An electrochemical biomimetic ATP-sensor

Sensors and Actuators B 104 (2005) 111–116

An electrochemical biomimetic ATP-sensor Wendelin Bücking a , Gerald A. Urban b , Thomas Nann a,∗ b

a Freiburg Materials Research Center (FMF), Stefan-Meier-Street 21, D-79104 Freiburg, Germany Institute for Microsystem Technology (IMTEK), Georges-Köhler-Allee 103, D-79110 Freiburg, Germany

Received 25 February 2004; received in revised form 19 April 2004; accepted 21 April 2004 Available online 11 June 2004

Abstract Molecular monolayers can be modified so that they mimic cell membranes. Two types of those biomimetic membranes were used in order to immobilize EF0 F1 -H+ -ATPase on a gold electrode. The work was motivated by the objective to measure the adenosin-triphosphate (ATP) concentrations by taking advantage of the ATPase activity. It was shown that one of the two forms of monolayers acted as a biosensor as the ATP concentration was directly proportional to the measured reduction current of the protons transported across the membrane by the ATPase. © 2004 Elsevier B.V. All rights reserved. Keywords: ATP-sensor; Biomimetic membrane; Amperometric sensor; ATPase; Electroanalytical chemistry

1. Introduction Adenosin-triphosphate (ATP) plays a key role in the energy turnover of the cell. It transports energy in the metabolism, and it can be seen as the energy unit in biology. Therefore, with the help of an ATP-sensor, biochemical processes can thus be studied and visualized directly. The cytoplasmatic membrane is a lipid bilayer. Generally, such membranes have proteins associated with them which may be integral (within the membrane) or peripheral (at the membrane surface). These proteins are involved in a wide range of cellular activity, e.g. in the mediated permeation of metabolites, signal transduction and immunoresponse [1]. Many of the mechanisms underlying these processes rely on the high specificity of ligand–protein binding events. This has prompted great interest in their use for the development of biosensors. Recently, many possible approaches to make use of the high specifity of such protein binding systems were performed. One could follow a “biomimetic” approach whereas a protein is placed within a phospholipid membrane or a natural membrane fragment attached to a solid support. The most straightforward approach is to attach a lipid film to a surface, adsorbed either directly on the substrate, or onto a thiopep∗ Corresponding author. Tel.: +49 761 203 4755; fax: +49 761 203 4768. E-mail address: [email protected] (T. Nann).

0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2004.04.115

tide or a self-assembled monolayer (SAM) modified surface [2–14]. The adsorbed lipid monolayers on SAMs have been examined by surface plasmon resonance, X-ray diffraction [15], impedance spectroscopy, cyclic voltammetry (CV) [16–19] and atomic force microscopy (AFM) [20,21]. The formation of lipid bilayers on SAMs showed that enzymes such as ATPase [22], bacteriorhodopsin [23], choline oxidase [24], cytochrome c oxidase [25], horseradish peroxidase [26], lipoamide dehydrogenase [27], pyruvate oxidase [28,29], tyrosinase [30] or uricase [31] could be incorporated into the lipid bilayer and retain enzymatic activity [32]. In this paper, we described the use of one of these membrane proteins, the EF0 F1 -H+ -ATPase. The EF0 F1 -H+ ATPase consists of two complexes, the F0 -complex, which is inside the membrane, and the F1 -complex, which is at the membrane surface. The EF0 F1 -H+ -ATPase catalyzes the oxidative and photosynthetic phosphorylation from adenosin-diphosphate (ADP) to ATP. The generation of ATP is driven by a transmembrane pH gradient. This process can be reversed: the ATPase transports protons actively through the membrane and hydrolyses thereby ATP to ADP [33]. The ATPases were integrated in liposomes, and these so-called proteoliposomes were added to a gold electrode, consisting of a SAM with terminal carboxygroups, which bore a phospholipid-monolayer. On this phospholipid-monolayer the liposomes were immobilized forming a phospholipid-bilayer. Electrochemical

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measurements, which exhibit the transport of protons by the ATPases through this membrane to the electrode, are presented [22]. Two different biomimetic membrane systems are investigated using CV and AFM.

2. Experimental methods 2.1. Non-covalent binding of biomimetic membranes First the gold electrodes, rinsed in caroic acid (30%-H2 O2 / H2 SO4 :3/7), were dipped for 2 h in a 5 mM ethanolic solution of 11-mercaptoundecanol. The electrodes were cleaned with ethanol and distilled water. Then they were immersed for 15 min in a suspension of liposomes and afterwards rinsed with liposome buffer. Liposomes with and without ATPases were prepared as described in ref. [34]. 2.2. Covalent binding of biomimetic membranes After rinsing in caroic acid, the gold electrodes were dipped for 2 h in a 5 mM ethanolic solution of 16-mercapto-hexadecaneacid. The electrodes were cleaned with ethanol and distilled water. Each of the SAM-layered electrodes were incubated over night in 10 ␮l diisopropylcarbodiimide (DIC), 5 ␮l ethylisopropylamine and 500 ␮l of the following solution: 5 ml dimethylformamide (DMF), 10 ml dichlormethane and 100 mg LiCl were mixed; 5 mg dimyristoylphosphatidylethanolamin (DMPE) were dissolved in 5 ml of this solution and stirred for 3 h; insoluble remains were centrifuged. After this incubation the electrodes were rinsed with ethanol and water. The liposomes were attached by immersing the electrodes for 15 min in the liposome suspension. Afterwards the electrodes were rinsed with liposome buffer.

2.3. Electrochemical setup All electrochemical measurements were made with 2 mm × 2 mm-gold electrodes, fabricated as described elsewhere [35]. A three-electrode setup was used, the reference-electrode was an Ag|AgCl-electrode. The supporting electrolyte was 0.1 M NH4 Cl. The scan rate was 0.1 V/s. For the characterization of the phosholipid membrane no additional electroactive species were used. The CVs were recorded between 0.2 and 0.3 V. For the measurement of the ATPase activity ATP concentrations between 2.5 and 60 mM were used. The voltammograms were recorded between 0.3 and −1.5 V.

3. Results and discussion In the following, the results with non-covalently and covalently bound membranes are discussed. Finally, the ATP-sensor is presented. 3.1. Non-covalently bound biomimetic membranes In the first series of experiments, a gold electrode was covered with a SAM with terminal hydroxy groups. Liposomes that contained the ATPases were non-covalently attached to this surface by hydrogen bonds. Fig. 1a shows the AFM-picture of a gold electrode, covered with a SAM of 11-mercaptoundecanol. In Fig. 1b the same SAM is represented bearing additional liposomes containing ATPases. The F1 -complex of the ATPases can be clearly seen as bright spots. Their diameter of 10 nm corresponds well with data given elsewhere [21]. This evinces very well, that the F1 -complex is on the outer side of the

Fig. 1. (a) AFM micrograph of a gold electrode, covered with a SAM of 11-mercaptoundecanol and (b) the same SAM-coated non-covalently with additional liposomes containing EF0 F1 -H+ -ATPases. The F1 -complexes of the ATPases can be clearly seen as bright points.

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membrane and thus on the top layer of the biomimetic setup at the electrode. Non-covalently bound biomimetic membranes were not used in further experiments, since they proved to be not stable. It was observed by means of AFM that the liposomes were removed after the electrodes remained for several hours in the electrolyte (these AFM pictures are not presented in this article). 3.2. Covalently bound biomimetic membranes In a second series of experiments, the gold electrode was covered with a SAM of 16-mercapto-hexadecaneacid. This SAM bore terminal carboxy groups, at which a phospholipid (DMPE) was covalently coupled forming a phospholipid monolayer. On this phospholipids monolayer liposomes were immobilised, the result was a phospholipid bilayer that was covalently bound to the gold electrode. The liposomes contained the ATPases, which thus are embedded in the artificial membrane. Fig. 2 shows schematically the biomimetic model system chosen. 3.3. Characterization by cyclic voltammetry Using cyclic voltammetry the building-up of the phospholipid membranes could be confirmed. The membrane served as dielectricum in a model condensator, the capacitance which was evaluated using the charge/discharge characteristics represented by CV diagrams. With increasing voltage the membrane is charged as a condensator by the anodic charging current. Therefore, from the CV the capacitance of the membrane can be calculated using

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the formula I C= 2Av

(1)

where C is the “capacitance” of the membrane, I the difference between anodic and cathodic charging current, A the surface of the electrode: 0.5 mm2 , and v the scan rate [22]. There are significant differences in the calculated capacitance of the layers between the consecutive steps of the membrane forming process. Adding the DMPE-layer results in a clear capacitance increase from 60 to 2.8 ␮F/cm2 . Adding the liposomes decreases the capacitance again distinctly to 500 nF/cm2 . We interpret this as a sign for the hydrophobicity of the layer surface; whereas the 16-mercapto-hexadecaneacid and the phospholipid bilayer have a hydrophilic polar surface, the lipid monolayer is hydrophobic. Table 1 summarizes the results. 3.4. ATP-sensor ATP-sensors were built on the basis of covalently bound biomimetic membranes as described above. The liposomes used in this setup contained the ATPases, which were thus immobilized in an artificial phospholipid environment on the electrode. As can be seen in the AFM pictures described above, the F1 -complex of the ATPase was on the top layer of the biomimetic setup. This results in an active transport of the protons through the membrane towards the electrode by the ATPase, whereby ATP is hydrolized. The protons were electrochemically reduced at the electrode according to the formula 2 H+ + 2e− ↔ H2 This reduction of the protons at the electrode is pH-dependent and the reduction potential E of the protons must be more cathodic with increasing pH in accordance with E = −0.059 V × pH. For the buffered system (100 mM NH4 Cl, pH 5.1) used in this study, the reduction peak of the protons should be measured at −0.1 V versus Ag|AgCl (EAg|AgCl = −0.22 V versus NHE). It is conspicuous that the reduction potential was found at approximately −1.1 V for all of our measurements (cf. Fig. 3). This effect can be explained by a decrease in pH at the electrode surface. The decrease is caused by a Table 1 Calculated capacitance of monomolecular and biomimetic layers according to Eq. (1) SAM

C (nF/cm2 )

Fig. 2. Schematic setup of biomimetic membrane covalently bound to a gold electrode.

16-mercapto-hexadecaneacid 16-mercapto-hexadecaneacid with DMPE (␮F/cm2 ) 16-mercapto-hexadecaneacid with DMPE and liposomes (nF/cm2 )

60 2.8 500

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As the electrolyte was buffered with 100 mM NH4 Cl and no change in the pH was considered, the pump capacity of the ATPase should therefore exclusively depend on the available amount of ATP in the electrolyte. The theoretical current density of the proton reduction at the electrode surface can be estimated in this way [12]: the maximum velocity of the ATP hydrolysis is v(ATP) = 100 mol ATP/ATPase for 1 mol ATP. Since the hydrolysis of one ATP transports four protons [34], the current density can be calculated I = 4v(ATP)ze

Fig. 3. Voltammograms showing the increasing reduction current of the protons at the electrode for different concentrations of ATP in the electrolyte. The supporting electrolyte was 0.1 M NH4 Cl. The covalently bound biomimetic membrane containing ATPases was used as described in the text.

fast proton reduction compared to the proton transport from the ATPase (in other words: the reduction rate exceeds the transport rate). The peak value of the reduction wave, which represents a Faradaic current, was directly linearly correlated to the proton concentration at the electrode surface. Fig. 4 depicts the reference experiments, whereas the membrane consisted of the same layers with the only difference that the liposomes contained no ATPases. An offset current, probably caused by protons diffusing through the membrane, was observed in these measurement. Oxygen reduction could not be observed since the SAM covered the electrode, and no oxygen could diffuse to the gold surface.

Fig. 4. Voltammograms taken with a biomimetic electrode, containing no ATPase at different ATP concentrations. The supporting electrolyte was 0.1 M NH4 Cl.

n(ATPase) A

with z = 1 as number of the electrons reduced per proton, e = 1.6 × 10−19 . As as elementary charge, and n(ATPase)/A the amount of ATPases per area, whereas an average value of 1.5 × 1010 cm−2 was considered. Therefore, the resulting current density should be 1 ␮A/cm2 for 1 mol ATP. Comparing this value with our results leads to the conclusion that the ATPase density on the electrode should be much higher than it is. On the other hand, a linear dependency of the reduction current density on the ATP concentration can be observed in Fig. 5—the same effect was found by other groups [22]. Future investigations should reveal the origin of this unusual increase in current. Furthermore, Fig. 5 shows, that the sensor is highly sensitive against the ATP stimulus. The sensitivity was determined to 50 ␮A mM−1 cm−2 . Biological cross-sensitivity could not be observed since the membrane enzymes are very attuned for their substrates. In Fig. 3, the voltammograms of aqueous solutions with increasing ATP concentration of an ATPase active electrode are presented. We used ATP concentrations between 2.5 and 60 mM, the physiological range is between 2 and 10 mM [33]. Therefore, this sensor is suitable for the use

Fig. 5. Plot of the peak currents from Fig. 4 vs. the ATP concentration in the electrolyte. (a) Squares (䊏) represent that the sensor is highly sensitive against the ATP concentration in the electrolyte and (b) triangles (䉱) represent ATPase-free electrode.

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in biological systems and new interesting metabolism processes can thus be studied.

4. Conclusion Fig. 5 shows two imposing results: on the one hand, the increase of the reduction current is definitely based on the activity of the ATPase, since the ATPase-free electrode shows only a small reduction current. On the other hand, there is a linear correlation between the peak-current and the ATP concentration in the concentration range studied. The sensor is highly sensitive for ATP, since membrane enzymes are very sensitive for their substrates and no cross-sensitivity was observed. A method was developed that allows an easy immobilization of membrane enzymes by conservation of their activity. On the basis of these experiments, new sensors could be developed with other transmembrane proteins.

Acknowledgements We thank Prof. P. Gräber (Institute for Physical Chemistry, Freiburg University) for providing us the EF0 F1 -H+ -ATPase integrated in liposomes and for interesting discussions.

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