ARTICLE pubs.acs.org/Langmuir
Cytochrome c/Polyelectrolyte Multilayers Investigated by E-QCM-D: Effect of Temperature on the Assembly Structure Christian Kepplinger,†,‡ Fred Lisdat,*,† and Ulla Wollenberger*,‡ † ‡
Biosystems Technology, Wildau University of Applied Science, Bahnhofstrasse 1, 15745 Wildau, Germany Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany ABSTRACT: Protein multilayers, consisting of cytochrome c (cyt c) and poly(aniline sulfonic acid) (PASA), are investigated by electrochemical quartz crystal microbalance with dissipation monitoring (E-QCM-D). This technique reveals that a four-bilayer assembly has rather rigid properties. A thickness of 16.3 ( 0.8 nm is calculated with the Sauerbrey equation and is found to be in good agreement with a viscoelastic model. The electroactive amount of cyt c is estimated by the deposited mass under the assumption of 50% coupled water. Temperature-induced stabilization of the multilayer assembly has been investigated in the temperature range between 30 and 45 °C. The treatment results in a loss of material and a contraction of the film. The electroactive amount of cyt c also decreases during heating and remains constant after the cooling period. The contraction of the film is accompanied by the enhanced stability of the assembly. In addition, it is found that cyt c and PASA can be assembled at higher temperatures, resulting in the formation of multilayer systems with less dissipation.
’ INTRODUCTION Polyelectrolyte multilayers (PMs) on surfaces have evolved into promising tools in biorecognition, biosensing, and bioelectronics over the last few years.15 Introduced by Decher and co-workers,6,7 the fast progress in fundamental research of PM turns out to be a solid base for biomaterial deposition and biocompatibility on surfaces for biotechnological application. PMs have also been used to create architectures that include electroactive biomolecules in order to build sensing devices based on electrochemistry.8,9 Signal transfer between proteins in redox chains plays an important role in living cells.10,11 Generally, it is of great interest to transform these complex biochemical reactions into artificial assemblies mimicking natural pathways, not only for basic research on redox processes and catalysis12 but also for sensors and energy conversion.13 One limitation of current sensing systems is given by the amount of immobilized protein on the electrode. Therefore, architectures including polyelectrolytes and proteins were fabricated in order to increase the amount of protein and to detect enzyme substrates on electrodes.1416 Beyond that, emphasis was placed on direct electron transfer from heme proteins to the electrode.17 The restriction of the inefficient electric communication of redox proteins with the electrode at a certain layer number18,19 was solved by alternating the layer-by-layer deposition of cytochrome c (cyt c) and weak anion poly(aniline sulfonic acid) (PASA). In this way, communication between layers becomes feasible.20 In previous reports, these multilayered films were used for the detection of superoxide anion radicals. Studies on protein multilayers have shown that with an increasing number of layers the r 2011 American Chemical Society
electroactive amount of cyt c on the electrode increases and that a temperature treatment of 45 °C leads to the stabilization of the multilayer.20 An analysis of the multilayer formation process by surface plasmon resonance (SPR), quartz crystal microbalance (QCM), and electrochemical measurements revealed information about the net mass transfer to the surface and the amount of electroactive cyt c, but morphological parameters could not be exactly determined.2022 Further structural information can be gained by means of electrochemical quartz crystal microbalance with dissipation monitoring (E-QCM-D) because the conventional QCM technique does not take the nature of the deposited material into account and gives only information about the net mass load.2326 For PM, the QCM-D technique has been used so far mostly to follow the assembly of PM in real time, to monitor structural changes of PM with increasing ionic strength of the bulk solution or to alter other parameters such as the concentration of the species.5,2729 The QCM-D technique is suitable for adsorption studies of biomolecules on a piezoelectric quartz crystal.30,31 It allows the simultaneous measurements of the layer resonance frequency, f, and dissipation, D. Two cases can be distinguished. In the first case, when the film is rigid, it does not slip on the surface and has negligible internal friction, and the adsorbed mass (Δm) can be derived from the Sauerbrey relation. Received: March 7, 2011 Revised: May 13, 2011 Published: June 02, 2011 8309
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In the second case, a soft film that does not fully couple to the crystal will be deformed and cause internal energy loss during the shear oscillatory motion. Simultaneous Δf and ΔD measurements are made while switching off the driving voltage of the resonator periodically and measuring the exponentially decaying signal. For the deposition of a soft film, D will increase and data are derived using a Voigt Kelvin-based viscoelastic model.32 To transform the dissipation into viscous and elastic features, several overtones of the fundamental frequency are required. To date, only a few papers have dealt with in situ E-QCM-D related to biosensing applications.3,3335 This article reports on the combination of simultaneous electrochemical measurement and mass adsorption that leads to deeper insight into the multilayer assembly of electroactive cytochrome c and sulfonated polyaniline. Besides that, temperature-induced rearrangements of the multilayer structure are discussed.
’ EXPERIMENTAL SECTION Materials. Horse-heart cytochrome c (cyt c; pI 10 10, 5 ), 11mercapto-1-undecanoic acid (MUA), 11-mercapto-1-undecanol (MU), and poly(aniline sulfonic acid) (PASA) were purchased from SigmaAldrich (Taufkirchen, Germany). All other chemicals were of the highest purity available. Water was received from a Milli-Q-system (Eschborn, Germany) with a resistance of 18 MΩ cm. Buffers. Potassium phosphate buffer was prepared from dipotassium phosphate and potassium diphosphate, with the pH adjusted with potassium hydroxide or phosphoric acid, respectively. Buffer 1 (5 mM potassium phosphate, pH 5.0) was used for the preparation of the cyt c and PASA solutions and for the immobilization of the cyt c monolayer. Buffer 2 (5 mM potassium phosphate, pH 7.0) was used for rinsing after the multilayer assembly and for all electrochemical measurements because the redox transformation of cyt c in the multilayers at pH 5.0 is not reversible. QCM-D Measurements. Measurements were made using a Q-Sense E4-instrument (Q-Sense, AB, G€oteborg, Sweden). As substrates, polished AT-cut piezoelectric quartz crystals with gold electrodes with a fundamental frequency of 5 MHz were used (Q-Sense, AB). Prior to use, crystals were immersed in a mixture of ammonia (25%), hydrogen peroxide (30%), and Milli-Q water (1:1:5), held at 70 °C for 10 min, rinsed extensively with Milli-Q water, and dried under a stream of nitrogen. Then the crystals were exposed to UV/ozone treatment for 20 min using a UV/ozone Pro Cleaner (BioForce Nanosciences, Ames, IA, USA). The sensors were modified with a mixture of 5 mM MUA and 5 mM MU with a volume ratio of 1:3 for 24 h at 4 °C.8,22 Then, the modified crystals were carefully rinsed with Milli-Q water, dried in a stream of nitrogen, and stored at 4 °C until used. Multilayer Electrode Preparation. The sensor was mounted inside the thermostatted flow cell, and 5 mM potassium phosphate (pH 5.0) was passed over the surface at a flow rate of 100 μL min 1. After a stable reading of the baseline, a cyt c solution (20 μM) was pumped over the modified surface until equilibrium was reached. Then buffer was again injected, followed by the injection of PASA (0.2 mg mL 1) and cyt c for 5 min with a 5 min buffer rinse in between each injection. After the buildup process, the multilayer was finally rinsed with pH 7.0 phosphate buffer for 5 min and the pump was stopped. For temperature experiments, the multilayer was heated after assembly to the final temperature for 30 min and then cooled to RT. Cyclic voltammetry (CV) scans were taken before and after the heating period and after the cooling period. In addition, multilayer formation was investigated at elevated temperatures. For this purpose, the quartz crystal and the solutions were preheated. The same procedure as for the room-temperature assembly of cyt c and PASA was used. Electrochemical Measurements. An electrochemical flow cell (Q-EM 401 module, Q-Sense, Sweden) was used for all measurements. 45
Figure 1. Time course of frequency (left) and dissipation during the assembly of [cyt c-(PASA/cyt c)4] on MUA/MU-modified Au by the addition of (A, C) 20 μM cyt c and (B) 0.2 mg mL 1 PASA and 5 mM potassium phosphate at pH 5.0 for rinsing. A represents cyt c monolayer formation. The frequency decreased (left axis) whereas the dissipation increased (right axis). Thereby the gold layer itself acts as a working electrode. A platinum foil, mounted on the upper side of the outlet tube, serves as a counter electrode. An Ag/AgCl 1 M KCl reference electrode is connected to the upper side of the flow cell. All cyclic voltammetric (CV) experiments were performed using a Gamry Instruments Reference 600 potentiostat/galvanostat (C3 Prozess- and Analysentechnik, Munich, Germany) and Gamry Framework Software. CV was performed using scan rates of between 0.1 and 3 V s 1. Phosphate buffer at pH 7.0 was used for all electrochemical experiments.
’ RESULTS AND DISCUSSION The main focus of this study is the characterization of cyt c-PASA multilayer films (hence denoted [cyt c-(PASA/cyt c)n]) by means of E-QCM-D. First, the formation of the multilayer was briefly followed and interpreted in terms of rigid or viscoelastic properties. Then, the electrochemical parameters were determined. Particular attention centered on the effect of different temperatures on the structural and functional properties. Multilayer Assembly Investigated by QCM-D. The frequency (Δf) and dissipation changes (ΔD) for three independent measurements of a four-layer electrode [cyt c-(PASA(cyt c)4] obtained at overtone numbers of n = 3 (15 MHz), n = 5 (25 MHz), and n = 7 (35 MHz) were recorded as a function of time. Figure 1 shows the Δf and ΔD shifts of the third overtone. The frequency decreased after each adsorption step and reached a maximum level of Δffin = 125 ( 6 Hz, which means that the total immobilized mass increased. The dissipation increased to a final value of ΔDfin = (4.1 ( 0.6) 10 6. The data for the three overtones are listed in Table 1. After each deposition of cyt c, the dissipation increased slightly. A buffer rinse removed excess cyt c molecules, and the dissipation decreased accordingly. In contrast, additional PASA after cyt c increased the dissipative, soft characteristics of the multilayer film. PASA is a weak polyanion, and counterions from solution may enter the polymer network, bringing in additional water molecules. Moreover, salt is known to contribute to an increase in the thickness of the multilayers.3638 Although we use only 5 mM phosphate buffer, this effect could contribute to a 8310
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Table 1. Frequency and Dissipation Shifts of the Third, Fifth, And Seventh Overtone for a Four-Layer Assembly of cyt c and PASA on cyt c Adsorbed to MU- and MUA-Modified Gold (Quartz Crystals)a
a
n (overtone)
Δf (Hz)
ΔD
mass (ng cm 2)
3
125.4 ( 6.2
(4.1 ( 0.6) 10 6
2219 ( 109
5
121.5 ( 6.5
(4.3 ( 1.1) 10 6
2151 ( 116
7
118.5 ( 6.7
(4.2 ( 1.1) 10 6
2089 ( 122
Δf changes were converted to mass.
Figure 2. Cyclic voltammograms showing oxidation and reduction peaks of cyt c in multilayers on thiol modified (Au/MU,MUA). Increasing order: [cyt c-(PASA/cyt c)4] and [cyt c-(PASA/cyt c)6]. (E vs Ag/AgCl, 1 M KCl, scan rate 100 mV s 1). Measurements performed in 5 mM phosphate buffer, pH 7.0.
small increase of mass after the addition of PASA and the buffer rinse. In addition, even the buffer change from pH 5 (for assembly) to pH 7 (for the electrochemical measurements) first caused a further decrease in Δf and an increase in ΔD for the multilayer (data not shown), followed by a rapid increase in Δf and a decrease in ΔD. Both values equilibrated slowly and reached nearly the same values as before. This indicates an equilibration in the new buffer solution. The total frequency shift of three independent measurements converted to mass by the Sauerbrey relation results in 2220 ( 110 ng cm 2 and a thickness of 16.3 ( 0.8 nm. The Δf to mass conversions for three investigated overtones are found in Table 1. It is obvious that the three overtones give rather similar results. By fitting the third, fifth, and seventh harmonics to a Voigt Kelvin viscoelastic model32 and taking a fixed density of 1.35 g cm 3 of cyt c as an input parameter,39 the thickness is estimated to be 17.3 ( 0.8 nm. Thus, it can be argued that the Sauerbrey relation is valid in the applied regime. The viscosity of the multilayer can be determined from the viscoelastic analysis. It is 6 ( 1 mPa s, indicating a less hydrated, more rigid structure compared to the viscosity of water (∼1 mPa s). Because of the correlation between high water content and low viscosity, this value can be used as a rough estimate of bound water, as has been shown by Larrson et al.39 From a combined SPR/QCM-D measurement, these authors also determined the amount of bound water for a protein layer (streptavidin) and a polyelectrolyte layer (DNA). They have found that the protein layer consist of 50% coupled water (still rigid properties)
whereas the water content of a DNA layer was much higher at about 90% (soft properties).39 With respect to the rigid properties of the protein polyelectrolyte system investigated here, a water content of not more than 50% can be assumed. Another indication of the presence of a compact structure of the multilayer might be the small difference between Δf and ΔD values at the three analyzed harmonics, respectively. From the data it is thus concluded that the Sauerbrey model is valid to be used in this regime. Obviously the small amount of surface bound water causes only a small viscous energy loss in the system. Electrochemical Characterization of the Multilayer Assembly. Electrochemical measurements were executed in buffer adjusted to pH 7, because it has been shown that peak separation, connected with the rate of electron transfer, is pH-dependent and shows a minimum at pH 7.20,44 At pH 5.0 the redox behavior of cytochrome c in the multilayer is irreversible (not shown). CV measurements reveal an increase in oxidation and reduction peak currents with growing layer number. Figure 2 shows a 4 and 6-layer electrode of cyt c and PASA corresponding to an enlargement of the amount of electroactive cyt c. The amount of a 4-layer electrode is found to be 49 ( 9 pmol cm 2 and 74 ( 14 pmol cm 2 for a 6-layer electrode, values that fit approximately to previously reported data,20,40 where less mass deposition on flat electrodes compared to needle electrodes was observed. Dronov et al. attributed the lower immobilization of mass to the difference in microroughness of these two surfaces.40 In addition the investigations are performed here in a flow system where lateral forces, induced by the buffer stream, could modify the layer assembly process. The formal potential Ef of [cyt c-(PASA/cyt c)4] is 20 ( 6 mV vs Ag/AgCl/ 1 M KCl (23 °C), which is slightly more positive then previously investigated.20 In order to correlate the deposited mass with the electroactive cyt c amount the cyt c adsorption during the assembly formation was evaluated. From the total mass increase of a 4-layer and 6-layer electrode a cyt c amount of 85 ( 12 pmol cm 2 and 152 ( 56 pmol cm 2 respectively can be calculated. However, if one assumes that approximately up to 50% of the detected mass increase is attributed to coupled water,21 these data correlate within the experimental error - with the amount of electrodeaddressable cyt.c. This result is also supported by a previous study where dried cytc/PASA assemblies with a different number of layers have been evaluated and a linear correlation between the cyt c mass and the electroactive protein amount was found.21 Influence of Temperature on the Multilayer Structure. In a previous report an increase of stability of multilayer electrodes after a temperature treatment at 45 °C for 30 min has been found.20,8 Thus, after overnight storage or repeated amperometric measurements no protein loss was detectable in contrast to untreated assemblies. The high stability was explained by structural changes within the assembly. Grochol et al. investigated multilayers by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and electrodes were also incubated at 45 °C. The data indicated a rearrangement of the polyelectrolyte network enhancing the interaction of both building blocks.41 In the study here, first a MUA-MU modified gold sensor was heated 45 °C for 30 min. (data not shown). This experiment was done in order to follow temperature-induced variations of the behavior of the thiol-modified resonator itself. f increased and D decreased during the increment of the temperature to 45 °C. This observation can be related to the variation of the viscosities and densities of the film and the bulk liquid (buffer) with increased temperature.42 Both baselines equilibrate after a short time period. 8311
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Figure 3. (A) cyt c monolayer (A), heated to 40 °C (B), and cooled to 23 °C (C). The Δf and ΔD values reach the same level as before when the temperature was increased. No cyt c molecules were desorbed from the surface. (B) Voltammograms of cyt c monolayer (Au/MU,MUA, cyt c) electrodes at 23 °C after heating to 40 °C and cooling to 23 °C again (E vs Ag/AgCl, 1 M KCl, v = 100 mV/s).
Table 2. Electrochemical Parameters of a cyt c Monolayer on MU- and MUA-Modified Gold (on Quartz Crystal), Incubated at 23 °C, Heated to 40 °C, and Cooled to 23°C Again surface density temperature
(pmol cm 2)
Ef (mV)
ΔEp (mV)
23 °C
8 ( 1.5
18 ( 5
37 ( 21
40 °C
9 ( 0.6
7 ( 1.4
56 ( 18
23 °C
8(1
17 ( 7.4
37 ( 7
When the sensor was cooled to 23 °C, f and D switched back to its original baselines which were nearly the same as before. Afterwards, a cyt c monolayer was assembled on the electrode at 23 °C (Figure 3a), and the frequency and dissipation were measured. Then the layer was heated to 40 °C and cooled to 23 °C. The final Δf and ΔD values reached the same level as the initial values after the cyt c deposition, indicating that no cyt c molecules were removed from the surface by the treatment. Figure 3b shows CV scans of a monolayer at three temperatures (23, 40, and 23 °C). The data demonstrate that the electroactive
Figure 4. (A) Assembly of [cyt c-(PASA/cyt c)4] on MU- and MUAmodified quartz crystals. The multilayer was heated to 45 °C and cooled to 23 °C. A C denote the assembly at 23 °C, heating to 45 °C, and annealing at 23 °C, respectively. CVs were taken at the end of each temperature step. (B) CV scans of [cyt c-(PASA/cyt c)4] at three different states, as described in part A. A C denote 23, 45, and 23 °C, respectively.
amount of cyt c remains unchanged on the electrode. Obviously, the protein retains its native structure and flexibility to a large extent (Table 2). It is noteworthy to mention that ΔEp increased at 40 °C, indicating changing rate of electron transport through the system. To analyze the kinetics of electron transfer at the three temperatures, the scan rate was varied. With increasing scan rate, the peak separation grows. Values for ks of 33 ( 9 s 1 at 23 °C, 65 ( 17 s 1 at 40 °C, and 44 ( 4 s 1 at 23 °C were calculated (n = 2) by the method of Laviron.43 The higher temperature resulted in a higher ks, but upon cooling, the initial value at 23 °C was not reached. To gain further insight into the system, multilayer assemblies were investigated by QCM-D and CV at different temperatures (30, 35, 40, and 45 °C). The buildup occurred at room temperature (23 ( 0.1 °C), and afterwards the temperature was raised. The temperature was held constant for 30 min, and the assembly was then cooled to 23 °C. CV scans were performed at 23 °C after incubation at higher temperature and after the cooling period. Figure 4a shows the procedure for a 45 °C treatment. Letters indicate the three points in time where CV scans were performed. 8312
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Table 3. Δf and ΔD Shifts of the Third Overtone and Corresponding Masses for the Assembly of [cyt c-(PASA/cyt c)4] on MUand MUA-Modified Quarz Crystals Prior to Temperature Treatment and after Cooling to RT Δf (Hz) after n 3
ΔD after
3
n 3
cooling
ΔD before
cooling
before
after cooling
T = 30 °C
to RT
T = 30 °C
to RT
T = 30 °C
to RT
2382 ( 493
1827 ( 386
134.6 ( 21.8
103.2 ( 27.9
4 ( 2.6 10
6
3.3 ( 1.2 10
3
6
ΔD after
mass
mass
(ng cm 2)
(ng cm 2)
Δf (Hz) before
cooling
ΔD before
cooling
before
after cooling
T = 35 °C
to RT
T = 35 °C
to RT
T = 35 °C
to RT
2896 ( 592
1954 ( 542
163.6 ( 33.5
109.9 ( 30.6
Δf (Hz) before
Δf (Hz) after cooling
ΔD before
T = 40 °C
to RT
T = 40 °C
167.1 ( 31.3
107 ( 30
3.6 ( 2 10
6
3.5 ( 1.6 10
6
ΔD after cooling to RT
4.6 ( 0.7 10
6
3.1 ( 0.6 10
Δf (Hz) after n
mass (ng cm 2)
Δf (Hz) before
Δf (Hz) after n
mass (ng cm 2)
6
ΔD after
mass
mass
(ng cm 2) before
(ng cm 2) after cooling
T = 40 °C
to RT
2957 ( 554
1896 ( 527
Mass
Mass
(ng cm 2)
(ng cm 2)
Δf (Hz) before
cooling
ΔD before
cooling
before
after cooling
T = 45 °C
to RT
T = 45 °C
to RT
T = 45 °C
to RT
2609 ( 153
1443 ( 199
147.4 ( 8.7
81.5 ( 11.2
5.1 ( 0.8 10
At all investigated temperatures, the frequency has increased after the cooling period, indicating some desorption of material from the surface. At 30 and 35 °C, the ΔD values are approximately the same before and after heating, indicating no changes in the composition of the film. Heating to 40 °C clearly induces a larger decrease in ΔD than observed before. This tendency is further continued at 45 °C, where the decrease in ΔD is at its highest level. Corresponding data of the third overtone of all temperature steps are listed in Table 3. These findings may lead to the conclusion that a thermal treatment of at least 40 °C contributes to a rearrangement of the film. This reorientation of the film is obviously attributed to the desorption of material and/or coupled water from the surface. For example, after a 45 °C treatment the “new” mass, obtained from the third overtone, is calculated to be 1443 ( 199 ng cm 2. In other words, 45% of the original deposited mass is lost and/or coupled water is released. As mentioned before, f increases and D decreases when the temperature rises because of changes in the viscosity and density of the film and the bulk fluid. In classical QCM-D operation, increasing positive frequency values indicate mass desorption from the crystal surface. At the blank chip, increasing temperature induces converse shifts in frequency and dissipation. However, the frequency and dissipation changes are larger for the quartz crystals with multilayer assemblies compared to crystals without them. Hence, these findings indicate a rearrangement of the multilayer building blocks, as has been observed before by SERR studies and electrode stability investigations.20,41 Effect of Temperature Treatment on Electrochemical Parameters. The CV scans in Figure 4b show a clear decrease in the electroactive amount of cyt c for an assembly heated to 45 °C This value does not change significantly after returning to 23 °C.
6
3.2 ( 1.4 10
6
Table 4. Electrochemical Parameters of [cyt c-(PASA/cyt c)4] Assemblies on MU- and MUA-Modified Gold Obtained from the CV Scans at Three States as Shown in Figure 4A C, Denoted by RT, Heating, and Cooling to RT T = 30 °C
surface density (pmol cm 2)
A
54 ( 10
21 ( 0.9
51 ( 12
B
40 ( 7
15 ( 2
38 ( 6
C
38 ( 12
20 ( 1
37 ( 5
T = 35 °C
surface density (pmol cm
2
)
Ef (mV)
ΔEp (mV)
Ef (mV)
ΔEp (mV)
A
61 ( 12
20 ( 7
45 ( 20
B
31 ( 10
3(9
45 ( 9
C
32 ( 11
16 ( 17
35 ( 14
T = 40 °C
surface density (pmol cm 2)
Ef (mV)
ΔEp (mV)
A
64 ( 20
22 ( 11
47 ( 25
B
38 ( 13
6(5
39 ( 16
C
42 ( 11
18 ( 9
36 ( 21
Ef (mV)
ΔEp (mV) 44 ( 29
T = 45 °C
surface density (pmol cm
2
)
A
51 ( 6
12 ( 7
B
31 ( 4
2 ( 16
41 ( 15
C
27 ( 6
18 ( 2
34 ( 18
This result supports the idea that heating the film leads to a structural change in the multilayer. Besides protein desorption, some cyt c molecules could also change their orientation, resulting in less electrode-addressable protein. This scenario would also contribute to a decrease in the electroactive amount that has been observed at all investigated temperatures. 8313
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Table 5. Electrochemical Parameters of [cyt c-(PASA/cyt c)4] Assemblies on MU- and MUA-Modified Gold Obtained from the CV Scans of the Multilayers at Elevated Temperatures and after Cooling to RT temperature
Figure 5. Assembly of [cyt c-(PASA/cyt c)4] on MU- and MUA-modified gold at 40 °C. (A) cyt c, (B) PASA, and (C) cyt c.
The electrochemical data of assemblies treated at different temperatures are listed in Table 4. Interestingly, Ef is between 10 and þ30 mV vs Ag/AgCl and always slightly smaller at higher temperatures than at room temperature, in accordance with other studies.44 The peak separation ΔEp becomes smaller (i.e., electrons from cyt c are transferred faster to the electrode). Unfortunately, it is not possible to determine ks exactly in the multilayer by voltammetric measurements because at higher scan rates not all cyt c molecules can take part in the redox conversion. Thus, the amount of electrodeaddressable cyt c is decreasing. This is not connected to a further loss of molecules because at slower scan rates all of the cyt c molecules can be detected again. To investigate the stability of the mulitlayer by heat treatment, repetitive CV scans were performed at 23 °C after a heating step at 40 °C and a cooling period. A stable signal has been obtained during the electrochemical measurement in contrast to that for an unheated multilayer film. The formal potential Ef of cyt c is 21 ( 3 mV, and the peak separation ΔEp is 28 ( 6 mV. The electroactive amount of cyt c remained constant during repetitive CV, that is, 25 pmol cm 2. Multilayer Assembly at Elevated Temperatures. In a second approach, we want to know if it would be possible to assemble a multilayer on a preheated sensor. Experiments were performed at the same temperatures formerly investigated. For all temperatures, the frequency decreased during the adsorption steps of cyt c and PASA in the same way as happens at room temperature, but the dissipation increased less (Figure 5). Generally, this shows that the formation of protein multilayer films is also possible at higher temperatures. The higher the temperature at the beginning of the assembly (up to 45 °C), the less the dissipation increased. During the cooling period to room temperature, the frequency decreased and the dissipation increased. This tendency thus corresponds to the behavior in the cooling period of the monolayers and multilayers that were heated after preparation. Table 5 shows resulting data from multilayer electrodes assembled at all temperatures. The measurements indicate that the assembly formation at higher temperature generally leads to a smaller amount of electroactive protein compared to assembly at room temperature; however, CV scans revealed approximately
surface density (pmol cm 2)
Ef (mV)
ΔEp (mV)
30 °C
53 ( 15
8(3
34 ( 3
RT
40 ( 14
14 ( 2
34 ( 14
35 °C
44 ( 15
4 ( 13
29 ( 11
RT
33 ( 10
19 ( 13
31 ( 6
40 °C
41
0.5
49
RT
30
3
40
45 °C RT
39 39
4 24
33 28
the same properties as measured for systems that have been assembled first and heat treated afterwards. It has been also analyzed whether the stability of the assembly is improved when the assembly process is performed at 45 °C compared to assemblies at room temperature. After the preparation and a waiting time of 1 hour (or a cooling time), electrodes are analyzed with respect to the peak height in repetitive cyclic voltammograms. Whereas the cytc/PASA system assembled at room temperature shows a decrease of 30% in peak current after 50 consecutive scans, the assemblies prepared at higher temperature still have 90% of the original value.
’ CONCLUSIONS In this study, we investigated the formation process of polyelectrolyte protein multilayers using E-QCM-D. By comparing the data analysis with the Sauerbrey model to the viscoelastic model, it is shown that cyt c/PASA assemblies, consisting of four bilayers, have still rigid features. CV measurements reveal an increase in the electroactive amount of cyt c by the multilayer buildup. A comparison of the deposited amount of protein and the determined electroactive amount supports the idea of a coupled water content of about 50%. Experiments with varying temperature enable the investigation of structural changes within the multilayer during the heating period. These changes are related to the loss of mass and a rearrangement of the film structure when heated to more then 40 °C. These characteristics also lead to higher stability of the film after heating, an idea that is confirmed with CV measurements. The multilayer buildup at elevated temperatures is also examined, and the results show that it is possible to assemble active multilayers up to 45 °C. The resulting properties are comparable to features of assemblies from heat treatment after preparation. This assembly platform can be extended to more sophisticated architectures, including other redox-active proteins and polyelectrolytes, where the combination of immobilized biomolecules and their catalytic and electrochemical conversion can provide the impetus for sensor applications and also insights into biological signal chains.
’ ACKNOWLEDGMENT This work was supported by the Ministerium f€ur Wissenschaft, Forschung und Kultur, Brandenburg, Germany (project 3508-14/13). We also acknowledge support from BMBF (IZIB 03IS2201B) and DFG (Unicat Cluster of Excellence, EXC 314). 8314
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dx.doi.org/10.1021/la200860p |Langmuir 2011, 27, 8309–8315
