. F.; Thomsen, D.; Papadimitrakopoulos, F.; Kawakami T.; Kunitake, T. Chem. Commun. 1998, 1229. (f) Liu, Y.; Wang A.; Claus, R. Appl. Phys. Lett. 1997, 71, 2265. Ichinose, I.; Tagawa, H.; Mozuki, S.; Lvov, Y.; Kunitake, T. Langmuir 1998, 14, 187. (g) Bekele, H.; Fendler, J. H.; Kelly, J. W. J. Am. Chem. Soc. 1999, 120, 7266. (h) Cassagneau, T.; Fendler, J. H. J. Phys. Chem. B 1999, 103, 1789. (5) (a) Lvov, Y. M.; Lu, Z.; Zu, X.; Schenkman, J. B.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073. (b) Rusling, J. F. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000; pp 337-354. (6) Brock, S. L.; Sanabria, M.; Suib, S. L.; Urban, V.; Thiyagarajan, P.; Potter, D. I. J. Phys. Chem. B 1999, 103, 7416.

10.1021/la000110j CCC: $19.00 © 2000 American Chemical Society Published on Web 06/13/2000

Films of Manganese Oxide with Polycations

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In this paper, we report novel films of manganese oxide nanoparticles fabricated with linear poly(dimethyldiallylammonium) (PDDA) ions and the globular cationic metalloprotein myoglobin. Films were characterized by quartz crystal microbalance, optical absorbance, voltammetry, and scanning electron microscopy. Films of manganese oxide or silica nanoparticles and myoglobin gave reversible electron exchange between rough graphite electrodes and up to 10 layers of the protein involving the heme FeIII/FeII redox couple. Experimental Section Chemicals and Materials. Preparation and characterization of colloids of lamellar manganese dioxide have been described in detail elsewhere.6 Briefly, a colloidal solution of manganese dioxide 0.1 M in Mn was prepared by adding 10 mmol of tetramethylammonium (TMA+) permanganate to a stirred mixture of 100 mL of water and 30 mL of 2-butanol at room temperature. After 30 min a dark red-brown sol was formed in the aqueous layer. This aqueous dispersion was separated from the upper, organic layer and then diluted to the final concentration of 0.02 or 0.01 M. Silica nanoparticles of average diameter 45 nm were a gift from Nissan Kagaku, Japan. Sodium poly(styrenesulfonate) (PSS, MW 70 000, Aldrich) was used at a concentration of 3 mg mL-1, and poly(dimethyldiallylammonium chloride) (PDDA, Aldrich) at a concentration of 2 mg mL-1, pH 12 adjusted by NaOH. Horse heart myoglobin from Sigma was filtered through a 30 000 MW cutoff filter.8 All other chemicals were reagent grade. Film Assembly. The method for preparation of nanoparticlePDDA films was similar to that described previously for polyionprotein films.5 Solid substrates with a negative surface charge, including quartz slides, silver-coated quartz crystal resonators, or rough pyrolytic graphite were immersed first in an aqueous solution of polycation at pH 12 for 15 min, then removed and washed for 1 min with pH 12 solution, and then immersed in dispersions of the negatively charged nanoparticles (0.01-0.02 M, pH 12) and washed. This sequence was repeated to obtain the desired number of layers. Protein films were prepared in a similar manner by sequential adsorption of myoglobin (3 mg mL-1, pH 5.2) and 0.01 M MnO2 (pH ca. 10) or SiO2 (pH 9) nanoparticles on a bed of PSS/PDDA adsorbed onto basal plane pyrolytic graphite electrodes with geometric area 0.16 cm2 roughened by abrading on medium emery cloth and then sonicated in water for 30 s, or on silver resonators. Cyclic voltammetry was done as described previously.5a For the CO binding studies, standard CO gas from a tank was bubbled through the solution to saturation. Samples were dried in a nitrogen stream before quartz crystal microbalance (QCM), UV-vis, voltammetric, or X-ray photoelectron spectroscopy (XPS) measurements. BET surface areas of films were measured with a Micromeritics ASAP 2010 system. About 0.2 g of sample was evacuated at 500 µmHg, and then immersed in liquid N2. The volume of a monolayer of adsorbed N2 gas was measured from isotherms via least-squares fitting of eight points. Quartz Crystal Microbalance. A quartz crystal microbalance (QCM, USI System, Japan) was used for time-dependent monitoring of film assembly in situ.2 Time-dependent adsorption measured by QCM was used to establish saturation adsorption times. QCM measurements were operated in two modes. In the stepwise measurement, the resonator was immersed in a polyelectrolyte solution for a given period and dried, and the frequency change was measured. For in situ monitoring of adsorption, only one side of the resonator was in contact with the solution and the frequency change was recorded continuously. The long-term stability (several hours) of quartz resonator frequency was (2 Hz. All experiments were carried out at ambient temperature of ca. 22 °C. QCM resonators were covered by evaporated silver electrodes (0.16 cm2) on both faces, and (7) Ressler, T.; Brock, S. L.; Wong, J.; Suib, S. L. J. Phys. Chem. B 1999, 103, 6407. (8) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386.

Figure 1. QCM frequency (∆F) dependence on time, showing kinetics of alternate adsorption of polycation PDDA and anionic MnO2 nanoparticles (1 and 3 days after preparation) on a silver resonator with intermediate rinsing in water and drying before measurement: pH 12, PDDA concentration 3 mg/mL, 0.02 M MnO2. resonance frequency was 9 MHz (AT-cut). We also monitored film assembly by UV absorption spectroscopy (Perkin-Elmer λ6). The Saurbrey equation provides a relation between adsorbed mass and frequency shift ∆F (Hz). Taking into account characteristics of the 9 MHz quartz resonators the film mass per unit area M/A (g cm-2) is given by:2

M/A ) -∆F/2(-1.83 × 108)

(1)

on one side of a resonator of A ) 0.16 ( 0.01 cm2. The thickness of a film may be calculated from its mass using film density, but the density of MnO2/PDDA is not known. We obtained direct scaling between ∆F and film thickness from scanning electron microscopy cross-section images. Scanning electron microscopy for a number of (MnO2/PDDA)10-18 film cross sections gave the following relation for adsorbed dry film thickness at both faces of the electrodes (d):

d (nm) ≈ -(0.016 ( 0.002)∆F (Hz)

(2)

X-ray Photoelectron Spectroscopy (XPS). Data were acquired using a Leybold-Heraeus LHS-10 XPS instrument equipped with an Al KR X-ray source at 200 W and a SPECS EA10MCD energy analyzer at pass energy of 29.6 eV at pressures less than 1 × 10-8 mbar. Scanning Electron Microscopy (SEM). Images were obtained for films constructed on silver-coated QCM resonators. Films were coated with 20 Å thick Pt by use of an ion-coater (Hitachi E-1030 ion sputter, 10 mA/10 Pa) under argon. Micrographs were obtained with a Hitachi S-900 scanning electron microscope at an acceleration voltage of 25 kV.

Results Film Assembly. Figure 1 shows a kinetic QCM frequency profile for representative consecutive steps of adsorption of PDDA and manganese dioxide (MnO2) nanoparticles. Adsorption reached saturation in 15 min for both materials. This time for the assembly of individual layers provided stable growth with repeatable steps as monitored by QCM at every adsorption cycle. In solution, QCM frequency shifts of about 150 Hz for PDDA layer adsorption and 500 Hz for MnO2 were found when using freshly prepared MnO2 dispersions, i.e., 6. To illustrate, E°′ was -0.275 V for n ) 6 and -0.294 V for n ) 9. These data can be compared to PSS/PDDA/MnO2(Mb/MnO2)n with average E°′ of -0.269 ( 0.006 V vs SCE (-0.025 V vs NHE), and E°′ values of -0.269 V for n ) 6 and -0.272 V for n ) 10. That is, nearly insignificant trends in E°′ appeared for Mb/ MnO2 films with increased number of layers. Clearly, Mb is a bit more easily reduced in the PSS/PDDA/MnO2(Mb/ MnO2)n films and less subject to effects of the number of layers. The integrated area under the cathodic CV peaks was used to estimate the amount of electroactive protein in mol cm-2 (Γ) using Faraday’s law.12 Γ for PSS/PDDA/MnO2(Mb/MnO2)n films increased linearly with layer number (14) Lvov, Y. M.; Rusling, J. F.; Thomsen, D. L.; Papadimitrakopoulos, F.; Kawakami T.; Kunitake, T. J. Chem. Soc., Chem. Commun. 1998, 1229.

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Figure 9. Influence of number of layers on amount of electroactive myoglobin in mol cm-2 (Γ) for films made with MnO2 and SiO2.

up to n ) 10 (Figure 9). Γ for PSS/PDDA/SiO2/(Mb/SiO2)n increased with layer number up to n ) 9, and then the amount of electroactive Mb reached a plateau. Experiments on films aged for several months showed insignificant decreases in charge estimated by CV. QCM frequency shifts for these two types of films on silver-coated resonators revealed significant differences in the mechanism of film formation. SiO2/(SiO2/Mb)n film growth on a precursor polyion underlayer showed regular film formation (Figure 10a) similar to that for PDDA/ MnO2 films (cf. Figures 2 and 3). Average ∆F was -532 ( 240 Hz for the Mb layers, compared to -536 ( 125 Hz for PDDA layers for the film in Figure 3. However, the Mb/MnO2 films changed frequency in an unusual way. Addition of the first MnO2 and Mb layers decreased the QCM frequency indicating a gain in mass, but subsequent insertion of films into MnO2 dispersions caused an increase in frequency by an average of 329 ( 15 Hz (Figure 10b).15 Films with “outer layers” of MnO2, after washing and insertion into Mb solutions, gave a reproducible decrease in frequency averaging -449 ( 11 Hz for 10 bilayers. Each bilayer of Mb/MnO2 added only -120 ( 16 Hz of mass equivalent, 330 ng cm-2 from eq 1. From eq 2, the total thickness of the n ) 9 SiO2/(SiO2/ Mb)n film was 350 nm. Total thickness of the n ) 10 MnO2(Mb/MnO2)n films was only 30 nm. The data on the MnO2(Mb/MnO2)n films suggests a complex but quite reproducible film growth process involving partial removal of the Mb layers each time they are immersed into the MnO2 dispersion. Significant removal of a fraction of each Mb layer upon immersion in the SiO2 dispersions may also occur. There is some evidence for this, since the first SiO2 layer on the polyion bed gave ∆F ) -2700 Hz, compared to the average -1970 Hz, and the first Mb layer on top of a SiO2 layer gave -1000 Hz, nearly twice as large as the average of -530 Hz. Introduction of oxygen into buffer solutions containing Mb films caused an increase in the FeIII reduction peak and disappearance of the FeII oxidation peak (Figures 11 and 12), consistent with catalytic reduction of oxygen by (15) Control experiments showed that Mb/MnO2 films with outer Mb layers were stable in solutions of pH 10 and 11 which did not contain MnO2.

Figure 10. QCM frequency shifts for films of myoglobin made with nanoparticles of (a) SiO2 and (b) MnO2 on silver resonators. (Negative F shift is upward.)

Figure 11. Cyclic voltammograms at 0.05 V s-1 of PG/PSS/ PDDA/MnO2(Mb/MnO2)10 films in pH 5.5 buffer (a) without oxygen, (b) with oxygen present, and (c) direct reduction of oxygen on the PG/PSS/PDDA/MnO2 electrode.

the film.5 The direct, uncatalyzed reduction of oxygen occurred at about -0.6 V on PG/PSS/PDDA/SiO2 and PG/ PSS/PDDA/MnO2 electrodes without Mb, with slightly smaller current on the latter electrode. The catalytic reduction peak (Icat) occurred at -0.24 V vs SCE on the MnO2(Mb/MnO2)n electrode and -0.35 V on the SiO2/(Mb/ SiO2)n film. Considering the amount of electroactive Mb in each film, the ratio of Icat/Γ was 35 µA cm-2/mol of Mb for MnO2(Mb/MnO2)n and 33 µA cm-2/mol of Mb for SiO2/ (Mb/SiO2)n, suggesting an equally efficient catalytic turnover in the two systems.

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Figure 12. Cyclic voltammograms at 0.05 V s-1 of PG/PSS/ PDDA/SiO2(Mb/SiO2)9 films in pH 5.5 buffer (a) without oxygen, (b) with oxygen present, and (c) direct reduction of oxygen on the PG/PSS/PDDA/SiO2 electrode.

Discussion Results above document the first layer-by-layer nanofabrication of films of inorganic particles with a functional protein to our knowledge. This assembly method was successful for making stable films of manganese dioxide nanoparticles in alternation with polycations PDDA or Mb. Film growth in regular layer-by-layer fashion is illustrated by QCM and UV-vis spectroscopy during film construction. (Figures 1-3). The manganese oxide/PDDA films made with fresh MnO2 nanoparticles have surface roughness of no more than 20 nm (Figure 4). MnO2/PDDA films can be made with a predetermined number of manganese oxide nanoparticle layers in thicknesses from tens to hundreds of nanometers. They retain nanoparticle characteristics which appear to be close packed by SEM. Observation of XPS peaks for Mn (2p1/2) at binding energy 653.30 eV and Mn (2p3/2) at binding energy 641.63 eV (Figure 5) is consistent with the mixed valent manganese oxide particles and is most likely due to Mn4+ and Mn3+.6 Films of Mb and aged manganese oxide nanoparticles, chosen because they adsorbed much more polycation, on rough pyrolytic graphite electrodes supported reversible redox conversion of the heme FeIII/FeII couple. These films had up to 10 electroactive layers of protein (Figures 6 and 9), considerably more than PSS/Mb on smooth gold, for which only the layer closest to the electrode and a fraction of the second layer were electroactive.5a Further, films made from globular PSS and Mb on rough PG had seven electroactive Mb layers, but additional layers added on top were not electroactive.13 It is noteworthy that E°′ values in Mb/MnO2 (-0.025 V vs NHE) and Mb/SiO2 (-0.037 V vs NHE) films at pH 5.5 are in the same range as for Mb in films of insoluble surfactants, in which the protein was shown by UV-vis and IR spectroscopy to retain a near-native structure.11 Values in the nanoparticle films are also close to the E°′ of 0.007 V vs NHE for multilayer Mb/PSS films on gold,5 for which the Soret absorbance band suggested the lack of extensive unfolding or denaturation. While a fraction of denaturation may occur in the nanoparticle films, the E°′ data suggest that extensive denaturation is absent. Complete denaturation would cause loss of the iron heme,16 resulting in a lack of electrochemical activity in the film. Reactions of Mb in the nanoparticle films with CO and (16) Nassar, A.-E. F.; Zhang, Z.; Hu, N.; Rusling, J. F.; Kumosinski T. F. J. Phys. Chem. 1997, 101, 2224-2231.

Lvov et al.

oxygen (Figures 7, 11, and 12) in similar ways to Mb in surfactant and polyion films are also consistent with a major fraction of structurally intact protein. QCM results suggested (Figure 10b) that the growth of Mb/MnO2 films did not proceed with a regular weight increase, as did films of MnO2/PDDA (Figures 2 and 3) and Mb/SiO2 (Figure 10a). The amount of Mb deposited at each step gave an average ∆F of -449 ( 11 Hz for Mb/MnO2 compared to -532 ( 240 Hz for Mb/SiO2. Application of eq 1 provides an estimate of 1330 ng cm-2 per layer of Mb on MnO2 and 1450 ng cm-2 per adsorbed layer of Mb on SiO2, which are quite comparable. However, attempts to add layers of MnO2 after the Mb adsorption resulted in an increase of frequency averaging +329 ( 15 Hz, suggesting a mass loss of about 900 ng cm-2. The standard deviations on ∆F for Mb and MnO2 steps show that the processes involved are quite reproducible. These results suggest a mass increase17 of 430 ng cm-2 per Mb/ MnO2 adsorption cycle. A similar negative-positive frequency shift pattern was also observed in assembling layered films of some proteins and polyions, e.g., glucose oxidase and poly(ethyleneimine).2b,18 Results suggest that for films with Mb on the top layer, the oppositely charged MnO2 nanoparticles in the aqueous dispersion compete with the surface for adsorbed Mb, removing some from the surface. This does not appear to be a major factor with SiO2 nanoparticles. However, the increase in charge from the Mb FeIII/FeII reduction with increasing number of Mb adsorption cycles (Figure 9) strongly suggests incorporation of an increased amount of Mb upon each pair of Mb/MnO2 adsorption steps. The result is a much thinner film than for SiO2 for an equivalent number of adsorption cycles. Consider the particles sizes of 2.5 × 3.5 × 4.5 nm for Mb,19 about 20 nm for MnO2, and 45 nm for SiO2. In the films discussed in this paper, we have globular protein molecules of about 4 nm adsorbing onto solid charged particles 4-10 times larger. Thus, it is probably more realistic to view these films as somewhat intimate mixtures of large and small particles. This view might better explain the high degree of electroactivity of Mb in the films and is in agreement with neutron reflectivity studies of layered linear polyion films which show significant mixing between adjacent layers.1a For Mb/SiO2 films, we can compare the estimated total amount of Mb (8.2 × 10-10 mol cm-2) in a nine-bilayer film with the total electroactive Mb from CV (3.9 × 10-10 mol cm-2) to conclude that about 48% of all the Mb in the film is electroactive. However, because the SiO2 deposition step may remove a fraction of each Mb layer as discussed above, the value of total Mb is probably overestimated and the fraction of electroactive protein may be >50%. Nevertheless, we can roughly estimate the total number of Mb molecules in this film as 4.9 × 1014 per cm2. From the total ∆F for SiO2 we estimate a total mass of SiO2 of 4.3 × 10-5 g cm-2. Estimating the mass of a single particle from its density, we find 4.1 × 1011 particles per cm2 in the film, suggesting that there are roughly 1200 Mb globules per SiO2 particle. The surface area of a single 45 nm SiO2 particle is 6400 nm2, and the approximate cross-sectional area of 1200 Mb molecules is 15 000 nm2, roughly a 2/1 ratio of areas of total Mb to SiO2. Possible biases discussed above make (17) While viscoelastic effects can bias estimates of mass by QCM,9 this is unlikely for these dry films since regular mass growth was observed for MnO2/PDDA and Mb/SiO2 films. (18) Lvov, Y.; Ariga, K.; Kunitake, T. Colloids Surf., A 1999, 146, 337. (19) Kendrew, J.; Phillips, D.; Stone, V. Nature 1960, 185, 422.

Films of Manganese Oxide with Polycations

Figure 13. Conceptual model of nanoparticle-myoglobin films with three adsorbed protein layers on rough pyrolytic graphite electrodes.

this ratio an upper limit. Nevertheless, these rough estimates suggest that Mb molecules coat the SiO2 particles rather completely and should be in close contact in the film (Figure 13). Assuming that Mb has restricted mobility within the film, we speculate that a close-packed interstitial protein network may be involved in “electron hopping”20 by selfexchange reactions between MbFeII and MbFeIII to propagate charge through the film during CV. The average electron self-exchange distance would then depend on the relative protein orientations in the film, which are not known, but should be on the order of the molecular diameter of Mb, about 4 nm.2a In the nine-bilayer Mb/ SiO2 film, the estimated fraction of electroactivity suggests that at least half of the Mb molecules in the film can participate in this process. This fraction is larger in films with less adsorption layers. We cannot do similar calculations for Mb/MnO2 films because the QCM data do not reflect the total masses of each component. However, it is reasonable to expect a similar mode of charge transport within these films. The shift in CV midpoint potential of Mb with CO in the solution (Figure 7) is consistent with significant porosity (20) For a review of electron transport in polymer films, see: Majda, M. In Molecular Design of Electrode Surfaces; Murray, R. W., Ed.; John Wiley & Sons: New York, 1992; pp 159-206.

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of the films to gas molecules, as also supported by the high surface area of 115 m2 g-1 found by the BET method. The effect of CO also confirms identification of the iron heme as the electroactive site in the film. The FeII form of heme proteins, but not the FeIII form, can form a complex with CO.11 The positive formal potential shift resulting from this complexation suggests the ready availability of CO throughout the film to react with the FeII heme as it is formed electrochemically, giving FeII-CO during the forward scan. Electrochemical catalysis of the reduction of oxygen has been reported for Mb in various types of films. The product hydrogen peroxide can activate the iron heme for enzymelike oxidative catalysis.11 Voltammetric observation of this catalysis for Mb/MnO2 and Mb/SiO2 films (Figures 11 and 12) opens the possibility of applications to enzyme-like catalysis of organic oxidations utilizing the iron heme center of the protein.5 These two types of films seem to provide equally efficient turnover for catalysis of reduction of oxygen. The Mb/MnO2 film catalyzed this reaction at an applied potential 110 mV less negative than Mb/SiO2 films, suggesting a slightly larger decrease in activation free energy for the process in Mb/MnO2 films. In summary, stable, porous films of MnO2 nanoparticles and PDDA or Mb were made by layer-by-layer adsorption. Films of Mb and MnO2 showed an unusual growth mechanism featuring competition for adsorption of Mb by the growing film surface and dispersed nanoparticles. Nevertheless, stable Mb/MnO2 films up to 30 nm thick featuring reversible redox conversion of the protein could be constructed. Electrochemical catalysis of the reduction of oxygen suggests that these films may be useful for enzyme-like oxidation of organic molecules. Porous nanoparticle-protein films may prove to be a generally useful new approach to biocatalytic materials. Acknowledgment. The authors thank the National Institute of Environmental Health Sciences (NIEHS Grant No. ES03154), NIH, and the US Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences for support of this research. Contents are solely the responsibility of the authors and do not necessarily represent official views of NIEHS, NIH, or DOE. The authors are grateful to Nissan Kagaku, Japan for the gift of silica nanoparticles. O.G. is grateful for support from Colsciencias, Columbia. LA000110J