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Assembly of Electroactive Layer-by-Layer Films of Myoglobin and Ionomer Poly(ester Sulfonic Acid)
Journal of Colloid and Interface Science 254, 257–265 (2002) doi:10.1006/jcis.2002.8574
Assembly of Electroactive Layer-by-Layer Films of Myoglobin and Ionomer Poly(ester Sulfonic Acid) Zhen Li and Naifei Hu1 Department of Chemistry, Beijing Normal University, Beijing 100875, China Received November 26, 2001; accepted July 9, 2002
Layer-by-layer films were assembled on solid substrates by alternate adsorption of negatively charged ionomer poly(ester sulfonic acid) or Eastman AQ55 from its aqueous dispersion and positively charged myoglobin (Mb) from its solution at pH 4.5. The film assembly process was monitored by cyclic voltammetry (CV), UV–vis spectroscopy, and quartz crystal microbalance (QCM). {AQ/Mb}n films grown on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible CV peaks at about −0.20 V vs Ag/AgCl in pH 5.5 buffers, characteristic of the Mb heme Fe(III)/Fe(II) redox couple. Although the amount of Mb adsorbed in each bilayer was essentially the same, the fraction of electroactive Mb decreased dramatically with an increase of bilayer number (n). Soret absorption bands of {AQ/Mb}n films on glass slides suggest that Mb in the films retains its native state in the medium pH range. Trichloroacetic acid, oxygen, and hydrogen peroxide were electrochemically catalyzed by {AQ/Mb}6 films with significant lowering of reduction overpotential. C 2002 Elsevier Science (USA) Key Words: myoglobin; poly(ester sulfonic acid); Eastman AQ; layer-by-layer assembly films; direct electrochemistry; electrocatalysis.
INTRODUCTION
Study of direct electron transfer between enzyme and electrode can provide a model for the study of enzyme-catalyzed reaction in biological systems and may establish a foundation for constructing new kinds of biosensors or enzymatic bioreactors (1). Achieving reversible, direct electron transfer between an enzyme and electrode without using any chemical mediators thus has great significance. Myoglobin (Mb) is a heme protein, which can store and transport oxygen in muscle cells in mammalians. Mb has a single polypeptide chain with an iron heme inside as a prosthetic group (2). Although Mb does not function as an electron carrier biologically, it is an ideal model for the study of electron transfer of heme enzymes such as cytochrome P450 because of its commercial availability, documented structure, and similar reactivity to that of cytochrome P450. However, direct electron exchange between Mb in solution and solid electrodes is usually very slow. 1 Author to whom correspondence should be addressed. E-mail: hunaifei@ bnu.edu.cn.
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Many researchers used promoters in Mb solution to enhance the electron transfer (3). A relative new avenue to realize direct electrochemistry of protein is to incorporate protein into films which are cast on the surface of solid electrodes (4). The materials of cast films containing Mb include water-insoluble surfactants (5, 6), amphiphilic polymer (7), and polyelectrolyte–surfactant (8–10) or clay–surfactant (11) complex composites. These cast films were easy to prepare and quite stable and provided a favorable microenvironment for Mb and greatly facilitated the rate of electron transport between Mb and electrodes. However, the final molecular architecture and thickness of the cast Mb films cannot be controlled precisely, and only a very small fraction of Mb in the films was electroactive. Recently, a novel technique of layer-by-layer assembly was developed and employed in fabricating ultrathin protein films (12). By alternate adsorption of polyions and oppositely charged proteins in solution at appropriate pH, multilayered protein assembly was achieved with precisely repeatable layer thickness. In this way, the design of ordered protein films at the molecular level and the precise control of film thickness on the nanometer scale can be realized. Layer-by-layer protein films can also provide the possibility of studying direct electrochemistry of redox proteins in their multilayer architecture (13). Take Mb as an example; Lvov et al. constructed layer-by-layer films of Mb with appropriately charged polyions on gold electrodes modified with mercaptopropanesulfonic acid (14). Cyclic voltammetry (CV) of the films in blank buffers showed a pair of direct, reversible peaks for the Mb Fe(III)/Fe(II) redox couple. Recently, we reported ultrathin films of Mb assembled layer by layer with poly(styrene sulfonate) (PSS) on pyrolytic graphite (PG) electrodes (15). Mb in {PSS/Mb}n films demonstrated direct electron transfer with underlying electrodes, and electroactivity was extended to seven {PSS/Mb} bilayers on a rough PG surface. An ionomer is an ionic polymer that contains only a fraction of ionizable groups with each monomer unit. In recent years, the ionomer poly(ester sulfonic acid), with the trade name Eastman AQ, has aroused interest among chemists as film-forming materials. AQ55 ionomer has 18 mol% aromatic sulfonate groups (see Scheme I) (16), which is ionizable when dispersed in water. This thermoplastic, amorphous ionomer gives translucent, lowviscosity dispersion in water without adding organic solvent. AQ films cast onto electrodes could bind hydrophobic cations 0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
All rights reserved.
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SCHEME I.
Chemical structure of Eastman AQ55.
preferentially and exclude negatively charged species (17, 18). In our previous work (19), Mb was mixed directly with aqueous dispersions of AQ ionomers and deposited onto solid surfaces to form films without denaturation. These cast Mb–AQ films on PG electrodes showed stable CV signals for the Mb Fe(III)/Fe(II) redox couple in buffer solutions, but only about 10% of the total Mb deposited was electroactive. In this work, layer-by-layer {AQ/Mb}n films were assembled onto solid substrates by alternate adsorption of the negatively charged AQ55 ionomer from its dispersion in water and the positively charged Mb from its aqueous solutions at pH 4.5. To our knowledge, this is the first time ionomers are used instead of regular polyions to assemble layer-by-layer protein films. The assembled {AQ/Mb}n films were characterized by voltammetry, UV–vis spectroscopy, and a quartz crystal microbalance (QCM). {AQ/Mb}n films on PG electrodes showed direct electrochemistry for Mb and were used to catalyze reduction of trichloroacetic acid, oxygen, and hydrogen peroxide.
ate water washing and nitrogen stream drying. This cycle was repeated periodically to make {AQ/Mb}n multilayer films on PG electrodes. Glass slides (1 × 4 cm, 1-mm thick) were first washed in a washing solution (60% ethanol + 39% water + 1% KOH) for 30 min at 50◦ C to induce negative charges on the surface and then carefully rinsed with water. A monolayer of positively charged precursor PEI was adsorbed on the glass surface by immersing the slides into PEI solutions (3 mg/mL, containing 0.5 M NaCl) for 20 min. The layer-by-layer films of {AQ/Mb}n were then assembled on the PEI-modified glass slides in the same way as on PG electrodes described above. Gold-coated resonator electrodes (geometric area 0.196 cm2 ) for a quartz crystal microbalance (QCM) were soaked in freshly prepared “piranha” solution (3 : 7 volume ratio of 30% H2 O2 and 98% H2 SO4 ) for 15 min at ca. 95◦ C and then washed in pure ethanol and water. (Caution: piranha solution should be handled with extreme care, and only small volumes should be prepared at any time.) The cleaned gold electrodes were immersed in 4 mM MPS ethanol solutions for 24 h to form MPS monolayer on gold electrodes and introduce negative charges on the surface. After being rinsed with pure ethanol and water and dried under a stream of highly purified N2 , the gold electrode resonators were then immersed into PEI solutions for 20 min to make the surface positively charged. The {AQ/Mb}n films were then assembled on the pretreated gold electrodes with the same method as on PG.
EXPERIMENTAL
Instruments and Procedures Chemicals Horse heart myoglobin (Mb, MW = 17,800) was from Sigma and used as received. Poly(ester sulfonic acid) or Eastman AQ55 (AQ, MW = 14,000, Tg = 55◦ C) was from Eastman Chemical Co. Poly(ethylenimine) (PEI, 90%, MW = 60,000) and 3mercapto-1-propanesulfonate (MPS, 90%) were from Aldrich. Trichloroacetic acid (TCA, 99%) was from Jinlong Chemical Reagent Co. Hydrogen peroxide (H2 O2 , 30%) was from Beijing Chemical Engineering Plant. Buffers were 0.05 M citric acid, 0.1 M acetate, 0.05 M sodium dihydrogen phosphate, or 0.05 M boric acid, all containing 0.1 M KBr. pH values were adjusted to desired values with HCl or KOH solutions. All other chemicals were reagent grade. Water used was twice distilled. Film Assembly The multilayer films of AQ and Mb were assembled onto solid substrates such as carbon electrodes, glass slides, and gold electrode resonators by the following methods. Prior to use, basal plane pyrolytic graphite (PG, Advanced Ceramics, geometric area 0.16 cm2 ) disk electrodes were polished on metallographic sandpaper while being flushed with water. The electrodes were then ultrasonicated in pure water for 30 s and dried in air. The PG electrodes were alternately placed for 20 min in aqueous dispersions of AQ (1%) and Mb solutions (3 mg/mL, in pH 4.5 buffers) with intermedi-
A CHI 420 electrochemical analyzer (CH Instruments) was used for both voltammetric and QCM studies. In electrochemical measurements, a regular three-electrode cell was used with an Ag/AgCl electrode as the reference electrode, a platinum wire as the counter electrode, and a PG disk electrode coated with films as the working electrode. Prior to electrochemical measurements, solutions were purged with purified nitrogen for at least 15 min, and a nitrogen atmosphere was maintained over solutions for exclusion of oxygen during experiments. In aerobic experiments, measured volumes of air were injected via a syringe into solutions in a sealed cell that had been previously degassed with purified nitrogen. For QCM studies, ATcut quartz resonators with fundamental resonant frequency of 8 MHz and coated by gold thin films on both sides were used. UV–vis absorption spectroscopy was measured for the films assembled on glass slides with a Cintra-10e UV–vis spectrometer (GBC). All experiments were performed at ambient temperature of 25 ± 2◦ C. RESULTS AND DISCUSSION
Assembly of Layer-by-Layer Films Monitored by CV and UV–vis Myoglobin, with its isoelectric point at pH 6.8 (20, 21), has positive surface charges at pH 4.5, while AQ has negative charges
ASSEMBLY OF ELECTROACTIVE LAYER-BY-LAYER FILMS
FIG. 1. Background-subtracted cyclic voltammograms for layer-by-layer {AQ/Mb}n films at 0.2 V s−1 in pH 5.5 buffers with a different number of bilayers (n).
on its backbone. Thus, layer-by-layer films of AQ and Mb could be assembled by Coulombic attraction between them on the surface of various substrates such as PG, pretreated glass slides, and MPS–PEI-modified Au by alternate adsorption from their dispersions or solutions (see Experimental section). The films were designated as {AQ/Mb}n , where n is the number of bilayers or adsorption cycles for the films. In this part, cyclic voltammetry (CV) and UV–vis spectroscopy were used to monitor the assembly of {AQ/Mb}n multilayer films. It is known that ordinary basal plane PG provides a surface with a partly hydrophobic character (22). AQ is a kind of amphiphilic ionomers containing a hydrophobic backbone. Thus, hydrophobic interaction between AQ and basal PG would be mainly responsible for spontaneous adsorption of AQ on the PG surface. Positively charged Mb from pH 4.5 buffer solutions was then adsorbed on the surface of a negatively charged AQ layer. CV was used here to monitor the growth of the {AQ/Mb}n films. After each adsorption cycle, the {AQ/Mb}n film electrode was placed in a pH 5.5 buffer containing no Mb, followed by CV scans (Fig. 1). A pair of well-defined, quite reversible CV peaks was observed at about −0.20 V vs Ag/AgCl, characteristic of the Mb heme Fe(III)/Fe(II) redox couple. Both reduction and oxidation peaks grew with the number of adsorbed AQ/Mb bilayers (n) at first. When n exceeded 6, however, no further increase in the reduction peak current was observed and the peak heights remained essentially the same, indicating that Mb in the bilayer of n > 6 was not yet electroactive. AQ monolayer adsorbed on PG electrodes had no CV signal at all in this potential window. As an ionomer, AQ has a much lower charge density compared with those of regular polyions such as PSS. The polyanion PSS has a linear aromatic backbone similar to AQ, but it has one negatively charged sulfonate group with every repeated monomer unit. For AQ, however, only a small fraction of its monomer units has a sulfonate group (see chemical structure of AQ55). This difference made a notable impact on the layer-bylayer assembly process. In the assembly of {PSS/Mb}n films, for example, pH 5.5 could be chosen for the Mb adsorbate
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solution, and {PSS/Mb}n films assembled on PG electrodes in this way demonstrated quite good CV responses (15). However, in the assembly of {AQ/Mb}n films, when pH 5.5 was selected for Mb adsorbate solutions, a much smaller CV peak response was obtained, indicating that much smaller amounts of Mb were adsorbed on the AQ layer surface compared with PSS at this pH. To enhance the adsorption of Mb on the AQ surface, the pH of Mb adsorbate solution in this work was adjusted to 4.5, further from the isoelectric point of Mb at pH 6.8 (20, 21) so that Mb could bear more positive surface charges and more Mb could be adsorbed onto the AQ surface layer. In the previous work of assembling {PSS/Mb}n layer-by-layer films (15), inorganic salt NaCl was usually added to the polyanion adsorbate solutions to enhance the adsorption of PSS. In the present work, however, since AQ is insoluble in water, only an aqueous dispersion of AQ could be obtained by a relatively long period of time of ultrasonication. If salt were added to this dispersion, AQ would precipitate immediately. Thus, in the assembly process of {AQ/Mb}n films, the AQ adsorbate dispersion was free of salt. Integration of the CV reduction peak for MbFe(III) can give the amount of reduction charge (Q) passed through the electrode, and according to the Q– ∗ relationship (23), the surface concentration of electroactive Mb ( ∗ , mol cm−2 ) can be deduced. The ∗ value increased nonlinearly with n up to 6 (Fig. 2a) and tended to level off afterward. The Mb molecule contains a heme prosthetic group, which has a sensitive absorption band at about 410 nm called the Soret absorption band. Thus, UV–vis spectroscopy was used here to monitor the growth of the AQ/Mb bilayer on PEI-modified glass slides. UV–vis spectra showed the Soret absorption band at 413 nm for {AQ/Mb}n films (Fig. 3). The Soret absorbance peak increased linearly with n (correlation coefficient 0.9979), indicating that the {AQ/Mb}n multibilayer is built up in a regular or linear mode, and the amount of Mb adsorbed in each bilayer is essentially the same. Assuming the amount of adsorbed Mb in the first bilayer closest to the PG surface is 100% electroactive (14),
FIG. 2. Influence of the number of bilayers (n) for {AQ/Mb}n films on the (a) surface concentration of electroactive Mb ( ∗ ) and (b) fraction of electroactive Mb. Data were from CVs at 0.2 V s−1 in pH 5.5 buffers.
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FIG. 3. UV–vis spectra on glass slides for an AQ monolayer and for layerby-layer {AQ/Mb}n films with a different number of bilayers.
according to the CVs, the fraction of electroactive Mb among all adsorbed Mb in each bilayer would decrease dramatically with n (Fig. 2b). This indicates that the distance between Mb and electrodes is crucial for efficient electron exchange. The further the distance, the lower the percentage of electroactive Mb in each bilayer. QCM Studies Quartz crystal microbalance (QCM) is a sensitive technique that can detect tiny mass changes on the QCM electrodes and was used here to monitor the layer-by-layer assembly of {AQ/Mb}n films. Based on the Sauerbrey equation (24), F =
−2 f 02 M , √ A µρ
A monolayer of MPS was first chemisorbed on the gold thin films coated on both sides of the quartz resonator so that the electrode surfaces became negatively charged. A precursor monolayer of PEI was then adsorbed onto the MPS surface to convert the surface to positively charged (see Experimental section). The {AQ/Mb}n multilayer films were then built up on the QCM electrode by alternate adsorption of AQ and Mb as described above. After each adsorption step, the electrode was washed thoroughly in water, dried under a nitrogen stream, and then measured by QCM. The results showed a roughly linear decrease of frequency with adsorption step (Fig. 4). Each AQ adsorption layer caused a frequency decrease of about 39 Hz, and each adsorbed layer of Mb resulted in a frequency decrease of about 250 Hz. AQ adsorption produced a smaller frequency shift than Mb, which was also observed in other layer-by-layer {polyion/protein}n films (25). Mb has a larger molecular weight and a relatively more rigid globular shape, while AQ is a flexible linear ionomer with small molecular weight for each of its repeated units. Roughly constant F for each Mb layer suggests that the amount of absorbed Mb in each cycle is approximately the same, as observed in UV–vis spectroscopic experiments. It is the same for the AQ layer. The QCM data were also used to estimate the thickness of {AQ/Mb}n films. The thickness, d (cm), can be expressed by d=
M , 2ρ A
[3]
where ρ is the density of the film material (g cm−3 ) and A is the area of QCM electrodes (0.196 cm2 ). Since two gold thin films are coated on both sides of the QCM resonator, the total mass adsorbed on Au electrodes should be divided by 2. Combined with Eq. [2] the relationship between d and F would be d = −3.4 × 10−9 F/ρ .
[4]
Since the density of AQ55 is about 1.33 g cm−3 (16), the average
[1]
a micromass change (M) can be converted to frequency shift (F) of the quartz crystal resonator. The following relationship between M (g) and F (Hz) is obtained by taking into account the characteristics of the quartz resonator used in this work, F = −7.40 × 108 M,
[2]
where f 0 is the resonant frequency of the fundamental mode of the quartz crystal (8 MHz), µ is the shear modulus of quartz (2.947 × 1011 g cm−1 s−2 ), ρ is the density of the crystal (2.648 g cm−3 ) and A is the geometric area of the QCM electrode (0.196 cm−2 ). Thus, 1 Hz of frequency decrease corresponds to 1.35 ng of mass increase with relatively good accuracy and reliability.
FIG. 4. Shift of QCM frequency with alternate adsorption step of AQ and Mb on MPS–PEI-modified Au electrodes. () AQ adsorption steps; () Mb adsorption steps.
ASSEMBLY OF ELECTROACTIVE LAYER-BY-LAYER FILMS
FIG. 5. Cyclic voltammograms at 0.2 V s−1 in pH 5.5 buffers for (a) an AQ monolayer and (b) layer-by-layer {AQ/Mb}6 films.
frequency decrease of 39 Hz for each AQ adsorption layer would correspond to the thickness of 1.0 nm. If the density of protein is assumed to be 1.3 g cm−3 (26), the frequency shift of 250 Hz for each adsorbed Mb layer would then correspond to the thickness of 6.5 nm. Thus, the total thickness of each AQ/Mb bilayer would be 7.5 nm. It should be pointed out, however, because of uncertainties in the estimation of film density and area, the calculated thickness here is just a rough estimation with reliability of about ±10%(25). The dimensions of Mb are 2.5 × 3.5 × 4.5 nm (27). Considering that the adsorbed Mb layer could not be arranged uniformly or perfectly and there existed experimental errors, the estimated thickness of 6.5 nm for each Mb layer thus shows reasonable agreement with the molecular dimensions of Mb, suggesting the formation of a monomolecular layer of Mb in the assembly process. Voltammetric Properties Since six AQ/Mb bilayers assembled on PG electrodes gave the largest CV peak currents (Fig. 1), most of the following voltammetric experiments were performed with the {AQ/Mb}6 films. CVs of {AQ/Mb}6 films in pH 5.5 buffers containing no Mb showed quite symmetric peak shapes and had nearly equal reduction and oxidation peak heights (Fig. 5). The peak current increased linearly with scan rate from 0.02 to 2.0 V s−1 , and integration of reduction peaks at different scan rates in this range gave nearly constant charge values. All these are characteristic of diffusionless, surface-confined voltammetric behavior (23), suggesting that all electroacotive MbFe(III) in the films is converted to MbFe(II) on the forward cathodic scan. The reverse anodic scan then converts all the MbFe(II) back to MbFe(III). The average surface concentration of electroactive Mb for {AQ/Mb}6 films was about 5.4 × 10−11 mol cm−2 . Bare PG electrodes could also adsorb Mb from its solution. After being immersed in a pH 4.5 buffer containing 3 mg mL−1 Mb for 120 min, the PG electrode was washed with water and
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then transferred into a blank buffer at pH 5.5. CV results showed a pair of redox peaks for adsorbed Mb at similar potentials but with much smaller peak heights compared with those for {AQ/Mb}6 films. This pair of peaks decreased with time in the blank buffers and totally disappeared after 30 min of soaking, indicating that smaller amounts of Mb are adsorbed directly onto the PG surface during the same period of immersing time in Mb solution, and Mb adsorbed directly on PG may not be stable in blank buffers. Adsorption of AQ on PG not only has a favorable effect on the adsorption of positively charged Mb at pH 4.5 and builds up more Mb layers on the electrode but also provides a favorable microenvironment for Mb to exchange electrons with underlying PG electrodes. Stability of {AQ/Mb}6 films was tested by two methods. In a solution study, PG electrodes coated with {AQ/Mb}6 films were stored in pH 5.5 blank buffers, and CV tests were carried out periodically. Alternately, with a “dry method”, {AQ/Mb}6 film electrodes were kept in dry form in air for most of the storing time and just returned to buffers occasionally for CV measurements. With both methods, the {AQ/Mb}6 films showed excellent stability. The peak potentials and currents essentially remained unchanged for at least 3 weeks. Square wave voltammetry (SWV) has a better signal-to-noise ratio and resolution than CV and, as a pulse electrochemical method, is easier to analyze theoretically and quantitatively than those sweep methods such as CV (28). SWV was thus used here to estimate the apparent heterogeneous electron transfer rate constant (ks ) and formal potential (E ◦ ). In the process, nonlinear regression analysis was employed for SWV forward and reverse curves, with the combination of the single-species surfaceconfined SWV model (29) and the formal potential dispersion model (30). The method was successfully used in various protein films, and a detailed description of the process was described previously in the literature (6, 30). Analysis of SWV data for {AQ/Mb}6 films demonstrated goodness of fit onto the model over a range of amplitudes and frequencies (Fig. 6). The average ks value obtained from fitting SWV data at pH 7.0 was 65 s−1 , and the average E ◦ was −0.325 V vs SCE (Table 1). Values obtained by the same method for Mb in other films are also listed in Table 1 for comparison. For {AQ/Mb}6 films, the relatively large ks value is close to that of {PSS/Mb}n films and also in the same magnitude with those in various cast Mb films. Both layer-by-layer and cast films provided a favorable microenvironment for Mb and enhanced the rate of electron transfer between Mb and underlying electrodes. For instance, cast Mb–AQ films showed a ks value of 52 s−1 , reasonably close to 65 s−1 for {AQ/Mb}6 films if the estimation error is considered. The advantage of {AQ/Mb}6 films here is the precise control of architecture and thickness with the layerby-layer method. The formal potential (E ◦ ) of the Mb Fe(III)/Fe(II) redox couple varies with different Mb films (Table 1). The E ◦ of {AQ/Mb}6 films is similar to those of {PSS/Mb}6 and cast
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Influence of pH
FIG. 6. Square wave forward and reverse current voltammograms for {AQ/Mb}6 films in pH 7.0 buffers at different frequencies. Solid lines represent the experimental background-subtracted SWVs, scattered points are the best fits to the 5-E ◦ dispersion model by nonlinear regression analysis. Amplitude, 60 mV; step height, 4 mV; frequencies (Hz): (a) 152, (b) 179, (c) 200, and (d) 228.
Mb–DHP–PDDA films and more positive than those for Mb–AQ and Mb–PAM films, but considerably more negative than those of cast films involving DDAB. This indicates a specific influence of the film environment on E ◦ of heme proteins, which had been reported previously (8, 19, 30, 31). Film components may change the formal potential through interaction with the protein or by their influence on the electrode double layer (8, 30). Even with the same components, cast Mb–AQ films and layer-by-layer {AQ/Mb}6 films demonstrated different E ◦ values, suggesting the assembly manner may also influence the formal potential since the molecular architecture and the microenvironment where Mb resides for two films may not be exactly the same.
TABLE 1 Apparent Heterogeneous Electron Transfer Rate Constants and Formal Potentials for Myoglobin Films on PG Electrodes in pH 7.0 Buffers Containing No Myoglobin
CVs of {AQ/Mb}6 films showed great dependence on pH of external buffers. An increase of pH in solution led to a negative shift in potential of both reduction and oxidation CV peaks. The changes of CV peak potentials and currents with pH were reversible in the range of pH 3.0 to pH 10.0. For example, a {AQ/Mb}6 film electrode was first placed in pH 5.0 buffers and tested by CV. It was then transferred to pH 7.0 buffers and examined by CV. When the film electrode was placed back into the pH 5.0 buffers again, CVs were quite reproducible and demonstrated almost the same peak potentials and heights as before. The surface concentration of electroactive Mb in {AQ/Mb}6 films estimated from integration of reduction peaks remained nearly constant between pH 3.0 and pH 11.0. The formal potential (E ◦ ), estimated as the midpoint of reduction and oxidation CV peak potentials of the Mb Fe(III)/Fe(II) redox couple, had a linear relationship with pH from pH 3.0 to pH 11.0 with a slope of −45.4 mV pH−1 (Fig. 7). This slope value is different from the theoretical value of −59 mV pH−1 at 25◦ C for a reversible proton-coupled single-electron transfer process (32, 33). The reason for this is not clear yet. But one thing for sure is that the electron transfer between Mb in {AQ/Mb}6 films and PG electrodes is accompanied by proton transportation. The shape and position of the Soret band can provide information about possible denaturation of heme proteins (34), and UV–vis spectroscopy was used here to detect the conformational change of Mb around the heme group for {AQ/Mb}6 films assembled on the glass slides. Dry films of pure Mb showed a Soret band at about 410 nm (Fig. 8a), while dry {AQ/Mb}6 films showed the Soret band at 413 nm (Fig. 8b). This suggests that the microenvironment provided by AQ in the layer-by-layer films has some influence on the position of the Soret band. Very good shape of the Soret band for dry {AQ/Mb}6 films also suggests that Mb may maintain its native secondary structure in this microenvironment. The influence of pH of external solution on the Soret band of the films was also tested. At a pH between 5.5 and 9.0, the Soret band appeared at 413 nm (Figs. 8c–8e), the same
Av. E ◦ (V vs SCE) Filmsa
Av. ks (s−1 )
CV
SWV
Ref.b
{AQ/Mb}6 {PSS/Mb}6 Mb–AQ (cast) Mb–DDAB (cast) Mb–PAM (cast) Mb–DDAB–PVS (cast) Mb–DHP–PDDA (cast) Mb–DDAB–clay (cast)
65 ± 16 53 ± 3 52 ± 6 31 ± 3 86 ± 19 58 ± 7 27 ± 3 44 ± 8
−0.311 −0.344 −0.362 −0.228 −0.335 −0.196 −0.323 −0.252
−0.325 −0.326 −0.340 −0.240 −0.357 −0.202 −0.326 −0.243
Tw 15 19 6 7 9 10 11
a AQ, poly(ester sulfonic acid); PSS, poly(styrenesulfonate); DDAB, didodecyldimethylammonium bromide; PAM, polyacrylamide; PVS, polyvinyl sulfate; DHP, dihexadecylphosphate; PDDA, poly(diallyldiethylammonium). b Tw: this work, reporting average values for analysis of eight SWVs at frequencies of 150–230 Hz, amplitudes of 60–75 mV, and a step height of 4 mV.
FIG. 7. Influence of pH on the formal potential for {AQ/Mb}6 film electrodes at a scan rate of 0.2 V s−1 .
ASSEMBLY OF ELECTROACTIVE LAYER-BY-LAYER FILMS
FIG. 8. UV–vis spectra on glass slides for (a) a dry Mb film, (b) a dry {AQ/Mb}6 film, and {AQ/Mb}6 films in buffers at different pH: (c) pH 9.0; (d) pH 7.0; (e) pH 5.5; (f) pH 4.0; (g) pH 11.0; (h) pH 3.0.
as that of dry {AQ/Mb}6 films, indicating that Mb in the films could retain its native state at medium pH. At pH 4.0 or 11.0, the Soret band showed blue shift and shape distortion (Figs. 8f and 8g). The Soret band even almost disappeared at pH 3.0 (Fig. 8h). All these indicate that Mb in {AQ/Mb}6 films may denature to a considerable extent under relatively extreme pH conditions. Catalytic Reactivity Layer-by-layer {AQ/Mb}6 film electrodes showed good catalytic reactivity toward various substrates. When trichloroacetic acid (TCA) was added to a pH 3.0 buffer, an increase in reduction peak of MbFe(III) at about −0.25 V at {AQ/Mb}6 film electrodes was observed, while the background current before reduction remained almost unchanged (Fig. 9). On an AQ monolayer, direct reduction of TCA was observed at the potential more negative than −0.8 V. Thus, {AQ/Mb}6 films lowered the reduction overpotential of TCA by at least 0.5 V, indicating a large decrease in activation energy for the system. Only a very small decrease of oxidation peak of MbFe(II) was observed after the addition of TCA if the obvious change of the re-oxidation background current was ignored. This seems different from the typical system of electrochemical catalysis. However, the lowering of reduction overpotential of TCA by {AQ/Mb}6 films, as well as the increase of reduction peak current with the concentration of TCA, supports the proposal that MbFe(II) produced at electrodes is chemically oxidized by TCA, and MbFe(III) is regenerated to propagate the catalytic cycle. This presumably results in the reductive dechlorination of the acid (35). In pH 3.0 buffers, the catalytic reduction current for TCA at the {AQ/Mb}6 film electrodes was much larger than that at pH 5.5; pH 3.0 was thus used here for the study of catalytic
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reduction of TCA. While Mb in {AQ/Mb}6 films might denature at pH 3.0 to a considerable extent (Fig. 8h), it retained quite good electrochemical activity at this pH (Fig. 9c). It is known that, at pH 3.0 in solution, Mb would rapidly and completely denature and lose its heme group (6, 36, 37). Good electrochemistry of {AQ/Mb}6 films at pH 3.0 thus suggests that the film environment may provide added stability to Mb, and the heme group of Mb in the films is not lost at pH 3.0. The pK a of TCA is 0.89 (38), indicating that, at pH larger than 2, TCA exists mainly as its conjugate base species. At pH 3.0, the availability of protons may rapidly form the more easily reduced conjugate acid species of TCA to react with MbFe(II), giving a larger catalytic current than that at pH 5.5 where proton availability is lower and the conjugate acid forms more slowly. The possibility of electrochemical catalytic reduction of oxygen by {AQ/Mb}6 films was also examined by CV with air present in the external solution. When a certain volume of air was passed through a pH 5.5 buffer in a sealed cell, an increase in reduction peak at about −0.3 V was observed at {AQ/Mb}6 film electrodes, accompanied by the disappearance of the oxidation peak of MbFe(II) (Fig. 10) because MbFe(II) had reacted with oxygen. Increasing the amount of injected air would result in the increase of the reduction peak. For the AQ monolayer without Mb, the peak for direct reduction of oxygen was observed at about −0.8 V. Thus, {AQ/Mb}6 film electrodes lowered the reduction overpotential of oxygen by about 0.5 V. Catalytic efficiency, expressed as the ratio of reduction peak current of MbFe(III) in the presence (Ic ) and absence (Id ) of oxygen, Ic /Id , decreased with increasing scan rate. All these are characteristic of electrochemical catalytic reduction of oxygen by {AQ/Mb}6 films (39, 40). Catalytic CV behavior was also observed for hydrogen peroxide at {AQ/Mb}6 film electrodes (Fig. 11), which is very similar to that of the oxygen system. When H2 O2 was added to a pH 5.5 buffer, an increase in reduction peak at about −0.3 V was seen
FIG. 9. Cyclic votalmmograms at a scan rate 0.1 V s−1 in pH 3.0 buffer solutions for (a) an AQ monolayer in buffers containing no TCA, (b) an AQ monolayer in buffers containing 8 mM TCA, (c) {AQ/Mb}6 films in buffers containing no TCA, (d) {AQ/Mb}6 films in buffers containing 4 mM TCA, and (e) {AQ/Mb}6 films in buffers containing 12 mM TCA.
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again and O2 . It is the production of O2 that makes the electrocatalytic CV behavior of H2 O2 similar to that of O2 at {AQ/Mb}6 film electrodes. It seems impossible that H2 O2 would undergo predisproportionation in solution when Mb is not present in the films since no signal was observed at the reduction potential of O2 at AQ monolayer film electrodes when H2 O2 was added in buffers (Fig. 11b). It was difficult to check the evolution of oxygen on the film surface during the catalysis, while vigorous degassing with N2 during the experiments caused the same results as those in Fig. 11. This indicates that the O2 produced by disproportionation of H2 O2 in the procedure is most probably from the catalysis by Mb. FIG. 10. Cyclic voltammograms at 0.2 V s−1 in 5 mL of pH 5.5 buffers for (a) an AQ monolayer in the absence of O2 , (b) an AQ monolayer after 5 mL of air was injected into the sealed cell, (c) an {AQ/Mb}6 film in the absence of O2 , (d) an {AQ/Mb}6 film after 5 mL of air was injected, and (e) an {AQ/Mb}6 film after 10 mL of air was injected.
with the disappearance of the oxidation peak for MbFe(II). The reduction peak current increased with the concentration of H2 O2 in solution. However, no direct reduction peak was observed on an AQ monolayer in the presence of H2 O2 in the studied potential range (Fig. 11b). The exact mechanism of catalytic reduction of hydrogen peroxide on {AQ/Mb}6 films is as yet unclear, but most probably similar to that of a horseradish peroxidase (HRP) film system (41) since Mb and HRP are all heme proteins and have similar electrochemical properties. It is known that, in the absence of reductant substrates and with excess hydrogen peroxide, HRP behaves like catalase, where H2 O2 acts either as an oxidant or as a reductant (42, 43). Thus, the oxidation product of Mb by H2 O2 , designated as Compound I, may be reduced by H2 O2 through a two-electron transfer pathway and produce native MbFe(III)
CONCLUSION
Negatively charged ionomer Eastman AQ55 can form ordered layer-by-layer films with positively charged myoglobin in pH 4.5 buffers by electrostatic interaction between them. An effective electron transfer rate involving a Mb heme Fe(III)/Fe(II) redox couple was greatly facilitated in the microenvironment of {AQ/Mb}n films compared with that of bare PG electrodes in Mb solutions. These layer-by-layer films had good stability, and Mb in the films essentially retained its native state at medium pH. {AQ/Mb}6 film electrodes could electrochemically catalyze reduction of trichloroacetic acid, oxygen, and hydrogen peroxide, suggesting that Mb in the films may act as a reductive enzyme for some substrates. This layer-by-layer film electrode demonstrates a promising approach to fabricate mediator-free biosensors or bioreactors. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (#29975003).
REFERENCES
FIG. 11. Cyclic voltammograms at 0.2 V s−1 in pH 5.5 buffers for (a) an AQ monolayer in buffers containing no H2 O2 , (b) an AQ monolayer in buffers containing 0.2 mM H2 O2 , (c) an {AQ/Mb}6 film in buffers containing no H2 O2 , (d) an {AQ/Mb}6 film in buffers containing 0.1 mM H2 O2 , and (e) an {AQ/Mb}6 film in buffers containing 0.2 mM H2 O2 .
1. Chaplin, M. F., and Bucke, C., “Enzyme Technology.” Cambridge Univ. Press, Cambridge, U.K., 1990. 2. Stryer, L., “Biochemistry,” 3rd ed. Freeman, New York, 1988. 3. Amstrong, F. A., Hill, H. A. O., and Walton, N. J., Acc. Chem. Res. 21, 407 (1988). 4. Rusling, J. F., Acc. Chem. Res. 31, 363 (1998). 5. Rusling, J. F., and Nassar, A.-E. F., J. Am. Chem. Soc. 115, 11891 (1993). 6. Nassar, A.-E. F., Zhang, Z., Hu, N., Rusling, J. F., and Kumosinski, T. F., J. Phys. Chem. B 101, 2224 (1997). 7. Shen, L., Huang, R., and Hu, N., Talanta 56, 1131 (2002). 8. Nassar, A.-E. F., Narikiyo, Y., Sagara, T., Nakashima, N., and Rusling, J. F., J. Am. Chem. Soc. Faraday Trans. 91, 1775 (1998). 9. Hu, Y., Hu, N., and Zeng, Y., Microchem. J. 65, 147 (2000). 10. Wang, L., and Hu, N., J. Colloid Interface Sci. 236, 166 (2001), doi: 10.1006/jcis.2000.7381. 11. Hu, N., Li, Z., and Ma, H., Gaodeng Xuexiao Huaxue Xuebao (Chem. J. Chinese Univ.) 22, 450 (2001). 12. Lvov, Y., in “Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology” (Y. Lvov and H. Mohwald, Eds.), p. 125. Dekker, New York, 2000.
ASSEMBLY OF ELECTROACTIVE LAYER-BY-LAYER FILMS 13. Rusling, J. F., in “Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology” (Y. Lvov and H. Mohwald, Eds.), p. 337. Dekker, New York, 2000. 14. Lvov, Y., Lu, Z., Schenkman, J., Zu, X., and Rusling, J. F., J. Am. Chem. Soc. 120, 4073 (1998). 15. Ma, H., Hu, N., and Rusling, J. F., Langmuir 16, 4969 (2000). 16. Raynolds, P. W., in “Proceedings of the Fine Particles Society Symposium” (M. K. Sharma and F. I. Micale, Eds.), p. 275. Plenum, New York, 1991. 17. Wang, J., and Golden, T., Anal. Chem. 61, 1397 (1989). 18. Gennett, T., and Purdy, W. C., Anal. Chem. 62, 2155 (1990). 19. Hu, N., and Rusling, J. F., Langmuir 13, 4119 (1997). 20. Bellellii, A., Antonini, G. M., Springer, B. A., and Sligar, S. G., J. Biol. Chem. 265, 18898 (1990). 21. Rossi-Fanelli, A., Antonini, E., and Povoledo, D., in “Symposium on Protein Structure” (A. Neuberger, Ed.), p. 144. Methuen Co., London, 1958. 22. Hill, H. A. O., Pure Appl. Chem. 59, 743 (1987). 23. Murray, R. W., in “Electroanalytical Chemistry” (A. J. Bard, Ed.), Vol. 13, p. 191. Dekker, New York, 1984. 24. Sauerbrey, G., Z. Phys. 155, 206 (1959). 25. Lvov, Y., Ariga, K., Ichinose, I., and Kunitake, T., J. Am. Chem. Soc. 117, 6117 (1995). 26. Creighton, T. E., Ed., “Protein Structure, a Practical Approach,” p. 43. IRL Press, New York, 1990.
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27. Kendrew, J. C., Philips, D. C., and Stone, V. C., Nature 185, 422 (1960). 28. Osteryoung, J. G., and O’Dea, J. J., in “Electroanalytical Chemistry” (A. J. Bard, Ed.), Vol. 14, p. 209. Dekker, New York, 1986. 29. O’Dea, J. J., and Osteryoung, J. G., Anal. Chem. 65, 3090 (1993). 30. Zhang, Z., and Rusling, J. F., Biophys. Chem. 63, 133 (1997). 31. Yang, J., Hu, N., and Rusling, J. F., J. Electroanal. Chem. 463, 53 (1999). 32. Meites, L., “Polarographic Techniques,” 2nd ed. Wiley, New York, 1965. 33. Bond, A. M., “Modern Polarographic Methods in Analytical Chemistry.” Dekker, New York, 1980. 34. George, P., and Hanania, G. I. H., Biochem. J. 55, 236 (1953). 35. Nassar, A.-E. F., Bobbitt, J. M., Stuart, J. D., and Rusling, J. F., J. Am. Chem. Soc. 117, 10986 (1995). 36. Shire, S. J., Hanania, G. I. H., and Gurd, F. R. N., Biochemistry 13, 2967 (1974). 37. Friend, S. H., and Gurd, F. R. N., Biochemistry 18, 4612 (1979). 38. Christian, G. D., “Analytical Chemistry,” 5th ed. Wiley, New York, 1994. 39. Bard, A. J., and Faulkner, L. R., “Electrochemical Methods,” Wiley, New York, 1980. 40. Andrienx, C. P., Blocman, C., Dumas-Bouchiat, J. M., M’Halla, F., and Saveant, J. M., J. Electroanal. Chem. 113, 19 (1980). 41. Huang, R., and Hu, N., Bioelectrochemistry 54, 75 (2001). 42. Nakajima, R., and Yamazaki, I., J. Biol. Chem. 262, 2576 (1987). 43. Arnao, M. B., Acosta, M., Del-Rio, J. A., Varon, R., and Garcia-Canovas, F., Biochim. Biophys. Acta 1041, 43 (1990).
Assembly of Electroactive Layer-by-Layer Films of Myoglobin and Ionomer Poly(ester Sulfonic Acid) Zhen Li and Naifei Hu1 Department of Chemistry, Beijing Normal University, Beijing 100875, China Received November 26, 2001; accepted July 9, 2002
Layer-by-layer films were assembled on solid substrates by alternate adsorption of negatively charged ionomer poly(ester sulfonic acid) or Eastman AQ55 from its aqueous dispersion and positively charged myoglobin (Mb) from its solution at pH 4.5. The film assembly process was monitored by cyclic voltammetry (CV), UV–vis spectroscopy, and quartz crystal microbalance (QCM). {AQ/Mb}n films grown on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible CV peaks at about −0.20 V vs Ag/AgCl in pH 5.5 buffers, characteristic of the Mb heme Fe(III)/Fe(II) redox couple. Although the amount of Mb adsorbed in each bilayer was essentially the same, the fraction of electroactive Mb decreased dramatically with an increase of bilayer number (n). Soret absorption bands of {AQ/Mb}n films on glass slides suggest that Mb in the films retains its native state in the medium pH range. Trichloroacetic acid, oxygen, and hydrogen peroxide were electrochemically catalyzed by {AQ/Mb}6 films with significant lowering of reduction overpotential. C 2002 Elsevier Science (USA) Key Words: myoglobin; poly(ester sulfonic acid); Eastman AQ; layer-by-layer assembly films; direct electrochemistry; electrocatalysis.
INTRODUCTION
Study of direct electron transfer between enzyme and electrode can provide a model for the study of enzyme-catalyzed reaction in biological systems and may establish a foundation for constructing new kinds of biosensors or enzymatic bioreactors (1). Achieving reversible, direct electron transfer between an enzyme and electrode without using any chemical mediators thus has great significance. Myoglobin (Mb) is a heme protein, which can store and transport oxygen in muscle cells in mammalians. Mb has a single polypeptide chain with an iron heme inside as a prosthetic group (2). Although Mb does not function as an electron carrier biologically, it is an ideal model for the study of electron transfer of heme enzymes such as cytochrome P450 because of its commercial availability, documented structure, and similar reactivity to that of cytochrome P450. However, direct electron exchange between Mb in solution and solid electrodes is usually very slow. 1 Author to whom correspondence should be addressed. E-mail: hunaifei@ bnu.edu.cn.
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Many researchers used promoters in Mb solution to enhance the electron transfer (3). A relative new avenue to realize direct electrochemistry of protein is to incorporate protein into films which are cast on the surface of solid electrodes (4). The materials of cast films containing Mb include water-insoluble surfactants (5, 6), amphiphilic polymer (7), and polyelectrolyte–surfactant (8–10) or clay–surfactant (11) complex composites. These cast films were easy to prepare and quite stable and provided a favorable microenvironment for Mb and greatly facilitated the rate of electron transport between Mb and electrodes. However, the final molecular architecture and thickness of the cast Mb films cannot be controlled precisely, and only a very small fraction of Mb in the films was electroactive. Recently, a novel technique of layer-by-layer assembly was developed and employed in fabricating ultrathin protein films (12). By alternate adsorption of polyions and oppositely charged proteins in solution at appropriate pH, multilayered protein assembly was achieved with precisely repeatable layer thickness. In this way, the design of ordered protein films at the molecular level and the precise control of film thickness on the nanometer scale can be realized. Layer-by-layer protein films can also provide the possibility of studying direct electrochemistry of redox proteins in their multilayer architecture (13). Take Mb as an example; Lvov et al. constructed layer-by-layer films of Mb with appropriately charged polyions on gold electrodes modified with mercaptopropanesulfonic acid (14). Cyclic voltammetry (CV) of the films in blank buffers showed a pair of direct, reversible peaks for the Mb Fe(III)/Fe(II) redox couple. Recently, we reported ultrathin films of Mb assembled layer by layer with poly(styrene sulfonate) (PSS) on pyrolytic graphite (PG) electrodes (15). Mb in {PSS/Mb}n films demonstrated direct electron transfer with underlying electrodes, and electroactivity was extended to seven {PSS/Mb} bilayers on a rough PG surface. An ionomer is an ionic polymer that contains only a fraction of ionizable groups with each monomer unit. In recent years, the ionomer poly(ester sulfonic acid), with the trade name Eastman AQ, has aroused interest among chemists as film-forming materials. AQ55 ionomer has 18 mol% aromatic sulfonate groups (see Scheme I) (16), which is ionizable when dispersed in water. This thermoplastic, amorphous ionomer gives translucent, lowviscosity dispersion in water without adding organic solvent. AQ films cast onto electrodes could bind hydrophobic cations 0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
All rights reserved.
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SCHEME I.
Chemical structure of Eastman AQ55.
preferentially and exclude negatively charged species (17, 18). In our previous work (19), Mb was mixed directly with aqueous dispersions of AQ ionomers and deposited onto solid surfaces to form films without denaturation. These cast Mb–AQ films on PG electrodes showed stable CV signals for the Mb Fe(III)/Fe(II) redox couple in buffer solutions, but only about 10% of the total Mb deposited was electroactive. In this work, layer-by-layer {AQ/Mb}n films were assembled onto solid substrates by alternate adsorption of the negatively charged AQ55 ionomer from its dispersion in water and the positively charged Mb from its aqueous solutions at pH 4.5. To our knowledge, this is the first time ionomers are used instead of regular polyions to assemble layer-by-layer protein films. The assembled {AQ/Mb}n films were characterized by voltammetry, UV–vis spectroscopy, and a quartz crystal microbalance (QCM). {AQ/Mb}n films on PG electrodes showed direct electrochemistry for Mb and were used to catalyze reduction of trichloroacetic acid, oxygen, and hydrogen peroxide.
ate water washing and nitrogen stream drying. This cycle was repeated periodically to make {AQ/Mb}n multilayer films on PG electrodes. Glass slides (1 × 4 cm, 1-mm thick) were first washed in a washing solution (60% ethanol + 39% water + 1% KOH) for 30 min at 50◦ C to induce negative charges on the surface and then carefully rinsed with water. A monolayer of positively charged precursor PEI was adsorbed on the glass surface by immersing the slides into PEI solutions (3 mg/mL, containing 0.5 M NaCl) for 20 min. The layer-by-layer films of {AQ/Mb}n were then assembled on the PEI-modified glass slides in the same way as on PG electrodes described above. Gold-coated resonator electrodes (geometric area 0.196 cm2 ) for a quartz crystal microbalance (QCM) were soaked in freshly prepared “piranha” solution (3 : 7 volume ratio of 30% H2 O2 and 98% H2 SO4 ) for 15 min at ca. 95◦ C and then washed in pure ethanol and water. (Caution: piranha solution should be handled with extreme care, and only small volumes should be prepared at any time.) The cleaned gold electrodes were immersed in 4 mM MPS ethanol solutions for 24 h to form MPS monolayer on gold electrodes and introduce negative charges on the surface. After being rinsed with pure ethanol and water and dried under a stream of highly purified N2 , the gold electrode resonators were then immersed into PEI solutions for 20 min to make the surface positively charged. The {AQ/Mb}n films were then assembled on the pretreated gold electrodes with the same method as on PG.
EXPERIMENTAL
Instruments and Procedures Chemicals Horse heart myoglobin (Mb, MW = 17,800) was from Sigma and used as received. Poly(ester sulfonic acid) or Eastman AQ55 (AQ, MW = 14,000, Tg = 55◦ C) was from Eastman Chemical Co. Poly(ethylenimine) (PEI, 90%, MW = 60,000) and 3mercapto-1-propanesulfonate (MPS, 90%) were from Aldrich. Trichloroacetic acid (TCA, 99%) was from Jinlong Chemical Reagent Co. Hydrogen peroxide (H2 O2 , 30%) was from Beijing Chemical Engineering Plant. Buffers were 0.05 M citric acid, 0.1 M acetate, 0.05 M sodium dihydrogen phosphate, or 0.05 M boric acid, all containing 0.1 M KBr. pH values were adjusted to desired values with HCl or KOH solutions. All other chemicals were reagent grade. Water used was twice distilled. Film Assembly The multilayer films of AQ and Mb were assembled onto solid substrates such as carbon electrodes, glass slides, and gold electrode resonators by the following methods. Prior to use, basal plane pyrolytic graphite (PG, Advanced Ceramics, geometric area 0.16 cm2 ) disk electrodes were polished on metallographic sandpaper while being flushed with water. The electrodes were then ultrasonicated in pure water for 30 s and dried in air. The PG electrodes were alternately placed for 20 min in aqueous dispersions of AQ (1%) and Mb solutions (3 mg/mL, in pH 4.5 buffers) with intermedi-
A CHI 420 electrochemical analyzer (CH Instruments) was used for both voltammetric and QCM studies. In electrochemical measurements, a regular three-electrode cell was used with an Ag/AgCl electrode as the reference electrode, a platinum wire as the counter electrode, and a PG disk electrode coated with films as the working electrode. Prior to electrochemical measurements, solutions were purged with purified nitrogen for at least 15 min, and a nitrogen atmosphere was maintained over solutions for exclusion of oxygen during experiments. In aerobic experiments, measured volumes of air were injected via a syringe into solutions in a sealed cell that had been previously degassed with purified nitrogen. For QCM studies, ATcut quartz resonators with fundamental resonant frequency of 8 MHz and coated by gold thin films on both sides were used. UV–vis absorption spectroscopy was measured for the films assembled on glass slides with a Cintra-10e UV–vis spectrometer (GBC). All experiments were performed at ambient temperature of 25 ± 2◦ C. RESULTS AND DISCUSSION
Assembly of Layer-by-Layer Films Monitored by CV and UV–vis Myoglobin, with its isoelectric point at pH 6.8 (20, 21), has positive surface charges at pH 4.5, while AQ has negative charges
ASSEMBLY OF ELECTROACTIVE LAYER-BY-LAYER FILMS
FIG. 1. Background-subtracted cyclic voltammograms for layer-by-layer {AQ/Mb}n films at 0.2 V s−1 in pH 5.5 buffers with a different number of bilayers (n).
on its backbone. Thus, layer-by-layer films of AQ and Mb could be assembled by Coulombic attraction between them on the surface of various substrates such as PG, pretreated glass slides, and MPS–PEI-modified Au by alternate adsorption from their dispersions or solutions (see Experimental section). The films were designated as {AQ/Mb}n , where n is the number of bilayers or adsorption cycles for the films. In this part, cyclic voltammetry (CV) and UV–vis spectroscopy were used to monitor the assembly of {AQ/Mb}n multilayer films. It is known that ordinary basal plane PG provides a surface with a partly hydrophobic character (22). AQ is a kind of amphiphilic ionomers containing a hydrophobic backbone. Thus, hydrophobic interaction between AQ and basal PG would be mainly responsible for spontaneous adsorption of AQ on the PG surface. Positively charged Mb from pH 4.5 buffer solutions was then adsorbed on the surface of a negatively charged AQ layer. CV was used here to monitor the growth of the {AQ/Mb}n films. After each adsorption cycle, the {AQ/Mb}n film electrode was placed in a pH 5.5 buffer containing no Mb, followed by CV scans (Fig. 1). A pair of well-defined, quite reversible CV peaks was observed at about −0.20 V vs Ag/AgCl, characteristic of the Mb heme Fe(III)/Fe(II) redox couple. Both reduction and oxidation peaks grew with the number of adsorbed AQ/Mb bilayers (n) at first. When n exceeded 6, however, no further increase in the reduction peak current was observed and the peak heights remained essentially the same, indicating that Mb in the bilayer of n > 6 was not yet electroactive. AQ monolayer adsorbed on PG electrodes had no CV signal at all in this potential window. As an ionomer, AQ has a much lower charge density compared with those of regular polyions such as PSS. The polyanion PSS has a linear aromatic backbone similar to AQ, but it has one negatively charged sulfonate group with every repeated monomer unit. For AQ, however, only a small fraction of its monomer units has a sulfonate group (see chemical structure of AQ55). This difference made a notable impact on the layer-bylayer assembly process. In the assembly of {PSS/Mb}n films, for example, pH 5.5 could be chosen for the Mb adsorbate
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solution, and {PSS/Mb}n films assembled on PG electrodes in this way demonstrated quite good CV responses (15). However, in the assembly of {AQ/Mb}n films, when pH 5.5 was selected for Mb adsorbate solutions, a much smaller CV peak response was obtained, indicating that much smaller amounts of Mb were adsorbed on the AQ layer surface compared with PSS at this pH. To enhance the adsorption of Mb on the AQ surface, the pH of Mb adsorbate solution in this work was adjusted to 4.5, further from the isoelectric point of Mb at pH 6.8 (20, 21) so that Mb could bear more positive surface charges and more Mb could be adsorbed onto the AQ surface layer. In the previous work of assembling {PSS/Mb}n layer-by-layer films (15), inorganic salt NaCl was usually added to the polyanion adsorbate solutions to enhance the adsorption of PSS. In the present work, however, since AQ is insoluble in water, only an aqueous dispersion of AQ could be obtained by a relatively long period of time of ultrasonication. If salt were added to this dispersion, AQ would precipitate immediately. Thus, in the assembly process of {AQ/Mb}n films, the AQ adsorbate dispersion was free of salt. Integration of the CV reduction peak for MbFe(III) can give the amount of reduction charge (Q) passed through the electrode, and according to the Q– ∗ relationship (23), the surface concentration of electroactive Mb ( ∗ , mol cm−2 ) can be deduced. The ∗ value increased nonlinearly with n up to 6 (Fig. 2a) and tended to level off afterward. The Mb molecule contains a heme prosthetic group, which has a sensitive absorption band at about 410 nm called the Soret absorption band. Thus, UV–vis spectroscopy was used here to monitor the growth of the AQ/Mb bilayer on PEI-modified glass slides. UV–vis spectra showed the Soret absorption band at 413 nm for {AQ/Mb}n films (Fig. 3). The Soret absorbance peak increased linearly with n (correlation coefficient 0.9979), indicating that the {AQ/Mb}n multibilayer is built up in a regular or linear mode, and the amount of Mb adsorbed in each bilayer is essentially the same. Assuming the amount of adsorbed Mb in the first bilayer closest to the PG surface is 100% electroactive (14),
FIG. 2. Influence of the number of bilayers (n) for {AQ/Mb}n films on the (a) surface concentration of electroactive Mb ( ∗ ) and (b) fraction of electroactive Mb. Data were from CVs at 0.2 V s−1 in pH 5.5 buffers.
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FIG. 3. UV–vis spectra on glass slides for an AQ monolayer and for layerby-layer {AQ/Mb}n films with a different number of bilayers.
according to the CVs, the fraction of electroactive Mb among all adsorbed Mb in each bilayer would decrease dramatically with n (Fig. 2b). This indicates that the distance between Mb and electrodes is crucial for efficient electron exchange. The further the distance, the lower the percentage of electroactive Mb in each bilayer. QCM Studies Quartz crystal microbalance (QCM) is a sensitive technique that can detect tiny mass changes on the QCM electrodes and was used here to monitor the layer-by-layer assembly of {AQ/Mb}n films. Based on the Sauerbrey equation (24), F =
−2 f 02 M , √ A µρ
A monolayer of MPS was first chemisorbed on the gold thin films coated on both sides of the quartz resonator so that the electrode surfaces became negatively charged. A precursor monolayer of PEI was then adsorbed onto the MPS surface to convert the surface to positively charged (see Experimental section). The {AQ/Mb}n multilayer films were then built up on the QCM electrode by alternate adsorption of AQ and Mb as described above. After each adsorption step, the electrode was washed thoroughly in water, dried under a nitrogen stream, and then measured by QCM. The results showed a roughly linear decrease of frequency with adsorption step (Fig. 4). Each AQ adsorption layer caused a frequency decrease of about 39 Hz, and each adsorbed layer of Mb resulted in a frequency decrease of about 250 Hz. AQ adsorption produced a smaller frequency shift than Mb, which was also observed in other layer-by-layer {polyion/protein}n films (25). Mb has a larger molecular weight and a relatively more rigid globular shape, while AQ is a flexible linear ionomer with small molecular weight for each of its repeated units. Roughly constant F for each Mb layer suggests that the amount of absorbed Mb in each cycle is approximately the same, as observed in UV–vis spectroscopic experiments. It is the same for the AQ layer. The QCM data were also used to estimate the thickness of {AQ/Mb}n films. The thickness, d (cm), can be expressed by d=
M , 2ρ A
[3]
where ρ is the density of the film material (g cm−3 ) and A is the area of QCM electrodes (0.196 cm2 ). Since two gold thin films are coated on both sides of the QCM resonator, the total mass adsorbed on Au electrodes should be divided by 2. Combined with Eq. [2] the relationship between d and F would be d = −3.4 × 10−9 F/ρ .
[4]
Since the density of AQ55 is about 1.33 g cm−3 (16), the average
[1]
a micromass change (M) can be converted to frequency shift (F) of the quartz crystal resonator. The following relationship between M (g) and F (Hz) is obtained by taking into account the characteristics of the quartz resonator used in this work, F = −7.40 × 108 M,
[2]
where f 0 is the resonant frequency of the fundamental mode of the quartz crystal (8 MHz), µ is the shear modulus of quartz (2.947 × 1011 g cm−1 s−2 ), ρ is the density of the crystal (2.648 g cm−3 ) and A is the geometric area of the QCM electrode (0.196 cm−2 ). Thus, 1 Hz of frequency decrease corresponds to 1.35 ng of mass increase with relatively good accuracy and reliability.
FIG. 4. Shift of QCM frequency with alternate adsorption step of AQ and Mb on MPS–PEI-modified Au electrodes. () AQ adsorption steps; () Mb adsorption steps.
ASSEMBLY OF ELECTROACTIVE LAYER-BY-LAYER FILMS
FIG. 5. Cyclic voltammograms at 0.2 V s−1 in pH 5.5 buffers for (a) an AQ monolayer and (b) layer-by-layer {AQ/Mb}6 films.
frequency decrease of 39 Hz for each AQ adsorption layer would correspond to the thickness of 1.0 nm. If the density of protein is assumed to be 1.3 g cm−3 (26), the frequency shift of 250 Hz for each adsorbed Mb layer would then correspond to the thickness of 6.5 nm. Thus, the total thickness of each AQ/Mb bilayer would be 7.5 nm. It should be pointed out, however, because of uncertainties in the estimation of film density and area, the calculated thickness here is just a rough estimation with reliability of about ±10%(25). The dimensions of Mb are 2.5 × 3.5 × 4.5 nm (27). Considering that the adsorbed Mb layer could not be arranged uniformly or perfectly and there existed experimental errors, the estimated thickness of 6.5 nm for each Mb layer thus shows reasonable agreement with the molecular dimensions of Mb, suggesting the formation of a monomolecular layer of Mb in the assembly process. Voltammetric Properties Since six AQ/Mb bilayers assembled on PG electrodes gave the largest CV peak currents (Fig. 1), most of the following voltammetric experiments were performed with the {AQ/Mb}6 films. CVs of {AQ/Mb}6 films in pH 5.5 buffers containing no Mb showed quite symmetric peak shapes and had nearly equal reduction and oxidation peak heights (Fig. 5). The peak current increased linearly with scan rate from 0.02 to 2.0 V s−1 , and integration of reduction peaks at different scan rates in this range gave nearly constant charge values. All these are characteristic of diffusionless, surface-confined voltammetric behavior (23), suggesting that all electroacotive MbFe(III) in the films is converted to MbFe(II) on the forward cathodic scan. The reverse anodic scan then converts all the MbFe(II) back to MbFe(III). The average surface concentration of electroactive Mb for {AQ/Mb}6 films was about 5.4 × 10−11 mol cm−2 . Bare PG electrodes could also adsorb Mb from its solution. After being immersed in a pH 4.5 buffer containing 3 mg mL−1 Mb for 120 min, the PG electrode was washed with water and
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then transferred into a blank buffer at pH 5.5. CV results showed a pair of redox peaks for adsorbed Mb at similar potentials but with much smaller peak heights compared with those for {AQ/Mb}6 films. This pair of peaks decreased with time in the blank buffers and totally disappeared after 30 min of soaking, indicating that smaller amounts of Mb are adsorbed directly onto the PG surface during the same period of immersing time in Mb solution, and Mb adsorbed directly on PG may not be stable in blank buffers. Adsorption of AQ on PG not only has a favorable effect on the adsorption of positively charged Mb at pH 4.5 and builds up more Mb layers on the electrode but also provides a favorable microenvironment for Mb to exchange electrons with underlying PG electrodes. Stability of {AQ/Mb}6 films was tested by two methods. In a solution study, PG electrodes coated with {AQ/Mb}6 films were stored in pH 5.5 blank buffers, and CV tests were carried out periodically. Alternately, with a “dry method”, {AQ/Mb}6 film electrodes were kept in dry form in air for most of the storing time and just returned to buffers occasionally for CV measurements. With both methods, the {AQ/Mb}6 films showed excellent stability. The peak potentials and currents essentially remained unchanged for at least 3 weeks. Square wave voltammetry (SWV) has a better signal-to-noise ratio and resolution than CV and, as a pulse electrochemical method, is easier to analyze theoretically and quantitatively than those sweep methods such as CV (28). SWV was thus used here to estimate the apparent heterogeneous electron transfer rate constant (ks ) and formal potential (E ◦ ). In the process, nonlinear regression analysis was employed for SWV forward and reverse curves, with the combination of the single-species surfaceconfined SWV model (29) and the formal potential dispersion model (30). The method was successfully used in various protein films, and a detailed description of the process was described previously in the literature (6, 30). Analysis of SWV data for {AQ/Mb}6 films demonstrated goodness of fit onto the model over a range of amplitudes and frequencies (Fig. 6). The average ks value obtained from fitting SWV data at pH 7.0 was 65 s−1 , and the average E ◦ was −0.325 V vs SCE (Table 1). Values obtained by the same method for Mb in other films are also listed in Table 1 for comparison. For {AQ/Mb}6 films, the relatively large ks value is close to that of {PSS/Mb}n films and also in the same magnitude with those in various cast Mb films. Both layer-by-layer and cast films provided a favorable microenvironment for Mb and enhanced the rate of electron transfer between Mb and underlying electrodes. For instance, cast Mb–AQ films showed a ks value of 52 s−1 , reasonably close to 65 s−1 for {AQ/Mb}6 films if the estimation error is considered. The advantage of {AQ/Mb}6 films here is the precise control of architecture and thickness with the layerby-layer method. The formal potential (E ◦ ) of the Mb Fe(III)/Fe(II) redox couple varies with different Mb films (Table 1). The E ◦ of {AQ/Mb}6 films is similar to those of {PSS/Mb}6 and cast
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Influence of pH
FIG. 6. Square wave forward and reverse current voltammograms for {AQ/Mb}6 films in pH 7.0 buffers at different frequencies. Solid lines represent the experimental background-subtracted SWVs, scattered points are the best fits to the 5-E ◦ dispersion model by nonlinear regression analysis. Amplitude, 60 mV; step height, 4 mV; frequencies (Hz): (a) 152, (b) 179, (c) 200, and (d) 228.
Mb–DHP–PDDA films and more positive than those for Mb–AQ and Mb–PAM films, but considerably more negative than those of cast films involving DDAB. This indicates a specific influence of the film environment on E ◦ of heme proteins, which had been reported previously (8, 19, 30, 31). Film components may change the formal potential through interaction with the protein or by their influence on the electrode double layer (8, 30). Even with the same components, cast Mb–AQ films and layer-by-layer {AQ/Mb}6 films demonstrated different E ◦ values, suggesting the assembly manner may also influence the formal potential since the molecular architecture and the microenvironment where Mb resides for two films may not be exactly the same.
TABLE 1 Apparent Heterogeneous Electron Transfer Rate Constants and Formal Potentials for Myoglobin Films on PG Electrodes in pH 7.0 Buffers Containing No Myoglobin
CVs of {AQ/Mb}6 films showed great dependence on pH of external buffers. An increase of pH in solution led to a negative shift in potential of both reduction and oxidation CV peaks. The changes of CV peak potentials and currents with pH were reversible in the range of pH 3.0 to pH 10.0. For example, a {AQ/Mb}6 film electrode was first placed in pH 5.0 buffers and tested by CV. It was then transferred to pH 7.0 buffers and examined by CV. When the film electrode was placed back into the pH 5.0 buffers again, CVs were quite reproducible and demonstrated almost the same peak potentials and heights as before. The surface concentration of electroactive Mb in {AQ/Mb}6 films estimated from integration of reduction peaks remained nearly constant between pH 3.0 and pH 11.0. The formal potential (E ◦ ), estimated as the midpoint of reduction and oxidation CV peak potentials of the Mb Fe(III)/Fe(II) redox couple, had a linear relationship with pH from pH 3.0 to pH 11.0 with a slope of −45.4 mV pH−1 (Fig. 7). This slope value is different from the theoretical value of −59 mV pH−1 at 25◦ C for a reversible proton-coupled single-electron transfer process (32, 33). The reason for this is not clear yet. But one thing for sure is that the electron transfer between Mb in {AQ/Mb}6 films and PG electrodes is accompanied by proton transportation. The shape and position of the Soret band can provide information about possible denaturation of heme proteins (34), and UV–vis spectroscopy was used here to detect the conformational change of Mb around the heme group for {AQ/Mb}6 films assembled on the glass slides. Dry films of pure Mb showed a Soret band at about 410 nm (Fig. 8a), while dry {AQ/Mb}6 films showed the Soret band at 413 nm (Fig. 8b). This suggests that the microenvironment provided by AQ in the layer-by-layer films has some influence on the position of the Soret band. Very good shape of the Soret band for dry {AQ/Mb}6 films also suggests that Mb may maintain its native secondary structure in this microenvironment. The influence of pH of external solution on the Soret band of the films was also tested. At a pH between 5.5 and 9.0, the Soret band appeared at 413 nm (Figs. 8c–8e), the same
Av. E ◦ (V vs SCE) Filmsa
Av. ks (s−1 )
CV
SWV
Ref.b
{AQ/Mb}6 {PSS/Mb}6 Mb–AQ (cast) Mb–DDAB (cast) Mb–PAM (cast) Mb–DDAB–PVS (cast) Mb–DHP–PDDA (cast) Mb–DDAB–clay (cast)
65 ± 16 53 ± 3 52 ± 6 31 ± 3 86 ± 19 58 ± 7 27 ± 3 44 ± 8
−0.311 −0.344 −0.362 −0.228 −0.335 −0.196 −0.323 −0.252
−0.325 −0.326 −0.340 −0.240 −0.357 −0.202 −0.326 −0.243
Tw 15 19 6 7 9 10 11
a AQ, poly(ester sulfonic acid); PSS, poly(styrenesulfonate); DDAB, didodecyldimethylammonium bromide; PAM, polyacrylamide; PVS, polyvinyl sulfate; DHP, dihexadecylphosphate; PDDA, poly(diallyldiethylammonium). b Tw: this work, reporting average values for analysis of eight SWVs at frequencies of 150–230 Hz, amplitudes of 60–75 mV, and a step height of 4 mV.
FIG. 7. Influence of pH on the formal potential for {AQ/Mb}6 film electrodes at a scan rate of 0.2 V s−1 .
ASSEMBLY OF ELECTROACTIVE LAYER-BY-LAYER FILMS
FIG. 8. UV–vis spectra on glass slides for (a) a dry Mb film, (b) a dry {AQ/Mb}6 film, and {AQ/Mb}6 films in buffers at different pH: (c) pH 9.0; (d) pH 7.0; (e) pH 5.5; (f) pH 4.0; (g) pH 11.0; (h) pH 3.0.
as that of dry {AQ/Mb}6 films, indicating that Mb in the films could retain its native state at medium pH. At pH 4.0 or 11.0, the Soret band showed blue shift and shape distortion (Figs. 8f and 8g). The Soret band even almost disappeared at pH 3.0 (Fig. 8h). All these indicate that Mb in {AQ/Mb}6 films may denature to a considerable extent under relatively extreme pH conditions. Catalytic Reactivity Layer-by-layer {AQ/Mb}6 film electrodes showed good catalytic reactivity toward various substrates. When trichloroacetic acid (TCA) was added to a pH 3.0 buffer, an increase in reduction peak of MbFe(III) at about −0.25 V at {AQ/Mb}6 film electrodes was observed, while the background current before reduction remained almost unchanged (Fig. 9). On an AQ monolayer, direct reduction of TCA was observed at the potential more negative than −0.8 V. Thus, {AQ/Mb}6 films lowered the reduction overpotential of TCA by at least 0.5 V, indicating a large decrease in activation energy for the system. Only a very small decrease of oxidation peak of MbFe(II) was observed after the addition of TCA if the obvious change of the re-oxidation background current was ignored. This seems different from the typical system of electrochemical catalysis. However, the lowering of reduction overpotential of TCA by {AQ/Mb}6 films, as well as the increase of reduction peak current with the concentration of TCA, supports the proposal that MbFe(II) produced at electrodes is chemically oxidized by TCA, and MbFe(III) is regenerated to propagate the catalytic cycle. This presumably results in the reductive dechlorination of the acid (35). In pH 3.0 buffers, the catalytic reduction current for TCA at the {AQ/Mb}6 film electrodes was much larger than that at pH 5.5; pH 3.0 was thus used here for the study of catalytic
263
reduction of TCA. While Mb in {AQ/Mb}6 films might denature at pH 3.0 to a considerable extent (Fig. 8h), it retained quite good electrochemical activity at this pH (Fig. 9c). It is known that, at pH 3.0 in solution, Mb would rapidly and completely denature and lose its heme group (6, 36, 37). Good electrochemistry of {AQ/Mb}6 films at pH 3.0 thus suggests that the film environment may provide added stability to Mb, and the heme group of Mb in the films is not lost at pH 3.0. The pK a of TCA is 0.89 (38), indicating that, at pH larger than 2, TCA exists mainly as its conjugate base species. At pH 3.0, the availability of protons may rapidly form the more easily reduced conjugate acid species of TCA to react with MbFe(II), giving a larger catalytic current than that at pH 5.5 where proton availability is lower and the conjugate acid forms more slowly. The possibility of electrochemical catalytic reduction of oxygen by {AQ/Mb}6 films was also examined by CV with air present in the external solution. When a certain volume of air was passed through a pH 5.5 buffer in a sealed cell, an increase in reduction peak at about −0.3 V was observed at {AQ/Mb}6 film electrodes, accompanied by the disappearance of the oxidation peak of MbFe(II) (Fig. 10) because MbFe(II) had reacted with oxygen. Increasing the amount of injected air would result in the increase of the reduction peak. For the AQ monolayer without Mb, the peak for direct reduction of oxygen was observed at about −0.8 V. Thus, {AQ/Mb}6 film electrodes lowered the reduction overpotential of oxygen by about 0.5 V. Catalytic efficiency, expressed as the ratio of reduction peak current of MbFe(III) in the presence (Ic ) and absence (Id ) of oxygen, Ic /Id , decreased with increasing scan rate. All these are characteristic of electrochemical catalytic reduction of oxygen by {AQ/Mb}6 films (39, 40). Catalytic CV behavior was also observed for hydrogen peroxide at {AQ/Mb}6 film electrodes (Fig. 11), which is very similar to that of the oxygen system. When H2 O2 was added to a pH 5.5 buffer, an increase in reduction peak at about −0.3 V was seen
FIG. 9. Cyclic votalmmograms at a scan rate 0.1 V s−1 in pH 3.0 buffer solutions for (a) an AQ monolayer in buffers containing no TCA, (b) an AQ monolayer in buffers containing 8 mM TCA, (c) {AQ/Mb}6 films in buffers containing no TCA, (d) {AQ/Mb}6 films in buffers containing 4 mM TCA, and (e) {AQ/Mb}6 films in buffers containing 12 mM TCA.
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LI AND HU
again and O2 . It is the production of O2 that makes the electrocatalytic CV behavior of H2 O2 similar to that of O2 at {AQ/Mb}6 film electrodes. It seems impossible that H2 O2 would undergo predisproportionation in solution when Mb is not present in the films since no signal was observed at the reduction potential of O2 at AQ monolayer film electrodes when H2 O2 was added in buffers (Fig. 11b). It was difficult to check the evolution of oxygen on the film surface during the catalysis, while vigorous degassing with N2 during the experiments caused the same results as those in Fig. 11. This indicates that the O2 produced by disproportionation of H2 O2 in the procedure is most probably from the catalysis by Mb. FIG. 10. Cyclic voltammograms at 0.2 V s−1 in 5 mL of pH 5.5 buffers for (a) an AQ monolayer in the absence of O2 , (b) an AQ monolayer after 5 mL of air was injected into the sealed cell, (c) an {AQ/Mb}6 film in the absence of O2 , (d) an {AQ/Mb}6 film after 5 mL of air was injected, and (e) an {AQ/Mb}6 film after 10 mL of air was injected.
with the disappearance of the oxidation peak for MbFe(II). The reduction peak current increased with the concentration of H2 O2 in solution. However, no direct reduction peak was observed on an AQ monolayer in the presence of H2 O2 in the studied potential range (Fig. 11b). The exact mechanism of catalytic reduction of hydrogen peroxide on {AQ/Mb}6 films is as yet unclear, but most probably similar to that of a horseradish peroxidase (HRP) film system (41) since Mb and HRP are all heme proteins and have similar electrochemical properties. It is known that, in the absence of reductant substrates and with excess hydrogen peroxide, HRP behaves like catalase, where H2 O2 acts either as an oxidant or as a reductant (42, 43). Thus, the oxidation product of Mb by H2 O2 , designated as Compound I, may be reduced by H2 O2 through a two-electron transfer pathway and produce native MbFe(III)
CONCLUSION
Negatively charged ionomer Eastman AQ55 can form ordered layer-by-layer films with positively charged myoglobin in pH 4.5 buffers by electrostatic interaction between them. An effective electron transfer rate involving a Mb heme Fe(III)/Fe(II) redox couple was greatly facilitated in the microenvironment of {AQ/Mb}n films compared with that of bare PG electrodes in Mb solutions. These layer-by-layer films had good stability, and Mb in the films essentially retained its native state at medium pH. {AQ/Mb}6 film electrodes could electrochemically catalyze reduction of trichloroacetic acid, oxygen, and hydrogen peroxide, suggesting that Mb in the films may act as a reductive enzyme for some substrates. This layer-by-layer film electrode demonstrates a promising approach to fabricate mediator-free biosensors or bioreactors. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (#29975003).
REFERENCES
FIG. 11. Cyclic voltammograms at 0.2 V s−1 in pH 5.5 buffers for (a) an AQ monolayer in buffers containing no H2 O2 , (b) an AQ monolayer in buffers containing 0.2 mM H2 O2 , (c) an {AQ/Mb}6 film in buffers containing no H2 O2 , (d) an {AQ/Mb}6 film in buffers containing 0.1 mM H2 O2 , and (e) an {AQ/Mb}6 film in buffers containing 0.2 mM H2 O2 .
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