Heme Protein−Clay Films: Direct ... - American Chemical Society

Langmuir 2002, 18, 211-219

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Heme Protein-Clay Films: Direct Electrochemistry and Electrochemical Catalysis Yinglin Zhou,† Naifei Hu,*,† Yonghuai Zeng,† and James F. Rusling‡ Department of Chemistry, Beijing Normal University, Beijing, 100875, China, and Department of Chemistry, University of Connecticut, U-60, Storrs, Connecticut 06269-3060 Received June 5, 2001. In Final Form: September 4, 2001 Stable protein-clay films were fabricated by casting an aqueous dispersion of protein and clay on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) in clay films gave a pair of well defined, quasi-reversible cyclic voltammetric peaks at about -0.28 V vs SCE in pH 5.5 buffers, characteristic of the protein heme FeIII/FeII redox couples. Square wave voltammograms (SWV) of the protein-clay films gave good fits by nonlinear regression analysis to a model that featured thin-layer SWV and formal potential dispersion, providing average apparent heterogeneous electrontransfer rate constants, ks, and average formal potentials, E°′. UV-vis and reflectance absorption infrared spectra showed that the proteins in clay films retained near-native secondary structures. X-ray diffraction revealed that Mb-clay and Hb-clay films feature ordered layered structures with Mb and Hb intercalated between clay layers. Incorporated HRP induced disorder in the clay films. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all three proteins in clay films.

Introduction Direct electron exchange between enzymes and electrodes can drive enzyme-catalyzed reactions as a basis for biosensors, biomedical devices, and enzymatic bioreactors.1,2 This approach simplifies such devices by removing the requirement for mediators and thus is of significance for fabricating the third generation biosensors,3as well as for mechanistic studies of biological electron transport. A long-term goal of our research is to develop general, stable electrode coatings for applications of enzyme chemistry. Successful approaches have included cast films of proteins with insoluble surfactants,4-6 hydrogel polymers,7-9 polyelectrolyte-surfactant composites,10-12 and films of proteins and polyions grown layer-by-layer.13-15 All these films facilitate much faster direct, reversible electron transfer between metalloproteins and electrodes compared to that on bare electrodes with the proteins in solution. Clays are layered aluminosilicates with cation-exchange properties. Clay colloids have a platelet shape and can be deposited on electrodes. The resulting films can incor† ‡

Beijing Normal University. University of Connecticut.

(1) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407. (2) Chaplin, M. F.; Bucke, C. Enzyme Technology; Cambridge University Press: Cambridge, U.K., 1990. (3) Gorton, L.; Lindgren, A.; Larsson, T.; Munteanu, F. D.; Ruzgas, T.; Gazaryan, I. Anal. Chim. Acta 1999, 400, 91. (4) Rusling, J. F.; Nassar, A.-E. F. J. Am. Chem. Soc. 1993, 115, 11891. (5) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363. (6) Yang, J.; Hu, N. Bioelectrochem. Bioenerg. 1999, 48, 117. (7) Hu, N.; Rusling, J. F. Langmuir 1997, 13, 4119. (8) Yang, J.; Hu, N.; Rusling, J. F. J. Electroanal. Chem. 1999, 463, 53. (9) Sun, H.; Hu, N.; Ma, H. Electroanalysis 2000, 12, 1064. (10) Sun, H.; Ma, H.; Hu, N. Bioelectrochem. Bioenerg. 1999, 49, 1. (11) Hu, Y.; Hu, N.; Zeng, Y. Talanta 2000, 50, 1183. (12) Wang, L.; Hu, N. J. Colloid Interface Sci. 2001, 236, 166. (13) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073. (14) Ma, H.; Hu, N.; Rusling, J. F. Langmuir 2000, 16, 4969. (15) Wang, L.; Hu, N. Bioelectrochemistry 2001, 53, 205.

porate electrochemically active cations from solutions. Clay films demonstrate several advantages: appreciable surface area, ordered structure, intercalation properties, low cost, high stability, and high exchange capacity.16 The interaction of proteins with clay has been studied for some time. For example, the activity, stability, and kinetic properties of the extracellular soil enzymes invertase and urease immobilized on different montmorillonite clays have been discussed.17 A glucose sensor was constructed by immobilizing glucose oxidase and methyl viologen into nontronite clay films on glassy carbon electrodes.18 Direct electrochemistry of cytochrome c,19 c-type cytochromes,20 hemoglobin,21 and cytochrome P45022 at clay-modified carbon electrodes was reported. Furthermore, clay films have been used to incorporate glucose oxidase with polycation polyethylene amine using layer-by-layer film growth.23 However, the structural features of the proteinclay films have not been fully investigated. We recently reported enhanced electron transfer of hemoglobin (Hb)24 and myoglobin (Mb)25 intercalated in surfactant-clay films on pyrolytic graphite (PG) electrodes. Increased interlayer clay spacing estimated by X-ray diffraction suggested that Hb and Mb were at least partly intercalated between clay platelets. The doublechain cationic surfactant in the films was self-assembled in an ordered bilayer structure between clay layers. (16) Macha, S. M.; Fitch, A. Mikrochim. Acta 1998, 128, 1. (17) Gianfreda, L.; Violante, A. In Environment Impact Soil Compound Interaction; Huang, P. M., Ed.; Lewis: Boca Raton, FL, 1995; Vol. 2, p 201. (18) Zen, J.-M.; Lo, C.-W. Anal. Chem. 1996, 68, 2635. (19) Lei, C.; Lisdat, F.; Wollenberger, U.; Scheller, F. W. Electroanalysis 1999, 11, 274. (20) Sallez, Y.; Bianco, P.; Lojou, E. J. Electroanal. Chem. 2000, 493, 37. (21) Fan, C.; Zhuang, Y.; Li, G.; Zhu, J.; Zhu, D. Electroanalysis 2000, 12, 1156. (22) Lei, C.; Wollenberger, U.; Jung, C.; Scheller, F. W. Biochem. Biophys. Res. Commun. 2000, 268, 740. (23) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (24) Chen, X.; Hu, N.; Zeng, Y.; Rusling. J. F.; Yang, J. Langmuir 1999, 15, 7022. (25) Hu, N.; Li, Z.; Ma, H. Gaodeng Xuexiao Huaxue Xuebao (Chem. J. Chinese Universities) 2001, 22, 450.

10.1021/la010834a CCC: $22.00 © 2002 American Chemical Society Published on Web 12/06/2001

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Electrochemical catalysis of some substrates at the protein-surfactant-clay film electrodes was realized. In the course of our work with surfactant-clay films,24 we found that simple, unmodified clay could also incorporate metalloproteins. In the present paper, we examine incorporation of three heme proteins, Mb, Hb, and horseradish peroxidase (HRP) into clay films on PG electrodes. All protein-clay films showed direct, reversible electrochemistry for their heme FeIII/FeII redox couples. The structure of the protein-clay films and the conformation of the proteins in the films were characterized by X-ray diffraction and optical techniques. Electrochemical reductions of oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalyzed by the proteins in these films. Experimental Section Chemicals. Horse heart myoglobin (Mb, MW 17 800) and human hemoglobin (Hb, MW 66 000) were from Sigma. Lyophilized horseradish peroxidase (HRP, MW 42 100) was from Shanghai Lizhu Dongfeng Biotechnology Co. Bentonite clay (Bentolite H) was from Southern Clay Products and had a cation exchange capacity of 80 mequiv/100 g. All other chemicals were reagent grade. NaNO2 and H2O2 were freshly prepared before being used. Buffers were 0.1 M sodium acetate, 0.05 M sodium dihydrogen phosphate, 0.05 M boric acid, or 0.05 M citric acid, all containing 0.1 M KBr. Buffer pH was adjusted with HCl or NaOH. Water was purified twice successively by ion exchange and distillation. Preparation of Films. Prior to coating, basal plane pyrolytic graphite (PG, HPG-99, Union Carbide, geometric area 0.16 cm2) electrodes were abraded by hand with metallographic sandpaper of 1200 grit while flushing with pure water. Electrodes were ultrasonicated in pure water for 30 s after each polishing step. Cloudy clay suspensions (1 mg mL-1) were prepared by dispersing clay in water with ultrasonication for about 2 h. This suspension was sonicated for another 15 min immediately before preparing the films. To get the best CV responses of protein-clay films, the concentration of proteins, the ratio of protein/clay, and the total volume of protein-clay dispersion were optimized. Typically, 10 µL of the dispersion containing 3.4 x 10-5 M Mb and 0.5 mg mL-1 clay was spread evenly onto PG electrodes for preparing Mbclay films. Protein concentrations in similar dispersion for the other films were 8.5 x 10-6 M Hb and 6.7 x 10-5 M HRP. A small bottle was fit tightly over the electrode so that water evaporated slowly and more uniform films were formed. Films were then dried in air overnight. Apparatus and Procedures. A CHI 660 electrochemical workstation (CH Instruments) was used for cyclic voltammetry (CV) and square wave voltammetry (SWV) as described previously.24 Potentials are reported vs saturated calomel electrode (SCE). In the experiments with oxygen, measured volumes of air were injected through solutions via a syringe in a sealed cell which had been previously degassed with purified nitrogen. UV-vis spectroscopy was done with a DMS 300 UV-visible spectrophotometer (Varian). Sample films for spectroscopy were prepared by depositing protein-clay films onto glass slides. Reflectance absorption infrared (RAIR) spectra were obtained by using a NEXUS 360 FT-IR spectrophotometer (Nicolet) with a DTGS detector at 2 cm-1 resolution. Films were prepared by depositing the protein-clay films onto Al disks with Au disk as reference. X-ray diffraction (XRD) was done with a D/MAX-RB powder diffractometer (Rigaku) using a Cu KR source at 40 kV and 120 mA with scan rate of 1 deg min-1. Films of clay and protein-clay for XRD were prepared on glass slides.

Results Cyclic Voltammetry (CV). Mb-clay, Hb-clay, or HRP-clay films on PG electrodes in protein-free pH 5.5 buffers gave pairs of well-defined, reversible CV peaks at about -0.28 V vs SCE at the steady state after multiple

Zhou et al.

Figure 1. Cyclic voltammograms at 0.2 V s-1 in pH 5.5 buffers for (a) clay film, (b) Mb-clay film, (c) Hb-clay film, and (d) HRP-clay film.

scans (Figure 1b-d). The peaks were located at the potentials characteristic of the heme FeIII/FeII redox couples of the proteins.4,26,27 No voltammetric peaks were observed for the clay-modified PG electrodes in the same potential window (Figure 1a). For Mb-clay or Hb-clay films, the peak pair quickly reached the steady state after several CV cycles in buffers, while for HRP-clay films, the peak currents increased with soaking time and reached the steady state in about 2 h. Thus, all following electrochemical experiments were reported at the steady state after multiple scans. CVs of protein-clay films had roughly symmetric peak shapes and nearly equal heights of reduction and oxidation peaks. The reduction peak currents increased linearly with scan rates from 0.01 to 5 V s-1. Integration of reduction peaks gave nearly constant charge (Q) values independent of scan rate. All these results are characteristic of thinlayer electrochemical behavior, in which all electroactive proteins in the films are reduced on the forward cathodic scan, with full conversion of the reduced proteins to their oxidized forms again on the reversed anodic scan.28 Using the integrals of reduction peaks and Faraday’s law,28 the average surface concentrations of electroactive proteins in the films (Γ*) were estimated (Table 1). While electroactive Mb and Hb in the clay films was about 10% of the total proteins deposited on the electrodes, the electroactive HRP in HRP-clay films accounted for only 2% of the total HRP cast on electrodes. To investigate the influence of film thickness on the fraction of electroactive protein, various amounts of Hbclay dispersion with the same Hb/clay ratio were deposited on PG electrodes. CVs were run to obtain the values of Γ*. Results showed that the fraction of electroactive Hb increased with decreasing film thickness (Table 2), suggesting that only those proteins in the inner layers of the films closest to electrodes can exchange electrons with the electrodes. Similar behavior was found earlier for protein-surfactant-clay films.24 The possibility of proteins entering the clay films from solution was also tested. Clay films on PG electrodes were placed into a pH 5.5 buffer containing Hb, and CVs were run periodically. CV scans at different times revealed the growth of the reversible peaks at about -0.3 V (Figure 2), indicating increasing amount of Hb entered the clay films. The peak current reached the steady state after 216 h of soaking. For Mb and HRP, a similar situation was observed but with the steady state reached after 214 and 188 h, respectively. Both cast and immersing methods showed very similar peak positions for protein-clay films at the (26) Huang, Q.; Lu, Z.; Rusling, J. F. Langmuir 1996, 12, 5472. (27) Ferri, T.; Poscia, A.; Santucci, R. Bioelectrochem. Bioenerg. 1998, 44, 177. (28) Murry, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1986; Vol. 13, p 191.

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Langmuir, Vol. 18, No. 1, 2002 213 Table 1. Electrochemical Parameters of Protein-Clay Films

molec wt Mb Hb HRP

17 800 66 000 42 100

isoelectric point 6.8c 7.4d 8.9e

pH used

deposited Γ, 10-11 mol cm-2

electroactive Γ*,a 10-11 mol cm-2

% electroactive protein

7.0 7.0 7.0

212 53.1 625

18.5 6.3 13.2

8.7 11.9 2.1

E°′, V vs NHE av ks

,b

s-1

50 ( 3 31 ( 2 74 ( 5

CV

SWVb

in solution

-0.100 -0.105 -0.122

-0.116 -0.118 -0.121

0.050f 0.137g -0.270h

a Estimated by integration of CV reduction peak. b Average values for analysis of eight SWVs at frequencies of 100-227 Hz, amplitudes of 60-75 mV, and a step height of 4 mV. c Bellelli, A.; Antonini, G.; Brunori, M.; Springer, B. A.; Sligar, S. G. J. Biol. Chem. 1990, 265, 18898. d Matthew, J. B.; Hanania, G. I. H.; Gurd, F. R. N. Biochemistry 1979, 18, 1919. e Welinder, K. G. Eur. J. Biochem. 1979, 96, 483. f King, B. C.; Hawkridge, F. M.; Hoffman, B. M. J. Am. Chem. Soc. 1992, 114, 10603. g Faulkner, K. M.; Bonaventura, C; Crumbliss, A. L. J. Biol. Chem. 1995, 270, 13604. h Harbury, H. A. J. Biol. Chem. 1959, 225, 1009.

Table 2. Surface Concentrations and Fractions of Electroactive Hb for Hb-Clay Films with Different Amounts of Clay and Hb (Area of PG Electrodes 0.28 cm2) clay, µg

Hb, µg

Hb, 10-11 mol

Γ*,a 10-11 mol cm-2

% electroactive Hb

2.5 3.5 5 7.5 10

2.9 4.0 5.7 8.6 11.4

4.3 6.0 8.5 12.8 17.0

4.2 5.0 5.6 6.0 7.0

27.4 23.6 18.4 13.0 11.6

a

From integration of CV reduction peaks in pH 5.5 buffers.

Figure 3. Square wave forward and reverse current voltammograms for Mb-clay films in pH 7.0 buffers at different frequencies. Points represent the experimental SWVs from which background has been subtracted. The solid line was the best fit by nonlinear regression onto the 5-E°′ dispersion model. SWV conditions: pulse height 75 mV, step height 4 mV, and frequencies (Hz) (a) 100, (b) 125, (c) 150, and (d) 175.

steady state, but the former was more convenient and quantitative and thus was used for preparing proteinclay films for the following studies. Stability of the protein-clay films was investigated by CV with two different methods. In the solution studies, a PG electrode coated with films was stored in buffers, and CVs were run periodically. Alternatively, the films were stored in air for most of the time and CVs were run periodically after returning the dry films into buffer solutions. Mb- and Hb-clay films demonstrated excellent stability with both methods. The CV peak potentials kept in the same positions and the peak currents showed little decrease for at least 2 months. Compared to these two protein-clay films, HRP-clay films seemed to be less stable. For example, the reduction peak current of HRPclay films decreased about 18% of its initial steady state after 1 month of storing in solution. Square Wave Voltammetry (SWV). SWV has better S/N and resolution than CV29 and thus was used to estimate average apparent heterogeneous electron-transfer rate constant (ks) and formal potential (E°′) for proteinclay films. The procedure employed nonlinear regression analysis for SWV forward and reverse curves, with a model combining a SWV model for monomolecular adsorbates30 with an E°′ dispersion model, as described in detail previously.31,32 Preliminary studies showed that the E°′

dispersion model with p ) 5 gave acceptable goodness of fit and consistency of parameters, as in other protein film systems to which this model has been applied.7,31 Thus, the p ) 5 model was used in the present study. The analysis of SWV data for the three protein-clay films showed good fits of the model to the data over a range of pulse amplitudes and frequencies (Figure 3). The average ks and E°′ obtained at pH 7.0 by this method for these three films are listed in Table 1. Although the ks values for various protein-clay films were different, they were of the same order or magnitude relatively large. E°′ values for the three protein-clay films were similar under the same conditions, consistent with the fact that all the proteins are heme proteins with the same electroactive prosthetic group. The E°′ value of heme FeIII/FeII couple for one protein in clay films was different than that for the same protein in surfactant or polymer films.4-9 This confirms a specific influence of the film environment on E°′ for heme proteins, as reported previously.5,7 Film components may change potentials via interactions with the protein or by their influence on the electrode double layer. Results of regression analysis gave surface concentration Γj* for each Ej°′, where j was the jth class of electroactive center for the 5-E°′ dispersion model. Γj* was plotted as a distribution diagram with each of five different Ej°′ values, where the relative amounts of redox center of electroactive protein were against different j. An example for Mb-clay films is shown in Figure 4. Roughly Gaussian distributions were returned from the fitting for all these three protein-clay films, as also found for proteins in other films.7,8,24 Influence of pH on Voltammetry. An increase in buffer pH caused a negative shift in potentials for both reduction and oxidation CV peaks for the protein-clay

(29) Osteryoung, J. G.; O’Dea, J. J. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1986; Vol. 14, pp 209-325. (30) O’Dea, J. J.; Osteryoung, J. G. Anal. Chem. 1993, 65, 3090.

(31) Nassar, A.-E. F.; Zhang, Z.; Hu, N.; Rusling, J. F.; Kumosinski, T. F. J. Phys. Chem. 1997, 101, 2224. (32) Zhang, Z.; Rusling, J. F. Biophys. Chem. 1997, 63, 133.

Figure 2. Cyclic voltammograms at 0.2 V s-1 for clay films immersed in pH 5.5 buffers containing 1.7 x 10-5 M Hb for (a) 0 min, (b) 5 min, (c) 2 h, (d) 25 h, (e) 96 h, and (f) 216 h.

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Figure 4. Distribution of relative fraction of electroactive Mb in Mb-clay films expressed as surface concentrations vs j for different Ej°′ values resulting from analysis of SWV data with the 5-E°′ dispersion model. SWV pulse height 75 mV; frequency 175 Hz; step height 4 mV. Figure 6. UV-vis absorption spectra of (a) dry Hb film, (b) dry Hb-clay film, and Hb-clay films in different pH buffers: (c) pH 5.5; (d) pH 7.0; (e) pH 10.0; (f) pH 3.5. The absorbance coordinate only reflects relative absorbance.

Figure 5. Influence of pH on the formal potential for (+) Mbclay film, (O) Hb-clay film, and (b) HRP-clay film from CV at 0.2 V s-1.

films. CV data were used to investigate the pH dependence of the formal potentials (E°′) of the heme FeIII/FeII redox couple for the films, estimated as the average of reduction and oxidation peak potentials. E°′ gave a linear relationship with pH with a slope of -48.7 mV pH-1 for Mb-clay films (pH 5.0-10.0), -48.7 mV pH-1 for Hb-clay films (pH 5.0-10.0), and -57.2 mV pH-1 for HRP-clay films (pH 6.0-11.0) (Figure 5). These slope values are reasonably close to the theoretical value of -57.6 mV pH-1 at 18 °C for a reversible one-proton coupled, reversible oneelectron transfer,33,34 which could be represented as

HemeFeIII + H+ + e- h HemeFeII An inflection point appeared in the E°′-pH plot at pH 5.0 for Mb- and Hb-clay films (Figure 5). At pH < 5.0, the variation of E°′ values with pH showed a much smaller slope. The position of the break in the E°′-pH plot suggests that protonatable sites associated with the electrode reaction have an apparent pKa′ values of 5.0. For HRPclay films, the inflection point appeared at pH 6.0, indicating an apparent pKa′ of 6.0. The different positions of the break probably reflect amino acid protonation of sites near the heme iron with different pKa’s in these proteins.31,35,36 UV-Vis Absorption Spectroscopy. Position of the Soret absorption band of prosthetic heme group provides information about possible denaturation of heme proteins.37,38 For example, the dry Hb-clay films cast on (33) Meites, L. Polarographic Techniques, 2nd ed.; Wiley: New York, 1965; pp 282-284. (34) Bond, A. M. Modern Polarographic Methods in Analytical Chemistry; Marcel Dekker: New York, 1980; pp 29-30. (35) Yang, A.-S.; Honig, B. J. Mol. Biol. 1994, 237, 602. (36) Bashford, D.; Case, D. A.; Dalvit, C.; Tennnant, L.; Wright, P. E. Biochemistry 1993, 32, 8045. (37) Theorell, H.; Ehrenberg, A. Acta Chem. Scand. 1951, 5, 823. (38) George, P.; Hanania, G. Biochem. J. 1952, 52, 517.

transparent glass slides showed the same peak positions at 412 nm as the corresponding dry protein films (Figure 6a,b), suggesting that Hb in clay films has a secondary structure similar to the native state of Hb. The dependence of Soret band position on pH of external solution was also tested. In pH 5.5 and 7.0 buffers, the Soret band appeared at 410 nm (Figure 6c,d), indicating that in the medium pH range, Hb essentially retained its native conformation in clay films. A small blue shift was observed when the pH of solution increased. For instance, the Soret band shifted to 408 nm at pH 10.0 (Figure 6e). On the other hand, as pH shifted in the acidic direction, the Soret band became smaller and broader. At pH 3.5, the Soret band was hardly observed (Figure 6f), suggesting denaturation of Hb at this pH. pH-dependent UV-vis spectroscopic properties of some heme proteins in solution were studied previously by Antonini39 and Mauk.40,41 Hb-clay films on glass slides were not as stable as on PG electrodes, mainly because of poorer adhesion between the films and the glass surface. Mb-clay films demonstrated similar spectroscopic behavior to Hb-clay films. Dry HRP-clay films showed a Soret band at the same position as for the dry HRP films. However, HRP-clay films on glass slides were very unstable in solution. Reflectance Absorption Infrared (RAIR) Spectroscopy. The shapes of amide I and amide II infrared absorbance bands of proteins provide detailed information on the secondary structure of polypeptide chain.42,43 The amide I band at 1700-1600 cm-1 is caused by the CdO stretching vibrations of the peptide linkage. The amide II band at 1600-1500 cm-1 results from a combination of N-H in-plane bending and C-N stretching of the peptide groups. Second derivative IR spectra can provide more detailed information on protein secondary structure than the original IR spectra. Negative second derivative bands are resolved from overlapped amide I and II bands in the original spectra, and frequencies have been assigned for R-helix, turns, disordered features and other secondary structural features of protein.44 Second derivative RAIR (39) Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in Their Reaction with Ligands; North-Holland: Amsterdam, 1971. (40) Bogumil, R.; Maurus, R.; Hildebrand, D. P.; Brayer, G. D.; Mauk, A. G. Biochemistry 1995, 34, 10483. (41) Hildebrend, D. P.; Tang, H.; Luo, Y.; Hunter, C. L.; Smith, M.; Brayer, G. D.; Mauk, A. G. J. Am. Chem. Soc. 1996, 118, 12909. (42) Kumosinski, T. F.; Unruh, J. J. In Molecular Modeling; Kumosinski, T. F.; Liebman, M. N., Eds.; ACS Symp. Ser. 576, American Chemical Society: Washington, DC, 1994; p 71. (43) Schlereth, D. D.; Mantele, W. Biochemistry 1992, 31, 7494.

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Figure 8. X-ray diffraction patterns for (a) Mb-clay film, (b) Hb-clay film, and (c) HRP-clay film. Table 3. X-ray Diffraction Results for Protein-Clay Films film clay (from organic solvent)56 clay (from aqueous dispersion) Mb-clay Hb-clay HRP-clay

2θ, basal plane deg spacing, Å 8.66 7.12 2.42 1.88 4.32

10.2 12.4 36.5 47.0 20.5

dimension of protein, Å

25 x 35 x 4557 50 x 55 x 6558 35 (diameter)59

Figure 7. Second derivative reflectance absorption infrared (RAIR) spectra of (a) Mb and Mb-clay films, (b) Hb and Hbclay films, and (c) HRP and HRP-clay films. The absorbance coordinate only reflects relative value.

spectra were used here to detect possible conformational changes of the three proteins in clay films (Figure 7). The second derivative RAIR spectra of protein-clay films had shapes and negative peak positions of amide I and II bands very similar to that of protein films alone. X-ray Diffraction (XRD). The lowest reflection angle 2θ of XRD for clay and protein-clay films was used to obtain the interlayer basal plane spacing of the films through Bragg’s law.45,46 A small-angle peak was observed at 7.12° for clay films in the 2-10° region of 2θ, showing a basal plane spacing of 12.4 Å. For Mb-clay and Hbclay films, 2θ peaks at 2.42° and 1.88° showed larger basal plane spacing of 36.5 and 47.0 Å (Figure 8a,b), respectively, indicating that Mb and Hb are intercalated into the clay layers and enlarge the interlayer spacing of the clay films. In addition to the main 2θ peaks, Mb-clay and Hb-clay films also showed other much smaller XRD peaks giving about one-half and one-third the Bragg’s spacing of the main peaks, which suggest the second- and third-order diffractions, respectively. The appearance of harmonic peaks suggests a high degree of order for Mb-clay and Hb-clay films. However, for HRP-clay films, the XRD (44) Rusling, J. F.; Kumosinski, T. F. Nonlinear Computer Modeling of Chemical and Biochemical Data; Academic Press: New York, 1996; pp 117-134. (45) Kamlet, M. J.; Abboud, J. L.-M.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1983, 49, 2877. (46) Shi, C.; Rusling, J. F.; Wang, Z.; Willis, W. S.; Winiecke, A. M.; Suib, S. L. Langmuir 1989, 5, 650.

Figure 9. Cyclic voltammograms at 0.2 V s-1 in 9 mL of pH 7.0 buffers: (a) clay film with no oxygen present, (b) clay film after 40 mL of air was injected into a sealed cell, (c) HRP-clay film with no oxygen present, (d) HRP-clay film after 40 mL of air was injected, and (e) HRP-clay film after 80 mL of air was injected.

pattern was very different. The 2θ peak at 4.32° giving basal plane spacing of 20.5 Å was so obscure that the ordered layer structure of the films was hardly observed (Figure 8c). XRD data of clay and protein-clay films were also listed in Table 3. Catalytic Reactivity. Electrochemical catalytic reduction of oxygen by the three protein-clay films was observed by CV. Taking HRP-clay films as an example, when a volume of air was passed through a pH 7.0 buffer, a significant increase in reduction peak at about -0.3 V was observed for the HRP-clay films (Figure 9d), compared to the reduction peak of HRPFeIII of the films without oxygen present (Figure 9c). This increase in the reduction peak was accompanied by the disappearance of the oxidation peak of HRPFeII, suggesting that HRPFeII had reacted with oxygen. An increase in the amount of oxygen

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Figure 10. Cyclic voltammograms at 0.2 V s-1 in pH 5.5 buffers for (a) clay film in buffers containing no TCA, (b) clay film in buffers containing 50 mM TCA, (c) Hb-clay film in buffers containing no TCA, and (d) Hb-clay film in buffers containing 50 mM TCA.

in solution increased the reduction peak current (Figure 9e). For clay films with no HRP, the peak for direct reduction of oxygen was observed at about -0.9 V (Figure 9b), far more negative than the catalytic peak potential. The catalytic efficiency expressed as the ratio of reduction peak current of HRPFeIII in the presence (Ic) and absence of oxygen (Id), Ic/Id, decreased with increase of scan rate. All these results are characteristic of electrochemical catalytic reduction47,48 of oxygen by HRP in clay films. Mb-clay and Hb-clay films showed similar catalytic properties toward oxygen. Electrocatalytic reduction of trichloroacetic acid (TCA) by the protein-clay films was also tested by CV. For example, when TCA was added to a pH 5.5 buffer, the HbFeIII reduction peak of Hb-clay film electrodes at about -0.35 V increased in height (Figure 10d), accompanied by decrease of HbFeII oxidation peak. These results are consistent with the reduction of TCA by HbFeII in a catalytic cycle, presumably resulting in the reductive dechlorination of the acid.49 Compared to direct reduction of TCA on clay films without protein (Figure 10b), Hbclay films lowered the overpotential for reduction of TCA by about 0.7 V. The catalytic efficiency decreased with increase of scan rate, and the reduction peak current for HbFeIII increased with concentration of TCA in solution. These are characteristic of electrochemical catalysis.47,48 Electrochemical catalytic reduction of TCA was also observed with Mb-clay and HRP-clay films. Proteins here are made to act like enzymes in this process, which presumably features stepwise reductive dechlorination to ultimately give acetic acid as documented for Mb in surfactant films.49 Farmer and co-workers studied electrocatalytic reduction of NO2- with Mb-DDAB films on PG electrodes.50 Electrochemical catalytic reduction of nitrite was also tested with the protein-clay films. For example, with Mbclay films, the addition of NaNO2 in a pH 5.5 buffer resulted in a new reduction peak at about -0.8 V (Figure 11d), and the further addition of NaNO2 caused an increase of the peak (Figure 11e). Direct reduction of NO2- at clay film electrodes was found at the potential more negative than -1.2 V (Figure 11b). Thus, Mb-clay films decreased reduction overpotential for NO2- by at least 0.4 V. The reduction products at -0.8 V is most likely N2O, which was detected previously by mass spectroscopy with MbDDAB films on electrolysis at -0.895 V in pH 7.0 buffers.50 (47) Andrieux, C. P.; Blocman, C.; Dumas-Bouchiant, J. M.; Saveant, J. M. J. Am. Chem. Soc. 1979, 101, 3431. (48) Andrieux, C. P.; Blocman, C.; Dumas-Bouchiant, J. M.; M’Halla, F.; Saveant, J. M. J. Electroanal. Chem. 1980, 113, 19. (49) Nassar, A.-E. F.; Bobbitt, J. M.; Stuart J. O.; Rusling, J. F. J. Am. Chem. Soc. 1995, 117, 10986. (50) Lin, R.; Bayachou, M.; Greaves, J.; Farmer, P. J. J. Am. Chem. Soc. 1997, 119, 12689.

Zhou et al.

Figure 11. Cyclic voltammograms at 0.1 V s-1 in pH 5.5 buffers for (a) clay film in buffers containing no NaNO2, (b) clay film in buffers containing 5 mM NaNO2, (c) Mb-clay film in buffers containing no NaNO2, (d) Mb-clay film in buffers containing 5 mM NaNO2, and (e) Mb-clay film in buffers containing 9 mM NaNO2.

Figure 12. Cyclic voltammograms at 0.2 V s-1 in pH 7.0 buffers for (a) clay film in buffers containing no H2O2, (b) clay film in buffers containing 0.22 mM H2O2, (c) HRP-clay film in buffers containing no H2O2, (d) HRP-clay film in buffers containing 0.22 mM H2O2, and (e) HRP-clay film in buffers containing 0.44 mM H2O2.

For Mb-clay films, the catalytic CV reduction peak of NO2- had a linear relationship with NO2- concentration in the range of 0.05-0.82 mM with detection limit of 0.03 mM and correlation coefficient of 0.999. Hb-clay films demonstrated very similar catalytic CV behavior and calibration curve for determining nitrite. However, HRPclay films showed much less sensitivity toward catalysis of NO2- with a detection limit of 5 mM. Electrochemical catalytic reaction of hydrogen peroxide (H2O2) was also observed for protein-clay films, using HRP-clay films as an example. When H2O2 was added to a pH 7.0 buffer, an increase in the reduction peak at about -0.3 V was seen with the disappearance of the oxidation peak for HRPFeII (Figure 12d). The reduction peak current increased with the concentration of H2O2 in solution (Figure 12e). However, direct reduction of H2O2 was not observed at clay film electrodes in the potential range of between 0.1 and -1.4 V (Figure 12b). The linear relationship of the electrocatalytic reduction peak current and H2O2 concentration was observed between 0.02 and 0.44 mM. The calibration curve tended to level off when the concentration of H2O2 became larger. When H2O2 concentration was larger than 0.7 mM, the catalytic peak current even decreased, suggesting a progressive enzyme inactivation in the presence of high concentration of reactant.51 Mb-clay and Hb-clay films showed similar catalytic behavior and calibration curve toward hydrogen peroxide. It is well-known that HRP can react with H2O2 and form a powerful enzymatic oxidizing agent, compound I, which is a two-equivalent oxidized form higher than the resting state of HRPFeIII.52 However, no direct reduction (51) Adeiran, S. A.; Lambeir, A. M. Eur. J. Biochem. 1989, 186, 571.

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Figure 13. Amperometric responses at various modified electrodes at constant potential of 0 V in 10 mL of pH 7.0 buffers with injecting 10 µL of 0.088 M H2O2 every 40 s: (a) clay film; (b) Mb-clay film; (c) Hb-clay film; (d) HRP-clay film. Table 4. Catalytic Efficiency or Detection Limit of Different Substrates with Protein-Clay Films substrate

film

O2a

H2O2b

TCAc

detection limit, mM NaNO2d

Mb-clay Hb-clay HRP-clay

3.0 3.1 5.2

3.0 3.6 3.8

2.0 2.0 1.1

0.03 0.03 5

catalytic efficiency (Ic/Id)

0.2 V s-1, 30 mL of air passed in 9 mL of solution. b 0.2 V s-1, 0.22 mM H2O2. c 0.2 V s-1, 50 mM TCA. d 0.1 V s-1. a

peak for compound I was observed by CV for HRP-clay films in the presence of H2O2. This is consistent with the previous work,53 in which direct electron transfer between compound I and electrodes was only observed by amperometry in the presence of hydrogen peroxide. For HRP-clay films, at a constant potential of 0 V vs SCE, at which the reduction of HRPFeIII does not interfere, the current was monitored while aliquots of H2O2 were injected into the pH 7.0 buffer every 40 s. The step increase of current with addition of H2O2 (Figure 13d) represents evidence for direct electron transfer between compound I and electrodes for the HRP-clay film system. Mb- and Hb-clay films showed similar amperometric behavior with injection of H2O2, but with lower sensitivity (Figure 13b,c). Catalytic efficiencies for different substrates (O2, H2O2, and TCA) with three protein-clay films are listed in Table 4 for comparison. The catalytic reduction peak potential of NO2- at protein-clay film electrodes is at about -0.8 V, very different from that of protein-clay films in the absence of NO2- (Figure 11); the ratio Ic/Id cannot be defined in this case. Thus, the detection limit values are listed in Table 4 for comparison for the NO2- system. In general, Mb-clay and Hb-clay films demonstrate similar catalytic behavior with all four substrates. However, HRP-clay films show different catalytic properties than the others. For oxygen and hydrogen peroxide, HRPclay films seem to be more active than Mb- and Hb-clay films, while for trichloroacetic acid and nitrite, HRPclay films are much less active than the other films in their catalytic reductions. Layer-by-Layer Protein-Clay Films. Since only the inner layers of protein-clay films were electroactive, ultrathin protein-clay films were fabricated on the surface of PG electrodes using a layer-by-layer growth technique.54 For example, films of the architecture (PG/PDDA/clay/ (52) Dunford, H. B., In Peroxidases in Chemistry and Biology; Everse, J., Everse, K. E., Grisham, M. B., Eds.; CRC Press: Boca Raton, FL, 1991; pp 1-24. (53) Ruzgas, T.; Csoregi, E.; Emneus, J.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1996, 230, 123. (54) Decher, G. Science 1997, 227, 1232.

Mb/clay) were made by immersing a fresh PG electrode into a poly(diallyldimethylammonium chloride) (PDDA) solution for 20 min so that a precursor layer of PDDA was formed on the PG surface, and a positively charged surface was obtained. This electrode was then dipped in clay dispersion for 20 min, and a negatively charged clay layer was assembled on the surface by Coulombic attraction. At pH 4.5, Mb has positive surface charges with its isoelectric point at pH 6.8 (Table 1). After being washed in pure water, the electrode was transferred into a Mb solution at pH 4.5 for 20 min. The adsorption of the Mb layer made the electrode surface become positive again. Another clay layer was then adsorbed on top of the Mb layer with the same immersing method, and the formed ultrathin layer-by-layer film was designated as (PG/ PDDA/clay/Mb/clay). In pH 5.5 buffers, the (PG/PDDA/clay/Mb/clay) film showed a pair of well-defined, reversible CV peaks at the same potential as for cast Mb-clay films at about -0.28 V, characteristic of the Mb heme FeIII/FeII redox couple. Surface concentration of electroactive Mb in the film was estimated at (6.9 ( 0.9) x 10-11 mol cm-2 by integrating the reduction peak. Discussion Electrochemical Properties. Nearly reversible cyclic voltammograms for all three heme proteins were obtained (Figure 1), indicating direct electron transfer between the heme proteins and PG electrodes in clay films. Electron communication was much faster for protein-clay films than that for the proteins in solution on bare PG. Thus, clay films have a large effect on 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 clay films inhibit adsorption of macromolecular impurities from protein solutions on the electrodes, which could otherwise block electron exchange for the proteins.55 Clay is a cationic exchanger featuring negative charges. At pH 5.5, considering their isoelectric points of Mb, Hb, and HRP (Table 1), the three proteins are all positively charged. Thus, the driving force for the proteins to enter clay films (Figure 2) would be mainly Coulombic. This interaction would be mainly responsible for the excellent stability of protein-clay films and the retention of the proteins in the films. Data in Table 2 suggest that only the inner layers of the proteins closest to the electrode surface are electrochemically addressable. This is consistent with the CV experiments of layer-by-layer (PG/PDDA/clay/Mb/clay) films. The adsorbed monolayer of Mb in the layer-by-layer films could be roughly considered as an equivalent of the first closest layer of Mb to the electrode surface for Mb-clay films. The surface concentration of 6.9 x 10-11 mol cm-2 for electroactive Mb in the first layer would then account for about 40% of the total electroactive Mb (18.5 x 10-11 mol cm-2) for Mb-clay films. This indicates that although there are thousands of layers for the cast protein-clay films, only a few first layers of protein closest to the electrode would make the greatest contribution to the direct electron transfer for the protein. The large uptake times of protein from solution (Figure 2) also suggest that the proteins are immobile during CV scans. Structural Features. Results of XRD of clay films cast from aqueous dispersions suggest ordered layer structures. The peak at 7.12° gave a basal plane spacing of 12.4 Å, (55) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386.

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larger than the value of 10.2 Å for clay films cast from organic solvent.56 This is understandable if the possibility of water intercalation between the clay layers in the former situation is considered. The value of 10.2 Å was thus used here as the thickness of dry clay platelets for a control (Table 3). XRD results of Mb-clay and Hb-clay films support an ordered lamellar structure (Figure 8a,b). The basal plane spacing increased greatly in Mb-clay films (36.5 Å) compared to plain clay films, suggesting Mb is intercalated between the clay layers and enlarges the interlayer spacing. The spacing corrected for the thickness of the dry clay platelets (10.2 Å) is 26.3 Å. The size of Mb is approximately 25 x 35 x 45 Å.57 Of the three dimensions of Mb, only the smallest one (25 Å) is smaller than the corrected interlayer spacing, suggesting that Mb intercalated in the clay films may have a specific orientation. Hb has larger size of 50 x 55 x 65 Å58 than Mb and reasonably shows a larger basal plane spacing in Hbclay films (47.0 Å) than in Mb-clay films. Hb-clay films give a corrected interlayer spacing of 36.8 Å, smaller than any of the three dimensions of Hb. Thus, the interlayer spacing is not large enough to accommodate the full Hb molecules. While extensive Hb denaturation can be considered, UV-vis and RAIR results are inconsistent with this view. Therefore, it can be reasonably assumed that Hb is not fully intercalated between the clay galleries. Hb may be partly intercalated between clay platelets and/ or on the outer surface of the clay layers. XRD of HRP-clay films suggest a considerable degree of disorder (Figure 8c). The layered structure of the clay films seems to be disrupted by the incorporation of HRP. Even if the obscure and small 2θ peak at 4.32° were considered as an indication of a layered structure, the corresponding basal spacing of 20.5 Å would have a corrected interlayer spacing of 10.3 Å, not only much smaller than those of Mb-clay and Hb-clay films but also much smaller than the average diameter of 35 Å for globular HRP.59 The reasons for the disruption by HRP are not entirely clear, but incorporation of HRP does not take place between clay layers. The second derivative RAIR spectra provided information of secondary structure of the three proteins in clay films (Figure 7). In general, second-derivative IR spectra of protein and protein-clay films are very similar. Mb films have a strong characteristic band at 1651 cm-1, consistent with 76% R-helix in the native structure. The Mb-clay films show a very small band at 1639 cm-1, which can be attributed to