Abstract. It is shown by capacitive monitoring that the self-assembly of alkanethiols on gold electrodes and desorption of these self-assembled monolayers.
Mikrochim. Acta 131, 29±34 (1999)
Electrical Control of Alkanethiols Self-Assembly on a Gold Surface as an Approach for Preparation of Microelectrode Arrays Michael Riepl, Vladimir M. Mirsky, and Otto S. Wolfbeis University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, 93040 Regensburg, Germany
Abstract. It is shown by capacitive monitoring that the self-assembly of alkanethiols on gold electrodes and desorption of these self-assembled monolayers from the electrodes are controlled by the electrode potential. At neutral pH, chemical adsorption of alkanethiols was observed at an electrode potential of 300 mV vs SCE, but only physical adsorption was detected when the electrode potential was ÿ1400 mV vs SCE. At electrode potentials between these values (ÿ300 mV, ÿ600 mV), chemical adsorption of alkanethiols occurred, but the alkanethiol monolayers were not stable in the absence of the alkanethiol in the bulk solution and were desorbed from the gold electrode. The desorption rate was higher at more negative electrode potentials. These results can be used in designing methods for electrically addressable immobilization of different receptors on (micro)electrode arrays. This has been demonstrated by deposition of two different types of alkanethiols onto a two-electrode array. Key words: self-assembly; electrical control; electrode capacitance; addressable immobilization, (bio)sensor array.
An intensive investigation of self-assembly of thiols on metal surfaces has already resulted in many technical and analytical applications [1±5]. In contrast to Langmuir±Blodgett ®lms, self-assembled monolayers of thiol compounds can be obtained without the use of expensive techniques but simply by chemical adsorption from aqueous or organic solutions. The nature of the gold±sulfur bond is not yet quite clear, To whom correspondence should be addressed
though electrochemical study has revealed oxidation of the sulfur atoms during self-assembly [6±11]. This fact means that self-assembly of thiols can be controlled by variation of the electrical potential of the metallic supports and this can be used for many exciting applications such as electrical control of wetting [12], electrochemical patterning [13, 14] and preparation of sensor arrays [15]. A number of techniques for addressable immobilization of reagents (receptors) to arrange sensor arrays have been described. Several groups [16±18] have used a precisely positioned microdrop dispensing system. This micromechanical technique provides a spatial resolution of about 100 nm. The Af®max company has developed a light-addressable immobilization technique based on photodissociation of caged compounds. This reaction releases thiol-terminated oligonucleotides to form a self-assembled receptor layer on a gold surface [19]. A lateral resolution better than 50 mm was achieved [20]. If the molecules to be immobilized are stable enough in organic phases, a lithographic technique may be used [21]. However, the requirement of a lithographic mask for each kind of immobilized molecules makes application of this technique for complicated sensor arrays rather expensive. Electrically addressable immobilization can provide a simpler solution. This method was used for immobilization by means of an electrochemical polymerization [22]. Electrical control of a self-assembly of thiols on a gold surface another technique for the electrically addressable immobilization which is suitable for preparation of highly ordered monolayer structures on electrodes. This technique, based on electrical control of self-assembly
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of alkanethiols on the gold surface, is described in this contribution. Experimental All measurements were performed at room temperature (22 C). A two-electrode circuit was used for capacitance measurements and a saturated calomel electrode served as a reference. Gold electrodes were prepared by rf-sputtering of a 250-nm thick gold layer with sublayers of titanium (50 nm) and palladium (50 nm) on a silicon support, and were generously provided by the group of Prof. V. Tvarozek in the Technical University of Bratislava. The macroscopic surface area of the sensitive electrode was 2.4 mm2. The electrodes were cleaned with a hot mixture of ``piranha'' solution (a 1:3 v/v mixture of 30% H2O2/conc. H2SO4) and then rinsed with water/ caution: piranha solution reacts violently with most organic materials and must be handled with extreme care. To study the electrically addressable immobilization, the simplest ``array'', consisting of two electrodes placed on the same piece of silicon, was used. Steady potentials from two independent voltage sources were applied to these electrodes. Potential values indicated are vs SCE. Capacitance was measured by registration of the 90 component of the capacity current by means of a lock-in ampli®er (PAR, Model 121) at 20 Hz. The amplitude of the sinusoidal voltage on the electrodes was 10 mV. A home-made current ampli®er was used, its typical ampli®cation being 104 V/A. At the last stage of the work, the capacitance measurements were performed with an electrochemical impedance spectrometer EIS-11 from Analytical m-Systems (Regensburg, Germany) for the same operating conditions. Contact angles were measured by means of a device G-1 from ERMA Optical Works (Tokyo, Japan). An electrolyte consisting of 100 mM potassium chloride and 5 mM phosphate buffer, adjusted to pH 5.7 or 12, was used. Demineralized water was additionally puri®ed by passing it through a Millipore-Milli-Q system, the ®nal resistivity being at least 18 MOhm cm. To prevent formation of air bubbles, the electrolyte was degassed under vacuum immediately before the experiment. The !-substituted alkanethiols were from Analytical m-Systems (Regensburg, Germany). Human serum albumin and 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were from Sigma. Unsubstituted alkanethiols and inorganic chemicals were from Merck. Further experimental details are described in [23].
Fig. 1. A principle of capacitive monitoring of adsorption and desorption processes. Cdl is the speci®c electrical capacitance of the ion double layer, Cads and are the speci®c electrical capacitance and fractional coverage, respectively, of the alkanethiol monolayer
Results and Discussion Electrical Control of Self-Assembly The method applied in the present work is based on measurement of electrode capacitance. The speci®c electrical capacitance of self-assembled monolayers is much less than that of an electrical double layer (i.e. the capacitance of a bare gold electrode). Therefore, the desorption of an organic monolayer results in an increase in the electrode capacitance (Fig. 1). Self-assembled thiol monolayers on a gold surface are stable only in an electrolyte at certain de®nite electrode potentials. At pH 5.7 no desorption of alkanethiols was observed at an electrode potential of
Fig. 2. Capacitive study of the stability of self-assembled monolayers of !-modi®ed alkanethiols at different electrode potential at pH 5.7 (a) and at ÿ300 mV at pH 12 (b). The electrode potentials in (a) 300 mV (curve 1), ÿ300 mV (curves 2, 4), ÿ1400 mV (curve 3). Type of monolayer: 16-mercaptohexadecanoic acid (1, 3, 4, 7), 16-mercaptohexadecanoic acid with immobilized bovine serum albumin (2), 1-hexadecanethiol (6), 16mercaptohexadecylamine (5)
Electrical Control of Alkanethiols Self-Assembly on a Gold Surface
300 mV (Fig. 2a, curve 1). Changing the electrode potential to ÿ300 mV led to a desorption of alkanethiols from the monolayer (Fig. 2a, curve 2). At more negative electrode potentials, the desorption rate increased (Fig. 2a, curve 3). For the monolayer crosslinked by a chemical immobilization of bovine serum albumin onto carboxy-groups of alkanethiols [23], the desorption rate was considerably lower (Fig. 2a, curve 4). The range of electrode potentials at which an alkanethiol monolayer is stable depends on pH. At basic pH desorption of the alkanethiols was also observed at 300 mV (Fig. 2a). The desorption rate of carboxy-terminated alkanethiol (curve 7) was higher than that of methyl- and amine-terminated alkanethiols (curves 5 and 6). The reason is probably the higher solubility of the carboxy-terminated alkanethiol at basic pH. A detailed investigation of pH effects on the stability of alkanethiol monolayers on gold electrodes will be presented elsewhere; all further results presented here were obtained at pH 5.7. Capacitive monitoring allows a real-time study of the self-assembly of alkanethiol monolayers on gold electrodes. With electrodes at either adsorption (300 mV) or desorption (ÿ1400 mV) potentials, a decrease of the electrode capacitance after addition of thiols was observed (Fig. 3). However, at 300 mV the kinetics of the capacitive effect was much faster and the magnitude greater. The stability of the monolayer was further tested by a short washing with water and chloroform. The capacitance effect due to the adsorption of alkanethiols at ÿ1400 mV was completely reversed (Fig. 3, ®lled circles), whereas no capacitance changes due to this washing were observed for the alkanethiol monolayer adsorbed at 300 mV (Fig. 3, open circles). Also, subsequent additions of thiol reversed the capacitance increase due to washing of the monolayer adsorbed at ÿ1400 mV, but had no capacitance effect on the monolayer adsorbed at 300 mV. These results suggest that only physical adsorption of the alkanethiols occurred at the electrode potential of ÿ1400 mV, but a strong (chemical) adsorption of alkanethiols occurred at the electrode potential of 300 mV. Similar experiments at the potentials ÿ300 and ÿ600 mV have shown that these potentials do not block chemical adsorption of the alkanethiols. Therefore, the boundary between the adsorption and desorption potentials of the alkanethiols depends on the presence of these alkanethiols in the bulk phase: in
31
Fig. 3. Electrical switching between physical and chemical adsorption. Additions (1) of 50 mM 6-mercaptohexanoic acid at the electrode potential ÿ1400 mV (®lled circles) and 300 mV (open circles) followed by washing with buffer and chloroform (2). pH 5.7
the absence of alkanethiols in the bulk phase, a stable chemically adsorbed monolayer can exist only at positive potentials (Fig. 2 a), but in the presence of a bulk concentration of alkanethiol as low as 50 mM, this stability range is expanded to potentials lower than ÿ600 mV. This applied-potential dependence of a self-assembly of alkanethiols on a gold surface provides an effective way to control this process by variation of the electrode potential. This is illustrated in Fig. 4. A potential of ÿ1400 mV applied to the gold electrode blocked a self-assembly of alkanethiols, and only a
Fig. 4. Electrically controlled chemical adsorption of 6-mercaptohexanoic acid. Adsorption potential 300 mV vs SCE, desorption potential ÿ1400 mV vs SCE. pH 5.7
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small capacitance decrease was observed after addition of an alkanethiol. Switching from the desorption potential to the adsorption potential leads to a fast self-assembly of alkanethiols on the electrode. The switching between the adsorption and desorption potential could be repeated many times (up to ®ve were tested) and always led to a corresponding decrease or increase in the electrode capacitance, i.e. to formation and desorption of a chemically adsorbed monolayer. Typical desorption potentials reported by others [6±11] lay in the range between ÿ700 and ÿ800 mV. However, we observed the loss of stability at a more positive potential (ÿ300 mV at neutral pH). The main reason for this difference is probably a difference in time scale. The earlier measurements [6±11] were performed with a potential sweep of 2 mV/s; therefore the desorption potential was reached in as little as 5±10 min. During this time we also did not observe any desorption at the electrode potential of ÿ300 mV (Fig. 2). Applications of the Electrical Control of Self-Assembly Control of the adsorption behaviour of alkanethiols by an electrode potential results in a kind of chemical memory which can be used for many applications. In particular, the possibility of governing the adsorption of functionalized alkanethiols may be used for electrically addressable immobilization of different species on a gold surface. This is a necessary step in a preparation of sensor arrays and other molecular devices. An electrically addressable immobilization can be reached according to the procedure shown in Fig. 5. Let us consider an array of n (micro) electrodes onto which thiols A, B, C, etc. are to be immobilized. These thiols may have different receptors already coupled or !-terminal groups (for example, carboxy- or aminogroups) which can be easily coupled with receptors. As the ®rst step, an adsorption potential is applied to the electrode E1 and a desorption potential to all other electrodes. An addition of the thiol type A results in a chemical adsorption of these molecules on only the electrode E1. The solution in the cell is then changed and, if necessary, a receptor is immobilized on the thiol coating the electrode. As the second step, an adsorption potential is applied to the electrode E2, and thiol B is added. The thiol will adsorb on only the electrode E2 because a desorption potential is applied
Fig. 5. A procedure for electrically addressable immobilization of different thiols (A, B, C,. . ., X) on an array of n electrodes. Details in text
to all other uncoated electrodes (E3±En). This procedure is repeated until the last electrode (En) is coated by the respective thiol. Electrically addressable immobilization was veri®ed by deposition of two different thiols onto two electrodes (Fig. 6). First, an adsorption potential was applied to the ®rst electrode and a desorption potential to the second. Capacitive monitoring during addition of 6-mercaptohexanoic acid and subsequent rinsing of the electrodes with chloroform and buffer showed that the thiol was chemically adsorbed on the ®rst electrode, while on the second electrode no chemical adsorption occurred. After exchange of the solution in the cell for the solution without alkanethiol, an adsorption potential was applied to the second electrode and octanethiol was added. This resulted
33
Electrical Control of Alkanethiols Self-Assembly on a Gold Surface
The minimal size of an array is probably limited by electrical breakdown and by electrostatic interferences between the electrodes. The latter effect can be minimized by a special geometry of the electrode array with a shielding reference electrode placed between neighboring working electrodes. Quality control during preparation of these complicated structures and detection of binding in applications of these sensor arrays can be exercised by means of direct capacitive monitoring, which has already been successfully applied to single sensors [23±28].
Fig. 6. Capacitive monitoring of electrically addressable immobilization of two thiols (6-mercaptohexanoic acid and octanethiol) on the two-electrode (E1 and E2) array. The arrows indicate additions of 250 mM of corresponding thiol. pH 5.7
Acknowledgements. We are grateful to Drs. V. Tvarozek, V. Rehacek and I. Novotny (Technical University of Bratislava, Slovakia) for providing us with gold electrodes. The research was supported by the German Science Foundation and the European Project COPERNICUS CIPA CT94-0231.
References in a strong decrease in the capacitance of the second electrode, indicating an adsorption of the octanethiol on this electrode. No change in the electrode capacitance on rinsing with chloroform and buffer indicated that the octanethiol had been chemically adsorbed. The values of the speci®c capacitance of the electrodes after the electrically addressable immobilization (4.2 mF/cm2 for the electrode covered by 6mercaptohexanoic acid and 1.4 mF/cm2 for the octanethiol covered electrode) were similar to those measured after adsorption of the corresponding thiols onto single electrodes. Measurements of contact angles con®rmed a difference in wetting of both electrodes, corresponding to hydrophilic coating of the ®rst electrode and hydrophobic coating of the second. A similar principle has been already applied for electrically addressable immobilization by means of electropolymerization [22] and for immobilization of thiols from a mixture of an extremely alkaline aqueous solution with ethanol, a ¯uorescent control of immobilization being used [14, 15]. We have performed an electrically addressable immobilization in the aqueous phase, in physiological conditions, with direct capacitive monitoring of the self-assembly in situ. This technique of electrically addressable immobilization can be applied for preparation of not only the simplest two-electrode arrays, but of much more complicated systems, and also with microelectrodes.
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