Electrochemical impedance measurements for the investigation of odorants interaction with thiol layer immobilized onto gold electrode

Sensors and Actuators B 75 (2001) 95±100

Electrochemical impedance measurements for the investigation of odorants interaction with thiol layer immobilized onto gold electrode Iwona SzymanÂska, Hanna Radecka, Jerzy Radecki* Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Division of Food Science, Tuwima 10, 10-747 Olsztyn, Poland Received 29 August 2000; received in revised form 06 December 2000; accepted 18 December 2000

Abstract The impedance measurements were used for the detection of the interaction of smell compounds with thiol layer deposited onto gold electrode. The following compounds were selected for the study: geraniol, coumarin, menthol, 1-octanol, b-ionone. The changes of the resistance and capacitance of the thiol layer, occurred under stimulation by odorants, calculated on the base of impedance measurements, were correlated with other parameters describing physico-chemical properties (partition coef®cients log P) and topological one such as total surface area (TSA) and van der Waals' volume (VDW). # 2001 Elsevier Science B.V. All rights reserved. Keywords: Impedance; Gold electrode modi®ed by thiol layers; Odorants

1. Introduction The sensitive and selective detection of molecular species within complicated matrices without sample preparation is the analytical goal of the bio- and biomimetic-sensors. A good model for such kind of analysis is the molecular recognition process occurring on the cell membrane surfaces. These processes are responsible for the regulation of the majority of biochemical reactions running in the living organisms. During the ®rst step of the molecular recognition phenomenon, the interaction between the receptor and target molecule is taking place. As the consequence, the same kind of complex is formed which is assisted by the change of the free energy. This change could be taken as an analytical signal and its sensitivity and selectivity will be governed by the chemistry of the observed event. Usually, the changes of the energy associated with each binding event are relatively small. This means that for the generation of the analytically useful signal many of them are necessary [1,2]. The second step involves the change of the physicochemical structure of the matrix in which the phenomenon of molecular recognition occurs. As the consequence, the secondary signal is observed. Its sensitivity is not limited by the chemistry, it depends rather on how much the * Corresponding author. Tel.: ‡48-89-523-4612; fax: ‡48-89-524-0124. E-mail address: [email protected] (J. Radecki).

environment of the matrix is altered by the recognition reaction. This ampli®cation phenomenon allows observing the results of relatively small number of binding events. The ion channel mimic sensors [3,4] are the representative examples. Recently, the biomimetic taste and odor sensing systems based on physico-chemical adsorption of target molecules on the polymer or biomembranes have been proposed by many investigators [5±12]. Continuing our research on the interaction of the neutral odorant compounds with the planar lipid bilayer [13] and supported BLMs [14], in this paper we presented the results on the attempt of the application of the electrochemical impedance measurements to investigate of the above phenomenon. The gold electrodes modi®ed by thiols were used as a sensory element of the sensing device investigated. The working principle of the sensor under study was as follows. The adsorption of the lipophilic odorant compounds in the hydrophobic thiol layer attached onto the gold electrode by covalent bounds, could changed its density and thickness. This in¯uenced on the resistance and the electrical capacitance of the electrode investigated. The dimension of these changes was related to the chemical structure of adsorbed compounds. The electrochemical impedance measurements have been used to study these phenomena. This technique allows to investigate many kinds of electrode reactions and can give detailed information on ligand-receptor interactions

0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 1 ) 0 0 5 4 1 - X

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Fig. 1. The chemical structures of odorants investigated together with their van der Waals' volume.

at functionalized surfaces, ion transport and electron transfer through the self-assembled monolayers [15±20]. The chemical structures of odorants selected for the study presented, together with their van der Waals' volume (VDW) were presented in Fig. 1.

Cleaned gold electrodes were immersed in 1 mM ethanol solutions of DDT for at least 18 h. Dodecanethiol monolayer-coated electrodes were rinsed with C2H5OH and dried before measurements.

2. Materials and methods

Electrochemical measurements were carried out using computer controlled electrochemical measurements system (AUTOLAB, Eco Chemie, Utrecht, The Netherlands) with Ag/AgCl as a reference electrode and Pt as an auxiliary electrode. Capacitance and resistance values were determined by impedance measurements in a supporting electrolyte solution: 0.1 M KCl, 10 mM K3Fe(CN)6, 10 mM BIS-TRIS, pH 8.2. A sinusoidal ac signal was applied at a frequency from 0.1 to 10000 Hz with 10 mV ac amplitude. The two impedance run, without the odorant in the solution was carried out to get the constant value of capacitance and resistance. Next, the ethanol solutions of odorant compounds: geraniol, coumarin, menthol, b-ionone and 1-octanol in the concentration: 10 4, 10 3, 10 2, 10 1 and 1 M were added to the supporting electrolyte to obtain the following concentration: 10 7, 10 6, 10 5, 10 4, 10 3 M. The stabile signal was generated after 5 min. The response time was found experimentally.

2.1. Chemicals 1-Dodecanethiol (DDT, 98%), potassium ferricyanide(III) (K3Fe(CN)6, 99%), bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane (BIS-TRIS, 98%), chloroform (CHCl3) and sulfuric acid (H2SO4) were purchased from Sigma, PoznanÂ, Poland. Potassium chloride (KCl), hydrogen peroxide (H2O2) and ethanol (C2H5OH) were obtained from POCH, Gliwice, Poland. All solutions were prepared with Milli-Q water (resistance of 18.2 MO). 2.2. Preparation of the samples The gold electrodes (MINERAL, Warsaw, Poland; diameter 1.6 mm, area 0.02 cm2) were used as working electrodes. To render hydrophobic surface of gold, before each covering by DDT, electrodes were carefully polished with 0.05 mm alumina slurry. Next, sonicated in CHCl3, cleaned with water, pretreated with ``pirania'' solution (hot 30% H2O2 and concentrated H2SO4, 1:1 volume ratio) for a few second. In the next step, the electrode potential was scanned between 0.3 and 1.5 V in freshly prepared 0.5 M H2SO4.

2.3. Measurements

3. Results and discussion Fig. 2 showed typical cyclic voltammograms of a bare gold electrode (curve 1) and coated with DDT (curve 2) in

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Fig. 2. Cyclic voltammograms of a freshly cleaned (1) gold electrode; (2) Au/DDT monolayer in 0.5 M H2SO4 solution. Scan rate: 100 mV/s.

0.5 M H2SO4 solution. The ratio of the oxide removal peak area of DDT/Au electrode to that of bare Au electrode was higher than those obtained by other authors [21,22], which may implied that the order of thiol monolayer is not perfect. However, the impedance spectra (Fig. 3) of the electrodes prepared in such a way before and after stimulation by geraniol in the concentration 10 7 and 10 3 M were consisted only with a semicircle in the high frequency domain indicating that the process is controlled only by electron transfer, with no ion diffusion. Thus, it might be concluded that the compactness of dodecanthiol monolayer is suf®cient for ion preventing to access the gold surface [22]. Impedance measurements allowing for the calculations of the resistance and the capacitance of DDT/Au electrode

Fig. 4. Changes of resistance of modified gold electrode upon the changes of the odorant compounds concentration; n ˆ 5, 0:005 < s < 0:011, measurements conditions: see Fig. 3.

showed that all hydrophobic odorant compounds investigated interacted with thiol monolayer deposited onto gold and changed its structure. Fig. 4 showed the run of the resistance changes of modi®ed gold electrode upon the changes of the odorant compound concentration. The increase of resistance was observed in the case of each compound investigated, but the dimension of the change of this parameter was differed and characteristic for each compound. The order of the odorant ef®ciency in the change of the gold electrode resistance was as follows: geraniol > coumarin > 1-octanol > menthol > b-ionone The increase of the lipid membrane resistance upon stimulation by taste compounds observed with using the impedance measurements was reported by Toko at al. [23,24]. They have pointed out that sucrose, non-electrolyte, adsorbed onto membrane surface and may increase its packing density and thickness. The relationship between the second parameter measured, the electrode capacitance after stimulation by odorants tested in the relation to their concentration was illustrated in Fig. 5. Generally, the capacitance decreased for all compounds investigated, but the run and the dimension was differed for each of them. It could be explained as follows. The capacitance is inversely proportional to the thickness of thiol layer [25]: Cm ˆ

Fig. 3. (1) (&) Typical impedance spectra of bare electrode; (2) (*) Au/ DDT monolayer; (3) (‡) Au/DDT monolayer after stimulation of geraniol in the concentration 10 7 M; (4) (*) Au/DDT monolayer after stimulation of geraniol in the concentration 10 3 M. Electrolyte composition: 0.1 M KCl, 10 mM K3Fe(CN)6, 10 mM BIS-TRIS pH ˆ 8 solution. The frequency range: from 0.1 to 10 kHz.

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e0 k d

where Cm is the specific capacitance, which is the capacitance normalized for the electrode area; e0 the permittivity in free space; k the dielectric constant of the material (for dodecanethiol monolayer k ˆ 2:25 [26,27]); d the thickness of a dielectric material. The electrical capacitance decrease observed after absorption of smell compounds is generally related to the thickness increase of the thiol layer. The changes of dielectric constant

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of thiol layer, which is mostly depended on the alkyl chain length [26,27], have weaker in¯uence on the above phenomenon. The general order of the compounds ef®ciency of the diminish of the thiol layer capacitance was as follows: geraniol > coumarin > 1-octanol > b-ionone > menthol

Fig. 5. Changes of capacitance of modified gold electrode upon the changes of the odorant compounds concentration; n ˆ 5, 0:30 < s < 2:40, measurements conditions: see Fig. 3.

This sequence was similar to this one connected with resistance changes (Fig. 4). Even thought all hydrophobic odorants interacted with sensing thiol layer in the differentiated way, it might be difficult to determine them with good selectivity on the base only one parameter measured. From the impedance spectra two parameters: resistance and capacitance could be measure. So, instead of one, these two were taken into account. In order to see the correlation between them, the regression analysis was done. Fig. 6 showed an example of the relationship between the odorant concentration and the capacitance and resistance changes (A: geraniol; B: b-ionone). It could be observed that each compound had its own run pattern of the phenomenon

Fig. 6. Changes of resistance and capacitance of modified gold electrode after stimulation by: (a) geraniol; (b) b-ionone.

I. SzymanÂska et al. / Sensors and Actuators B 75 (2001) 95±100 Table 1 The physico-chemical and topological parameters of smell compoundsa Compound

Ê 3) VDW (A

Ê 2) TSA (A

Log P

Menthol Geraniol b-Ionone 1-Octanol Coumarin

141 127.5 170 121.9 109.6

138.9 130.8 161.5 128.5 106.4

2.78 4.68 3.47 ± 2.03

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These results are very promising. They indicated that thiol modi®ed gold electrode could be applied as a selective detector for neutral compounds. 4. Conclusions

a

VDW: van der Waals' volume, calculated by CHEM-X; TSA: total surface area calculated by CHEM-X; log P: partition coefficients (octanol/ water).

discussed. So, it means that molecular recognition process between the thiol layer and odorant compounds run with some selectivity, which could be improve by taking into account more than one parameter measured. The olfactory properties of odorants are determined by the shape, size and type of functional groups [28,29]. Thus, in order to establish the relationship between the value of generated signals and the structure of the stimulant compound we have taken under consideration the following parameters describing the molecular properties: van der waals' volume (VDW), total surface area (TSA) and partition octanol/water coef®cients (Table 1). The correlation between mentioned above parameters and the capacitance and resistance changes measured by impedance, under the particular concentration level of geraniol (the strongest stimulant), was calculated using the multiple regression. The results obtained were collected in Table 2. Generally, the relatively high correlation between the parameter measured was observed for VDW and log P, the lower in the case of TSA, especially in the relation to the changes of the electrical capacitance of the electrode investigated. Statistically, the in¯uence of the parameters considerate was valid in six cases with the con®dence level 0.1 (P < 0:1) and in three cases with the con®dence level 0.05 (P < 0:05).

It has been demonstrated that electrochemical impedance measurements could be applied for the observations of signals induced by thiol layer covalently attached onto gold electrode upon stimulation by odorants. The parameters measured: the resistance and capacitance correlated in some extent with other, describing the physico-chemical and topological properties of analyzed molecules. The sensing mode presented gives the possibility to determine simultaneously two parameters, the resistance and the capacitance, induced by the target molecules. This feature improves the selectivity of the odorant recognition process. The correlation between the parameters measured and others describing the physico-chemical properties of the analyte led to creation of so-called pattern recognition, which could make the detection more effective. Such sensors, with a multidimensional analysis of analytical signals and/or multiple assemblies of linked devices, might extend further the limits of sensitivity and selectivity, and facilitate the determination of many components in a complex sample matrix. Acknowledgements This research was sponsored by State Committee of Science (KBN) grant 5P06 G 026615. The authors thank D. Kikut-Ligaj (Economy Department, Agriculture University, PoznanÂ, Poland) for CEM-X calculations and T. Jelinski (Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland) for statistical calculations.

Table 2 The correlation between changes of measured (capacitance and resistance) and physico-chemical parameters of geraniol calculated by multiplied regression (n ˆ 5)a Correlation factors VDW DC7 DC6 DC5 DC4 DC3 DR7 DR6 DR5 DR4 DR3 a

0.65718 0.60112 0.61101 0.62691 0.71947 0.46848 0.67732 0.70420 0.62064 0.61058

P-values

<0.1 <0.1

<0.1

TSA 0.47761 0.49157 0.53271 0.59167 0.73111 0.40110 0.69905 0.69471 0.63556 0.61037

P-values

<0.1 <0.1

<0.1

Log P 0.68785 0.58366 0.51464 0.42236 0.26025 0.50185 0.21961 0.32120 0.31483 0.36509

P-values

Mix

P-values

<0.1

0.99688 0.96528 0.96193 0.97620 0.99834 0.86652 0.94396 0.99253 0.99065 0.99840

<0.1

<0.1 <0.1

<0.05

<0.05 <0.05 <0.1 <0.1 <0.05

DC: changes of capacitance and DR: changes of membrane resistance under particular concentration of geraniol; 7,6,5,4,3 describe the geraniol concentration 10 7, 10 6, 10 5, 10 4 10 3 M, respectively.

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Biography Iwona SzymanÂska was born in April 10, 1974. She obtained her MSc degree at the Department of Chemistry of Technical University of GdanÂsk in 1998. Since 1998, she has been working at the Institute of Animal Reproduction and Food Research (Analytical Department) of Polish Academy of Sciences in Olsztyn. She is co-author of three papers and communications.