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Langmuir 2004, 20, 722-729
Driving Forces for Layer-by-Layer Self-Assembly of Films of SiO2 Nanoparticles and Heme Proteins Pingli He,† Naifei Hu,*,† and James F. Rusling‡ Department of Chemistry, Beijing Normal University, Beijing, 100875, People’s Republic of China, Department of Chemistry, University of Connecticut, U-60, Storrs, Connecticut 06269-3060, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032 Received June 9, 2003. In Final Form: October 16, 2003 Heme protein hemoglobin (Hb) or myoglobin (Mb) and silica nanoparticles in a variety of charge states were assembled layer-by-layer into films on solid surfaces to investigate the driving forces for film assembly. Cyclic voltammetry (CV), quartz crystal microbalance (QCM), X-ray photoelectron spectroscopy (XPS), and UV-vis and reflectance absorption infrared (RAIR) spectroscopy were used to characterize the different {SiO2/protein}n films. Even when the proteins and silica were both negatively charged, stable layer-bylayer {SiO2/protein}n films were successfully fabricated, although amounts of protein were smaller than when nanoparticles and proteins had opposite charges. Results suggest the importance of localized Coulombic attractions between the negative nanoparticle surface and positively charged amino acid residues on the Mb or Hb surfaces in the assembly and for the stability of {SiO2/protein}n films.
Introduction Beginning in the 1990s, development of a layer-by-layer method of assembly with precise thickness control on the nanometer scale has opened up a new approach to organizing proteins and polyions or nanoparticles in ultrathin films according to a predesigned architecture.1 Layer-by-layer film assembly is based on adsorption of oppositely charged polyions and proteins from their solutions in alternate steps. The major stabilizing interaction is usually assumed to be electrostatic. Mechanically stable films constructed by this method have been successfully designed for enzyme-catalyzed synthesis,2 for biosensors,3 and for fundamental electrochemical and biochemical studies of proteins.4 Our interest in the layer-by-layer film assembly derives partly from the fact that direct electron exchange between redox sites of proteins in the films and underlying electrodes can be achieved.5 Direct electrochemistry of †
Beijing Normal University. University of Connecticut and University of Connecticut Health Center. ‡
(1) (a) Lvov, Y. M.; Decher, G. Crystallog. Rep. 1994, 39, 628. (b) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwald, H., Eds.; Marcel Dekker: New York, 2000; pp 125-166. (c) Lvov, Y. In Handbook of Surfaces and Interfaces of Materials, Vol. 3. Nanostructured Materials, Micelles and Colloids; Nalwa, R. W., Ed.; Academic Press: San Diego, CA, 2001; pp 170-189. (2) Ariga, K.; Kunitake, T. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwald, H., Eds.; Marcel Dekker: New York, 2000; pp 169-192. (3) For selected examples, see (a) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708. (b) Zhang, X.; Sun, Y.; Shen, J. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwald, H., Eds.; Marcel Dekker: New York, 2000; pp 229-250. (c) Zhou, L.; Rusling, J. F. Anal. Chem. 2001, 73, 4780. (d) Mugweru, A.; Rusling, J. F. Anal. Chem. 2002, 74, 4044. (e) Dennany, L.; Forster, R. J.; Rusling, J. F. J. Am. Chem. Soc. 2003, 125, 5213. (f) Zhou, L.; Yang, J.; Estavillo, C.; Stuart, J. D.; Schenkman, J. B.; Rusling, J. F. J. Am. Chem. Soc. 2003, 125, 1431. (4) (a) Rusling, J. F. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwald, H., Eds.; Marcel Dekker: New York, 2000; pp 337-354. (b) Rusling, J. F.; Zhang, Z. In Handbook of Surfaces and Interfaces of Materials, Vol. 5. Biomolecules, Biointerfaces, and Applications; Nalwa, R. W., Ed.; Academic Press: San Diego, CA, 2001; pp 33-71.
redox proteins or enzymes in films can provide models for fundamental studies as well as a basis for fabricating new biosensors, bioreactors, and biomedical devices without the use of electron mediators.6,7 While we have developed various types of films featuring direct protein electron transfer by using surfactant bilayers,8,9 hydrogel polymers,10,11 biopolymers,12,13 surfactant-polyion conjugates,14,15 and surfactant-clay composites,16,17 these methods do not provide nanometer-scale thickness control. Further, except for the clay-based films, stability of cast films in hydrodynamic environments can be an issue. Lvov, Rusling, and co-workers assembled films of heme proteins with oppositely charged DNA, poly(styrene sulfonate) (PSS), or poly(ethylenimine) (PEI) as the “polyion glue” layer-by-layer on gold electrodes.5 Reversible FeIII/FeII electrochemistry of myoglobin (Mb) or cytochrome P450cam (Cyt P450) in these films was achieved and used to drive enzyme-catalyzed epoxidation of styrene.5,18 Moreover, several Cyt P450 enzymes were shown to adsorb to both positively and negatively charged polyion layers as a consequence of differently signed charge patches on different sides of the enzyme.19 We also achieved direct (5) Lvov, Y.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073. (6) Chaplin, M. F.; Bucke, C. Enzyme Technology; Cambridge University Press: Cambridge, U.K., 1990. (7) Armstrong, F. A.; Wilson, G. S. Electrochim. Acta 2000, 45, 2623. (8) Rusling, J. F.; Nassar, A.-E. F. J. Am. Chem. Soc. 1993, 115, 11891. (9) Yang, J.; Hu, N. Bioelectrochem. Bioenerg. 1999, 48, 117. (10) Hu, N.; Rusling, J. F. Langmuir 1997, 13, 4119. (11) Sun, H.; Hu, N.; Ma, H. Electroanalysis 2000, 12, 1064. (12) Huang, H.; Hu, N.; Zeng, Y.; Zhou, G. Anal. Biochem. 2002, 308, 141. (13) Liu, H.; Hu, N. Anal. Chim. Acta 2003, 481, 91. (14) Wang, L.; Hu, N. J. Colloid Interface Sci. 2001, 236, 166. (15) Sun, H.; Ma, H.; Hu, N. Bioelectrochem. Bioenerg. 1999, 49, 1. (16) Chen, X.; Hu, N.; Zeng, Y.; Rusling, J. F.; Yang, J. Langmuir 1999, 15, 7022. (17) Hu, N.; Li, Z.; Ma, H. Gaodeng Xuexiao Huaxue Xuebao (Chem. J. Chin. Univ.) 2001, 22, 450. (18) (a) Zu, X.; Lu, Z.; Zhang, Z.; Schenkman, J. B.; Rusling, J. F. Langmuir 1999, 15, 7372. (b) Munge, B.; Estavillo, C.; Schenkman, J. B.; Rusling, J. F. ChemBioChem 2003, 4, 101. (19) Schenkman, J. B.; Jansson, I.; Lvov, Y. M.; Rusling, J. F.; Boussaad, S.; Tao, N. J. Arch. Biochem. Biophys. 2001, 385, 78.
10.1021/la035006r CCC: $27.50 © 2004 American Chemical Society Published on Web 12/20/2003
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electrochemistry of Mb in layer-by-layer films with PSS on pyrolytic graphite (PG) electrodes.20 Surface roughness of PG and the high salt concentration of PSS adsorbate solutions provided up to seven electroactive Mb layers, while fewer layers were electroactive on smooth electrodes. We have also reported reversible voltammetry of hemoglobin (Hb) in layer-by-layer films with poly(diallyldimethylammonium) (PDDA)21 and poly(vinyl sulfonate) (PVS)22 at PG electrodes. These layer-by-layer protein films were fabricated under conditions in which proteins and synthetic polyions had opposite charges. They exhibited electrochemical properties similar to those of cast protein films but had better mechanical stability under hydrodynamic conditions. Layer-by-layer assembly has been extended to fabricate ultrathin protein films with nanoparticles. Kunitake and co-workers23 achieved spatially controlled films of cytochrome c (Cyt c) with TiO2 nanoparticles by the stepwise deposition/adsorption procedure. Willner and co-workers24 assembled multilayered microperoxidase-11/Au-nanoparticle films which acted as electrochemical catalysts for the reduction of H2O2. Lvov et al.25 made Mb films assembled layer-by-layer with MnO2 or SiO2 nanoparticles. Recently, we reported heme protein films assembled layerby-layer with clay nanoparticles.26,27 Direct reversible electrochemistry and electrochemical catalysis with various heme proteins in these films at PG electrodes were achieved. Inorganic nanoparticles, with their structural stability and small size, provided a favorable microenvironment for redox proteins in the films and facilitated the direct electron transfer of up to 10 layers of iron heme proteins with underlying electrodes.4 Again, in all these films, proteins and nanoparticles had opposite charges. The main driving force in alternate layer-by-layer film assembly is thought to be electrostatic interaction between oppositely charged species.28 However, driving forces such as hydrogen bonding and hydrophobic attraction have received recognition.29 Stockton and Rubner30 showed that layer-by-layer assembly of polyaniline with a variety of different nonionic water-soluble polymers could be realized by using hydrogen-bonding interactions. The amount of adsorbed polyaniline assembled with nonionic poly(vinylpyrrolidone), poly(vinyl alcohol), poly(acrylamide), or poly(ethylene oxide) was actually greater than that with ionic PSS. Layer-by-layer films made from poly(omethoxyaniline) (POMA) and poly(ethenesulfonic acid) (PVS) were also reported,31 whose formation was driven mainly by hydrogen-bonding interactions. Formation of layer-by-layer films was also achieved by electrostatic attraction between charged TiO2 or SiO2 nanoparticles and oppositely charged PSS or PDDA species.32,33 However, Kotov34 reported that both electro(20) Ma, H.; Hu, N.; Rusling, J. F. Langmuir 2000, 16, 4969. (21) He, P.; Hu, N.; Zhou, G. Biomacromolecules 2002, 3, 139. (22) Wang, L.; Hu, N. Bioelectrochemistry 2001, 53, 205. (23) Kimizuka, N.; Tanaka, M.; Kunitake, T. Chem. Lett. 1999, 12, 1333. (24) Patosky, F.; Gabriel, T.; Willner, I. J. Electroanal. Chem. 1999, 479, 69. (25) Lvov, Y.; Munge, B.; Giraldo, O.; Ichinose, I.; Suib, S.; Rusling, J. F. Langmuir 2000, 16, 8850. (26) Zhou, Y.; Li, Z.; Hu, N.; Zeng, Y.; Rusling, J. F. Langmuir 2002, 18, 8573. (27) Li, Z.; Hu, N. J. Electroanal. Chem. 2003, 558, 155. (28) Decher, G. Science 1997, 227, 1232. (29) Hammond, P. T. Curr. Opin. Colloid Interface Sci. 2000, 4, 430. (30) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717. (31) Raposo, M.; Oliverira, O. N., Jr. Langmuir 2002, 18, 6866. (32) Liu, Y.; Wang, A.; Claus, R. J. Phys. Chem. B 1997, 101, 1385. (33) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (34) Kotov, N. A. Nanostruct. Mater. 1999, 12, 789.
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static and hydrophobic interactions should be taken into account in the multilayer formation. Hydrophobic interactions between the hydrocarbon groups of (3-aminopropyl)trimethoxysilane-modified yttrium iron garnet (YIG) nanoparticles and polyelectrolytes PDDA or PSS significantly increased the attractive component of the nanoparticle/polyelectrolyte interactions.35 In this paper, layer-by-layer films of heme proteins (Hb and Mb) and SiO2 nanoparticles were constructed on solid substrates under different conditions to investigate the nature of the driving forces for assembly, which has not been studied systematically up to now to the best of our knowledge. Results of cyclic voltammetry (CV), quartz crystal microbalance (QCM), X-ray photoelectron spectroscopy (XPS), and UV-vis absorption spectroscopy suggested that the localized electrostatic forces between the charged surface residues of the proteins and nanoparticle surface of silica play a significant role in film formation and stability. Experimental Section Chemicals. Horse heart myoglobin (Mb, MW 17 800) was from Sigma, and bovine hemoglobin (Hb, MW 66 000) was from Shanghai Chemical Reagent Co. They were used as received. Poly(ethylenimine) (PEI, 90%, average MW 60 000), poly(sodium styrenesulfonate) (PSS, average MW 70 000), and 3-mercapto1-propanesulfonate (MPS, 90%) were from Aldrich. Nanosized SiO2 (15 ( 5 nm) was from Zhoushan Nanoparticle Technology Co.. All other chemicals were reagent-grade. Water was purified by successive ion exchange and distillation. Film Assembly. The method for layer-by-layer assembly of {SiO2/protein}n films on PG was similar to that described previously.20 Here, a typical procedure for assembly of PEI/{SiO2/ Hb}n films on PG is described briefly as an example. Prior to use, basal plane pyrolytic graphite disk (Advanced Ceramics, geometric area 0.16 cm2) electrodes were abraded on a metallographic sandpaper of 400 grit while being flushed with water. The electrodes were then ultrasonicated in water for 30 s and dried in air. A layer of positively charged PEI was adsorbed by immersing the PG electrodes into PEI solutions (3 mg mL-1 containing 0.5 M NaCl) for 20 min. After being washed in water for 1 min, the PG electrodes with the PEI precursor monolayer were alternately immersed for 20 min in aqueous dispersion of SiO2 (5 mg mL-1 containing 0.1 M KBr) at pH 9.0 and aqueous Hb solution (1 mg mL-1) at pH 5.0 with intermediate water washing and drying in a nitrogen stream. This cycle was repeated to obtain the desired number of bilayers of SiO2/Hb. For UV-vis spectroscopic study of layer-by-layer {SiO2/ protein}n films, glass slides (1 × 4 cm, 1 mm thick) were washed in 60% ethanol/39% water/1% KOH for 30 min at 50 °C, carefully rinsed with water, and dried with a nitrogen stream. The slides with negative charges on the surface were then immersed into PEI solutions for 20 min. The following procedure to fabricate {SiO2/protein}n films was the same as on PG electrodes. For QCM study, gold-coated quartz crystal resonator electrodes (geometric area 0.196 cm2, fundamental frequency 8 MHz) were soaked in a freshly prepared “piranha” solution (3:7 volume ratio of 30% H2O2 and 98% H2SO4) for 10 min (Caution: the piranha solution should be handled with extreme care, and only small volumes should be prepared at any time) and then washed in pure water and ethanol successively. The cleaned gold electrodes were immersed in MPS ethanol solutions (4 mM) for 24 h to form MPS monolayers on gold electrodes and introduce negative charges on the surface. The procedure to make PEI/{SiO2/ protein}n films on the Au/MPS surface was the same as on PG electrodes. After each adsorption step, the gold resonator electrode was washed in water, dried in a N2 stream, and measured by QCM. The {SiO2/protein}n films on Au resonator electrodes were also used for microscopic reflection absorption infrared (RAIR) spectroscopy. (35) Ostrander, J. W.; Mamedov, A. A.; Kotov, N. A. J. Am. Chem. Soc. 2001, 123, 1101.
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Figure 1. TEM image of SiO2 nanoparticles from aqueous dispersions deposited onto a carbon-coated copper grid. Apparatus and Procedures. A CHI 420 electrochemical workstation (CH Instruments) was used for cyclic voltammetry (CV) and QCM studies. For electrochemical study, a threeelectrode cell was used with a saturated calomel electrode (SCE) as the reference electrode, a platinum wire as the counterelectrode, and a PG disk modified by layer-by-layer films as the working electrode. CVs on multilayer film electrodes were run in buffers containing no proteins. Buffers were purged with a highly purified nitrogen for at least 15 min prior to a series of experiments. A nitrogen environment was then kept in the cell by continuously bubbling N2 during the whole experiment. A Cintra 10e UV-visible Spectrophotometer (GBC) was used for UV-vis spectroscopy. Microscopic RAIR spectroscopy was done with a Magna-IR 670 spectrometer (Nicolet). XPS was performed with an Escalab MKII photoelectron spectrometer (VG). A thin PG disk with adsorbed films was fixed on the XPS mounting stage by a two-sided adhesive tape. Transmission electron microscopy (TEM) image was taken with an H 600 TEM instrument (Hitachi) operating at 100 kV. All experiments were done at ambient temperature of 18 ( 2 °C.
Results Particle Size of SiO2. Transmission electron microscopy (TEM) was used to observe the actual dimension of SiO2 particles in the aqueous dispersions, showing that the diameter of SiO2 particles was in the range of 30-50 nm with an average of 35 nm (Figure 1). Layer-by-Layer Assembly of {SiO2/Hb}n by Strong Electrostatic Attraction. SiO2/Hb films were assembled on the surface of PG electrodes with the usual electrostatic growth mode, using alternate adsorption of oppositely charged layers. An initial “precursor” layer of PEI was adsorbed onto the PG surface, making the surface positively charged. Considering the isoelectric point of SiO2 is at pH 236 or 3,37 negatively charged SiO2 nanoparticles from their aqueous dispersions at pH 9.0 were adsorbed onto the PG/PEI surface. With the isoelectric point of Hb at pH 7.4,38 positively charged Hb from its solution at pH 5.0 was then adsorbed onto the PG/PEI/ SiO2 surface. This cycle was repeated to assemble multilayer {SiO2/Hb}n films on the PG/PEI surface. Here, both PEI/SiO2 and SiO2/Hb bilayers were expected to be assembled mainly by the electrostatic attraction between the oppositely charged species. CV was used to monitor the growth of the {SiO2/Hb}n films by placing the film electrode into pH 5.5 buffers after each assembly cycle (36) Franks, G. V. J. Colloid Interface Sci. 2001, 249, 44. (37) Fisher, M. L.; Colic, M.; Rao, M. P.; Lange, F. F. J. Am. Ceram. Soc. 2001, 84, 713. (38) Matthew, J. B.; Hanania, G. I. H.; Gurd, F. R. N. Biochemistry 1979, 18, 1919.
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(Figure 2). A pair of chemically reversible reductionoxidation peaks was observed at about -0.26 V vs SCE, characteristic of Hb heme FeIII/FeII redox couples. Peak currents increased with the number of SiO2/Hb bilayers (n) up to 6. Hb in the seventh bilayer assembled on the surface of {SiO2/Hb}6 films essentially showed no further increase in CV peak heights, indicating that Hb in the bilayers of n > 6 was almost not electroactive. The peak current of the {SiO2/Hb}6 films in pH 5.5 buffers depended linearly on the scan rate from 0.05 to 2.0 V s-1, and the reduction and oxidation peak currents were almost equal, consistent with the diffusionless thin-layer voltammetry.39 The {SiO2/Hb}6 films on PG electrodes showed excellent stability. The peak potentials and currents were essentially unchanged for at least 1 month when the film electrodes were stored in buffers. The layer-by-layer growth of {SiO2/Hb}n films on glass slides was monitored via the UV-vis Soret adsorption band of Hb at about 408 nm (see Supporting Information). The absorbance of the Hb Soret band increased linearly with the number of bilayers, suggesting that in each bilayer deposition nearly the same amount of SiO2/Hb was adsorbed onto the film surface, as was observed previously for other layer-by-layer Hb films.21,22 A quartz crystal microbalance (QCM) was also used to monitor the assembly of {SiO2/Hb}n films. Based on the Sauerbrey equation,40 the following relationship is obtained between adsorbed mass, ∆M (grams), and frequency shift, ∆F (hertz), by taking into account the properties of quartz resonator used in this work:
∆F ) (-1.45 × 108)∆M/A
(1)
QCM data were also used to estimate the nominal thickness of adsorbed layer for dry {SiO2/Hb}n films. The thickness, d (centimeters), can be expressed by26
d ) (-3.4 × 10-9)∆F/F
(2)
where A is the geometric area of QCM electrode (0.196 cm2), F is the density of the film material (grams per cubic centimeter). For the protein, the density was estimated to be 1.3 ( 0.1 g cm-3 41 while the density of SiO2 nanoparticles was about 2.2 ( 0.1 g cm-3.33 QCM results for dry films showed a roughly linear decrease of frequency with the adsorption step number (Figure 3). Each SiO2 adsorption layer caused a nearly constant frequency decrease of 491 Hz, corresponding to the thickness of 7.6 nm, while each Hb adsorption layer resulted in a roughly constant frequency shift of 736 Hz, indicating the thickness of 19 nm for each Hb layer (Table 1). Because of uncertainties in the estimation of film density, area, and smoothness, the estimated thicknesses are considered nominal values.42 For the films of {SiO2/Hb}n on PG electrodes, integration of the CV reduction peak gave the charge (Q) used for protein reduction, and the charge was used to estimate the surface concentration of electroactive Hb (Γ*) in the films. Γ* for {SiO2/Hb}n films increased nonlinearly with n up to 6 (Figure 4a) and then reached a nearly constant value. The surface coverage of the first monolayer of Hb on Au/MPS/PEI/SiO2 measured by QCM was about 2.4 × 10-11 mol cm-2 (Figure 3), while Γ* of electroactive Hb in (39) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 191-368. (40) Sauerbrey, G. Z. Phys. 1959, 155, 206. (41) Creighton, T. E. Protein Structure, A Practical Approach; IRL Press: New York, 1990; p 43. (42) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117.
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Figure 2. (A) Original and (B) background-subtracted cyclic voltammograms for layer-by-layer PEI/{SiO2/Hb}n films on PG electrodes at scan rate of 0.2 V s-1 in pH 5.5 buffer solutions with different numbers of bilayers. Table 1. QCM Results for Each Layer of {SiO2/Protein}n Films on Au/MPS/PEI Surface under Different Conditions frequency decrease (∆F)/Hz
amts adsorbed (∆M/A)/ng cm-2
nominal thickness (d)/nm
films
SiO2
protein
SiO2
protein
SiO2
protein
{SiO2(pH 9.0)/Hb(pH 5.0)}10 {SiO2(pH 9.0)/Mb(pH 5.0)}10 {SiO2(pH 9.0)/Mb(pH 9.0)}10
491 ( 98 443 ( 89 334 ( 53
736 ( 160 634 ( 79 353 ( 68
3380 3050 2300
5070 4370 2430
7.6 6.8 5.5
19.0 16.5 9.2
Figure 3. Shift of QCM frequency with alternate adsorption steps of SiO2 (pH 9.0) and Hb (pH 5.0) on Au/MPS/PEI electrodes: (b) SiO2 adsorption steps; (4) Hb adsorption steps.
Figure 4. Influence of the number of bilayers (n) for PEI/ {SiO2(pH 9.0)/Hb(pH 5.0)}n films on (a) surface concentration of electroactive Hb (Γ*) and (b) fraction of electroactive Hb. Data are from cyclic voltammograms at 0.2 V s-1 in pH 5.5 buffers.
the first SiO2/Hb bilayer on Au/MPS/PEI surface estimated from CV was 2.5 × 10-11 mol cm-2, suggesting that nearly 100% of Hb in this first adsorbed bilayer was electroactive. While Hb in the first bilayer is about 100% electroactive, the fraction of electroactive Hb in the following bilayers decreased significantly with n (Figure 4b), although the total amount of adsorbed Hb in each bilayer was almost the same (Figure 3, Table 1). Thus, the distance between Hb and electrode, and the possible film structure changes over this length scale, are important for efficient electron exchange.
Figure 5. (A) XPS patterns of (a) SiO2 monolayer and (b) PSS/ SiO2 films on PG disks for element of silicon. (B) XPS patterns of (c) SiO2/Hb bilayer and (d) PSS/SiO2/Hb films on PG disks for element of nitrogen. Number of scans: 40.
Adsorption of SiO2 on PG and PG/PSS. A freshly abraded PG disk electrode without the PEI precursor layer was immersed into SiO2 dispersions (pH 9.0) for 20 min. After being washed with H2O thoroughly and dried, the PG disk was tested by XPS in order to monitor the characteristic element of silicon. A characteristic Si2p peak was observed at about 104 eV (Figure 5A), suggesting that SiO2 nanoparticles can be directly adsorbed onto the PG surface. It is known that the basal plane PG surface has a partly hydrophobic character,43 with some negative charges on its rough surface on which some “edgelike” regions functionalized by oxygen are present. Roughening and polishing of carbon surfaces (e.g., PG and glassy carbon) also promotes surface oxide sites.44 Thus, PG adsorbs both positively and negatively charged organic polyions.20-22 Although the exact nature of the driving force of direct adsorption of SiO2 on the bare PG surface is not very clear, Coulombic attraction can be ruled out since SiO2 has negative charges at pH 9.0. SiO2, with many siloxane (Si-O-Si) surface groups, exhibits rather hydrophobic character.45,46 Thus the driving force may be related to hydrophobic interaction between siloxane groups on the silica surface and the PG surface. (43) Hill, H. A. O. Pure Appl. Chem. 1987, 59, 743. (44) Kamau, G. N.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1985, 57, 545. (45) Bolis, V.; Cavenago, A.; Fubini, B. Langmuir 1997, 13, 895. (46) Pelmenschikov, A.; Leszczynski, J.; Pettersson, L. G. M. J. Phys. Chem. A 2001, 105, 9528.
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Table 2. Cyclic Voltammetry Parameters of {SiO2/Mb}6 Films on Various Underlying Layers Modified on PG Electrodes and Assembled under Different Conditionsa PG/PEI
PG
PG/PSS
films
Ε°′/V
-Ipc/µA
Γ*/(mol cm-2)
Ε°′/V
-Ipc/µA
Γ*/(mol cm-2)
Ε°′/V
-Ipc/µA
Γ*/(mol cm-2)
{SiO2(pH 9.0)/Mb(pH 5.0)}6 {SiO2(pH 9.0)/Mb(pH 9.0)}6
-0.258 -0.234
3.85 1.71
2.50 × 10-10 1.11 × 10-10
-0.296 -0.284
2.03 0.807
1.32 × 10-10 5.26 × 10-11
-0.267 -0.282
1.87 0.865
1.22 × 10-10 5.23 × 10-11
a
Data from CVs at 0.2 V s-1 in pH 5.5 buffers. E°′ vs SCE.
The adsorbed SiO2 layer on PG could adsorb Hb from its solution at pH 5.0. XPS showed the characteristic nitrogen peak (N1s) at 401.5 eV (Figure 5B), confirming that Hb was adsorbed onto the SiO2 layer. The precursor film of linear polyions on solid substrates was thought to be necessary for assembly of layer-by-layer {nanoparticle/ protein}n films.47 However, our results showed that SiO2 nanoparticles could be adsorbed directly onto the PG surface, and {SiO2/Hb}n films were successfully assembled on PG without any polyion precursor. The polyion-free PG/{SiO2/Hb}n films also showed a pair of well-defined, reversible CV peaks in pH 5.5 buffers with similar peak potentials but smaller peak heights compared with the PG/PEI/{SiO2/Hb}n films at the same n value. The unique properties of SiO2 nanoparticles demonstrated above stimulated us to further investigate the possibility of assembling SiO2 on a layer of negatively charged PSS. A PG disk was first immersed into a PSS solution for 20 min, then washed with H2O thoroughly, and dried. XPS results showed a characteristic sulfur peak (S2p) at 169 eV, confirming that PSS was adsorbed onto the PG surface. The PG/PSS film was then soaked into SiO2 dispersions at pH 9.0 for 20 min. After the washing and drying procedure, an XPS peak of Si2p was observed at 104 eV (Figure 5A), although the peak was smaller than that for silica on PG under the same condition. If only electrostatic interaction was taken into account, the negatively charged SiO2 nanoparticles should not be adsorbed onto the surface of the polyanionic PSS layer because of their repulsive interaction. However, our experiments indicate that this repulsive interaction between PSS and SiO2 could be overcome by some stronger effect, and the adsorption of SiO2 nanoparticles on the PSS layer was realized. This effect would most probably be the hydrophobic interaction between Si-O-Si groups of SiO2 nanoparticles and hydrocarbon chains of PSS. The XPS Si2p peak height for PG/PSS/SiO2 films was smaller than that for PG/SiO2 films under the same condition (Figure 5A). This is understandable since the electrostatic repulsion between PSS and SiO2 may cause less adsorption of SiO2 on the PSS layer. The adsorption of Hb on the surface of PG/PSS/SiO2 films was also realized and confirmed by XPS. A N1s peak at 401.5 eV for PG/ PSS/SiO2/Hb films was observed but smaller than that for PG/SiO2/Hb films for a similar reason (Figure 5B). In pH 5.5 buffers, PG/PSS/{SiO2/Hb}n films showed quite good CV signals for the Hb heme FeIII/FeII couples, which grew with the number of bilayer (n) when n < 7 (see Supporting Information). With the same adsorption bilayer number (n), the CV reduction peak currents showed a sequence of PG/PEI/{SiO2/Hb}n > PG/{SiO2/Hb}n > PG/ PSS/{SiO2/Hb}n. Layer-by-Layer Assembly of SiO2 with Negatively Charged Hb. The isoelectric point of Hb is at pH 7.4.38 Thus, the surface charge of Hb is positive at pH 5.0 and negative at pH 9.0. The SiO2 nanoparticles in the pH 9.0 dispersion are negatively charged. Layer-by-layer {SiO2/
Hb(pH 5.0)}n and {SiO2/Hb(pH 9.0)}n films were assembled on PG. CVs were performed after each SiO2/Hb bilayer deposition cycle. Both {SiO2/Hb(pH 5.0)}n and {SiO2/Hb(pH 9.0)}n films showed a pair of nearly reversible reduction-oxidation peaks at about -0.26 V, and the reduction peak increased with n when n < 7. The {SiO2/ Hb(pH 9.0)}n films showed a smaller reduction in peak height than the {SiO2/Hb(pH 5.0)}n films at the same n (see Supporting Information) since a smaller amount of Hb was adsorbed onto SiO2 layers at pH 9.0 due to repulsive interaction between SiO2 and Hb. Although SiO2 and Hb have the same negative surface charges at pH 9.0, the {SiO2/Hb(pH 9.0)}n films could still be successfully assembled layer by layer. This indicates that in multilayer assembly involving proteins, oppositely charged species are favorable but not absolutely necessary. Layer-by-Layer Assembly of SiO2 with Mb under Different Conditions. Mb, with a 4-fold smaller molecular weight than Hb, was also used to assemble layerby-layer films with SiO2 nanoparticles. In general, Mb showed very similar properties to Hb during assembly with SiO2 nanoparticles, suggesting a generality of the interaction between proteins and nanoparticles. With the isoelectric point at pH 6.8,48 Mb has a positive surface charge at pH 5.0 and a negative surface charge at pH 9.0. SiO2 nanoparticles are negatively charged at pH 9.0. Thus, layer-by-layer {SiO2/Mb}n films assembled under different conditions, designated as {SiO2(pH 9.0)/ Mb(pH 5.0)}n and {SiO2(pH 9.0)/Mb(pH 9.0)}n, respectively, were fabricated on the surface of PG/PEI and PG/ PSS, as well as bare PG. Under all selected conditions, the assembly of layer-by-layer {SiO2/Mb}n films was successfully realized. In pH 5.5 buffers, both CV reduction and oxidation peaks centered at about -0.26 V, characteristic of Mb heme FeIII/FeII redox couple, grew with the number of bilayers (n) up to 6. Some of the CV results for {SiO2/Mb}6 films assembled under different conditions and with different precursor layers are listed in Table 2 for comparison. For example, with the same underlying layer of PG/PEI, the reduction peak current of {SiO2(pH 9.0)/Mb(pH 5.0)}6 films was larger than that of {SiO2(pH 9.0)/Mb(pH 9.0)}6 films (Table 2, Figure 6). For the same {SiO2(pH 9.0)/Mb(pH 5.0)}6 films on different underlying layer surfaces, the CV reduction peak currents and Γ* demonstrated an order of PG/PEI > PG > PG/PSS, the same as for the {SiO2/Hb}n film system. All the {SiO2/ Mb}n films on PG electrodes showed a very good stability. QCM was used to monitor the assembly process of {SiO2/ Mb}n films deposited on the surface of Au/MPS/PEI. A roughly linear frequency decrease with n was observed for both {SiO2(pH 9.0)/Mb(pH 5.0)}n and {SiO2(pH 9.0)/ Mb(pH 9.0)}n film systems, indicating the amount of each adsorbed SiO2/Mb bilayer is almost constant, respectively, as observed for the Hb system. The QCM results for {SiO2/ Mb}n films assembled under different conditions are also listed in Table 1 for comparison. As expected, the amount of SiO2 and Mb adsorbed for the {SiO2(pH 9.0)/Mb(pH
(47) Kotov, N. A.; Haraszti, T.; Turi, L.; Zavala, G.; Geer, R. E.; Dekany, I.; Hendler, J. H. J. Am. Chem. Soc. 1997, 119, 6821.
(48) Bellelli, A.; Antonini, G.; Brunori, M.; Springer, B. A.; Sligar, S. J. J. Biol. Chem. 1990, 265, 18898.
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Figure 6. Cyclic voltammograms at 0.2 V s-1 in pH 5.5 buffers for (a) PEI/{SiO2 (pH 9.0)/Mb(pH 9.0)}6 films and (b) PEI/{SiO2(pH 9.0)/Mb(pH 5.0)}6 films modified on PG electrodes.
Figure 7. UV-vis spectra on glass slides for (a) dry Mb films, (b) dry PEI/{SiO2/Mb}6 films, and PEI/{SiO2/Mb}6 films in different pH buffers: (c) pH 5.0, (d) pH 7.0, (e) pH 9.0, (f) pH 11.0, and (g) pH 4.0.
5.0)}n films is larger than that for {SiO2(pH 9.0)/Mb(pH 9.0)}n films, respectively. For oppositely charged assembly of {SiO2(pH 9.0)/Mb(pH 5.0)}n films, each Mb adsorption layer resulted in a frequency decrease of 634 Hz, indicating the nominal layer thickness of about 16.5 nm, about twice larger than that of {SiO2(pH 9.0)/Mb(pH 9.0)}n films (9.2 nm). This result is consistent with that of CV experiments, in which the surface concentration of electroactive Mb (Γ*) for the first SiO2(pH 9.0)/Mb(pH 5.0) bilayer was about twice as large as that of {SiO2(pH 9.0)/Mb(pH 9.0)}1 films. Mb has a dimension of 2.5 × 3.5 × 4.5 nm.49 In both situations, the nominal thickness of Mb layer is larger than the monolayer thickness of Mb, suggesting the multilayer adsorption or possible aggregation of Mb on the surface of SiO2 nanoparticles. UV-Vis Spectroscopy of {SiO2/Protein}n Films. UV-vis spectroscopy50 was used to check conformational integrity of the heme proteins in {SiO2/protein}n films. For example, both dry cast Mb films and dry {SiO2/Mb}6 films on glass slides showed Soret bands at 410 nm (Figure 7a,b), suggesting that Mb in dry {SiO2/Mb}6 films has a secondary structure nearly the same as the native state of Mb in dry Mb films alone. The position of the Soret band depended on pH when {SiO2/Mb}6 films were immersed into buffer solutions. At pH 5.0 and 7.0, the Soret band appeared at 410 nm (Figure 7c,d), while at pH 9.0, the Soret band was at 411 nm (Figure 7e). This suggests that Mb in {SiO2/Mb}6 films essentially retains a secondary structure similar to its native state in the (49) Kendrew, J.; Phillips, D.; Stone, V. Nature 1960, 185, 422. (50) George, P.; Hanania, G. I. H. J. Biochem. 1953, 55, 236.
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Figure 8. (A) Original and (B) second-derivative microscopic RAIR spectra of (a) monolayer SiO2 adsorbed on Au/MPS/PEI surface, (b) cast Mb films onto Au surface, (c) layer-by-layer {SiO2(pH 9.0)/Mb(pH 9.0)}10 films assembled on Au/MPS/PEI surface, (d) layer-by-layer {SiO2(pH 9.0)/Mb(pH 5.0)}10 films assembled on Au/MPS/PEI.
medium pH range. When pH was changed toward more basic or more acidic direction, the Soret band became smaller and broader (Figure 7f,g), indicating Mb in {SiO2/ Mb}6 films denatures to a considerable extent at these relatively extreme pHs. Hb in {SiO2/Hb}6 films showed similar spectra. Reflectance Absorption Infrared (RAIR) Spectroscopy of {SiO2/Protein}n Films. The shape and position of amide I (1600-1700 cm-1) and amide II (15001600 cm-1) infrared bands provides detailed information on the secondary structure of polypeptide chain of proteins.51,52 Microscopic RAIR spectroscopy was used here to detect conformational change of proteins in {SiO2/ protein}n films (Figure 8A). Amide bands of proteins are composites of many overlapped bands, so we used secondderivative RAIR spectra to enhance resolution (Figure 8B).52 A cast Mb films showed an IR amide I band at 1652 cm-1, which was caused by CdO stretching vibration, and an amide II band at 1540 cm-1, which was assigned to a combination of N-H in-plane bending and C-N stretching vibration. The RAIR spectrum of SiO2 monolayer was also showed in Figure 8 as a control. Results show that the IR peaks at 1651 and 1543 cm-1 for both {SiO2(pH 9.0)/Mb(pH 9.0)}10 and {SiO2(pH 9.0)/Mb(pH 5.0)}10 films would be solely attributed to Mb amide I and II bands, respectively. Both amide I and II bands for {SiO2/Mb}10 films in RAIR spectra had very similar shapes and positions to those of Mb film alone. Furthermore, the strongest bands in the second-derivative spectra near 1660-1655 cm-1 corresponding to R-helices, which comprise 76% of Mb’s secondary structure, are similar for all the films. These findings support the view that Mb in {SiO2/Mb}10 films retains a secondary structure close to the native form. Discussion Driving Forces for Film Assembly. The above experimental results confirm that Coulombic attraction between oppositely charged nanoparticles and proteins plays an important role in the assembly of layer-by-layer {SiO2/protein}n films (Figures 2 and 3), as observed previously.25,26 However, multilayer assembly was also realized by alternate adsorption of negatively charged Mb or Hb on negatively charged SiO2 nanoparticles (Tables (51) Dong, A.; Huang, P.; Caughey, W. S. Biochemistry 1990, 29, 3303. (52) Rusling, J. F.; Kumosinski, T. F. Nonlinear Computer Modeling of Chemical and Biochemical Data; Academic Press: New York, 1996; pp 117-134.
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1 and 2, Figures 6 and S3), suggesting that the interaction between SiO2 and the proteins is more complicated than simple Coulombic attraction. Short-range hydrophobic interactions between polymers or between polymers and proteins play a significant role in layer-by-layer film assembly.34 SiO2 nanoparticles have many siloxane surface groups and demonstrate rather hydrophobic properties.45,46 This may explain why negatively charged SiO2 were successfully adsorbed onto PG or a polyanionic PSS layer (Figure 5), which both have some hydrophobic character. However, it is unlikely that hydrophobic interaction becomes a predominant driving force in the assembly of {SiO2/protein}n films, since watersoluble Mb and Hb demonstrate very hydrophilic character, especially on their surface. Hydrogen bonding is recognized as a relatively important secondary force to fabricate films in a layer-by-layer manner.30,31 However, hydrogen-bonding interactions between nanoparticles and proteins seem not to have been reported. The surface of silica has many silanol groups when it contacts water. Above the isoelectric point (pH 2-3),36,37 the silica surface becomes negatively charged due to the dissociation of these surface hydroxyl groups.53 Thus, at pH 9.0, the negative surface charges on the surface of SiO2 nanoparticles are from the silanol ionization (SiOH f Si-O- + H+). Mb and Hb have a considerable number of side-chain amine or imine groups on their surface residues. Thus, Mb or Hb might interact with SiO2 nanoparticles through N-H‚‚‚O hydrogen bonds between amine or imine residues on the proteins and Si-O- groups on the nanoparticle surface. While we have initiated RAIR studies of these interactions, they have yet to produce convincing and reproducible evidence of strong H-bonding. Given this background, it is difficult to consider hydrogen bonding as a predominant driving force in the film assembly. Schenkman et al.19 studied the influence of surface charge of amino acid residues of Cyt P450 enzymes CYP101 and CYP2B4 on binding to polyion surface and other proteins by atomic force microscopy and QCM. They found that the binding was profoundly influenced by the asymmetrical distribution of surface charges on Cyt P450s. Cyt P450 enzymes had “charge patches” of different signs on different sides, which imparted a sidedness to these protein molecules. We thus also considered the influence and locations of surface charges of amino acid residues of Mb and Hb. The surface number of all amino acid residues and the corresponding pKa values in horse heart Mb and bovine Hb are known (see Supporting Information). Taking Mb as an example, aspartic acid (Asp) and glutamic acid (Glu) are residues with carboxyl groups. There are eight Asp (pKa ) 2-4) and 12 Glu (pKa ) 2-4) on the Mb surface, and the total number of negatively charged surface residues is 20 at both pH 5.0 and 9.0. Lysine (Lys), arginine (Arg), and histidine (His) are residues that all have amine or imine groups. There are 19 Lys (pKa ) 10-12) and two Arg (pKa ) 12-13) on the Mb surface, and the total number of positively charged surface Lys and Arg residues is 21 at both pH 5.0 and 9.0. Among the total 11 His residues in Mb, only six are located at the surface, while the other five are buried in the internal hydrophobic pockets of Mb.54 These six surface His residues with pKa of 6-7 will be protonated at pH 5.0 and noncharged at pH 9.0. Thus, at pH 5.0, there are 20 negative surface charges and 27 positive surface charges for Mb, while at pH 9.0, the (53) de Keizer, A.; van der Ent, E. M.; Koopal, L. K. Colloids Surf. A 1998, 142, 303. (54) Stigter, D.; Alonso, D. O. V.; Dill, K. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4176.
He et al.
negative surface charges remain at 20 but the positive surface charges decrease to 21. Some of these residues on the protein surface may be involved in electrostatic interactions with silica nanoparticles. With the isoelectric point of Mb at pH 6.8,48 the net surface charge of Mb is negative at pH 9.0. However, at this pH, there is still quite a number of positively charged groups at the Mb surface. Thus, electrostatic attraction between the positively charged surface side chains of Mb residues and the negatively charged nanoparticle surface of SiO2 may be the main driving forces for the assembly. At pH 5.0, there is a net positive charge (+7)54,55 on the Mb surface, and stronger electrostatic attraction increases the amount of adsorbed Mb. The surface charge distribution of Mb and Hb at different pH and on different regions of the protein surface can be represented in space-filling models (see Supporting Information). Considerable binding of overall negative Mb and Hb molecules to like-charged surfaces during film formation is likely to result from the presence of localized electrostatic interactions between the negative nanoparticle surface and positive residues on the protein surface that exist at all pH values used. This is similar to what has been observed with Cyt P450 enzymes on polyion surfaces,19 but in that case different sides of the enzyme retained localized charged patches of opposite sign. Mb and Hb do not seem to have such patches of charge on their surfaces, having a more even distribution of surface charges. Yet the interactions are strong enough for them to participate in film formation even at pH 9.0 with the negatively charged nanoparticles. Theoretical studies have also been carried out to investigate interactions in charged colloid, protein, macroion, or polydisperse systems.56-59 For example, Prausnitz and co-workers56 performed Monte Carlo simulations of the simultaneous orientation-averaged interaction between anisotropic charged colloids in electrolyte solution. The results demonstrated that the colloids with a dipole moment could carry asymmetrically distributed charged groups, and the angle-averaged interaction between the colloids could be strongly attractive. This electrostatic multipolar force may be largely responsible for attractions between proteins even when they are like-charged.59 These theoretical studies qualitatively support our interpretation based on localized attractions of proteins with like-charged SiO2 nanoparticles. Electrochemical Properties. Nearly reversible cyclic voltammograms for the two heme proteins, Hb and Mb, were observed for layer-by-layer {SiO2/protein}n film electrodes (Figures 2 and 6), indicating direct electron transfer between the heme proteins and PG electrodes in the film environment. Electron communication was much faster for {SiO2/protein}n films than for the proteins in solution on bare PG. Thus, SiO2 nanoparticle films greatly enhance the electron-transfer rates and provide a favorable microenvironment for the proteins to transfer electrons with underlying PG electrodes. While the exact nature of this effect is not yet clear, it is probable that the adsorbed layer of SiO2 on PG electrodes selectively adsorbs Hb or Mb from the protein solutions and prevents the adsorption of macromolecular impurities onto electrodes, which could otherwise block electron exchange for the proteins.60 (55) Goto, Y.; Fink, A. L. J. Mol. Biol. 1990, 214, 803. (56) Striolo, A.; Bratko, D.; Wu, J. Z.; Elvassore, N.; Blanch, H. W.; Prausnitz, J. M. J. Chem. Phys. 2002, 166, 7733. (57) Piech, M.; Walz, J. Y. J. Colloid Interface Sci. 2002, 253, 117. (58) Stevens, M. J.; Falk, M. L.; Robbins, M. O. J. Chem. Phys. 1996, 104, 5209. (59) McClurg, R. B.; Zukoski, C. F. J. Colloid Interface Sci. 1998, 208, 529.
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Data in Figure 4 suggest that, for {SiO2/Hb}n films, the innermost layers of Hb closest to the electrode surface are electrochemically addressable and the fraction of electroactive Hb in each bilayer decreased dramatically with n. The electroactive Hb could only extend to about six SiO2/Hb bilayers, demonstrating the key role of distance between the electroactive center of proteins and the surface of electrodes. Mb in {SiO2/Mb}n films demonstrated similar behavior. It is unlikely that the protein molecules are mobile in these films, and electron transport is probably facilitated by a charge-hopping mechanism as suggested for polyion-protein films.4 Structural Features. The average size of SiO2 particles in their aqueous dispersions is about 35 nm (Figure 1), larger than that of the original ones at about 15 nm, indicating a certain degree of aggregation of SiO2 in the dispersions. QCM results show that the nominal thickness of the SiO2 layer measured in the process of layer-bylayer assembly of {SiO2/protein}n films was in the range of 5-8 nm (Table 1), much smaller than the size of SiO2 particles measured by TEM, and even smaller than the original size of SiO2. This nominal thickness corresponds with only 1/7-1/4 the monolayer thickness of SiO2 layer. Since the thickness estimated by QCM is based on the assumption that the adsorbed particles are packed tightly with each other, the smaller nominal thickness of SiO2 layers compared with the size of SiO2 particles seems to suggest that, in the adsorbed SiO2 layer, the SiO2 particles are arranged loosely on the surface. The nominal thickness of the adsorbed protein layer estimated by QCM is about 19 nm for Hb and 9-17 nm for Mb (Table 1), larger than the molecular sizes of Hb (5.0 × 5.5 × 6.5 nm)61 and Mb (2.5 × 3.5 × 4.5 nm),49 respectively. It is probable that the protein molecules are adsorbed around the surface of the SiO2 particles and fill in spaces between the SiO2 particles, as suggested by Lvov et al.25 (60) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386. (61) Perutz, M.; Muirhead, H.; Cox, J.; Goaman, L.; Mathews, L.; Mcgandy, E.; Webb, L. Nature 1968, 219, 29.
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The UV-vis and RAIR spectra of {SiO2/protein}n films (Figures 7 and 8) indicate that Mb and Hb in layer-bylayer films with SiO2 nanoparticles essentially retain the secondary structure similar to that of their native state in their dry and “wet” forms at medium pH. Conclusions Electrostatic interaction between oppositely charged species is an important driving force for the layer-by-layer film assembly of SiO2 nanoparticles and proteins. However, the localized Coulombic attraction between positively charged surface residues of the proteins and the negatively charged nanoparticle surface also plays a significant role. This localized electrostatic attraction may lead to a stable film assembly even when the proteins have net negative charges on their surface the same as the silica nanoparticles. Acknowledgment. Financial support from the National Natural Science Foundation of China (NSFC, 20275006, 29975003) and the State Key Laboratory of Electroanalytical Chemistry of Changchun Institute of Applied Chemistry, the Chinese Academy of Sciences (N.H.), and National Institute of Environmental Health Sciences (NIEHS), NIH, under PHS Grant ES03154 (J.F.R.) is gratefully acknowledged. Supporting Information Available: Four figures showing layer-by-layer growth of {SiO2/Hb}n films monitored by UV-vis spectroscopy, layer-by-layer growth of PSS/{SiO2/Hb}n films on PG electrodes monitored by CV, influence of the number of bilayers (n) on CV reduction peak current for {SiO2(pH 9.0)/ Hb(pH 5.0)}n and {SiO2(pH 9.0)/Hb(pH 9.0)}n films, and spacefilling models for Mb and Hb at different pHs; and two tables showing the total and surface number of amino acid residues and corresponding pKa values in horse heart Mb and bovine Hb. This information is available free of charge via the Internet at http://pubs.acs.org. LA035006R
