Atomic Force Microscope: A Tool for Studying Ionophores

The cantilever and tip were then immersed in 10 μL of the homogeneous membrane solution and centrifuged at 160g. To centrifuge the probe, the .... If...
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Anal. Chem. 2000, 72, 3689-3695

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Atomic Force Microscope: A Tool for Studying Ionophores Prisca Zammaretti,† Alphons Fakler,† Frank Zaugg,*,‡ and Ursula E. Spichiger-Keller*,†

Centre for Chemical Sensors/Biosensors and bioAnalytical Chemistry, Department of Pharmacy, Swiss Federal Institute of Technology, Zu¨rich (ETHZ) CH-8005 Zu¨rich, Switzerland, and Zyonix, San Francisco, California 80419

The aim of the investigations was to show the analytical use of an atomic force microscopy (AFM) tip coated with an ion-selective membrane and to show that the ionselective boundary potential is detectable as a force induced by ion-selective electrostatic interactions, which are more pronounced than double-layer forces. This new technique allows the area-specific ion exchange over boundaries to be displayed with a destruction-free technique by AFM in an aqueous buffer. From experiments with ISEs (ion-selective electrodes), a boundary potential for valinomycin was assumed to be established in close vicinity to a K+-releasing surface. To trace this boundary potential, an AFM tip was coated with a potassiumselective polymer film containing valinomycin as used in preparing ISEs. The K+-releasing substrate was prepared by incorporating a lipophilic potassium salt into a plasticized PVC layer. In contact with an electrolyte such as sodium chloride solution, an ion exchange takes place. Analogue experiments were performed using a sodiumselective ionophore, DD16C5, incorporated into the coating of the AFM tip, with a Na+-releasing substrate. The boundary potential was traced and investigated with the help of force vs distance curves. The resulting adhesion forces for a valinomycin-coated tip in a 150 mM NaCl solution were 9.8 ( 3.275 nN using a blank PVC substrate and 330.15 ( 113.0 nN using a substrate containing 10 wt % potassium tetrakis(4-chlorophenyl) borate. The selectivity of the ion-selective AFM tips was measured on sodium relative to potassium-releasing substrates and studied in different salt solutions with concentrations between 10 mmol L-1and 1 mol L-1. For valinomycin, a force selectivity coefficient log KfK,Na of -2.5 ( 0.5 for K+ against Na+ and a selectivity coefficient log KfNa,K of -4 ( -0.5 for DD16C5 were measured. In addition, the surface of the polymer substrate was imaged by AFM in contact mode with and without lipophilic potassium salt. The modulation of the force-distance curves induced by the ion exchange was compared to the experimental change in elasticity of the blank and ionexchanging substrate. The Young’s moduli measured with strain force analysis on a potassium-containing substrate 10.1021/ac000084u CCC: $19.00 Published on Web 07/08/2000

© 2000 American Chemical Society

were 5 times smaller than the ones registered with nanoindentation and did not explain the modulation of the force vs distance curves. At present, no technique is known that enables the ion-selective detection of the exchange density of ions on a membrane surface under in vitro conditions. Atomic force microscopy (AFM) is an attractive tool for in vitro monitoring since this technique allows nondestructive imaging of surfaces. The AFM can provide resolution within nanometer range without destroying hydrated samples. Therefore, it seems a promising technique for imaging a cell surface in three dimensions. A number of techniques have been developed to detect ion exchange over biological membranes and between cells and cell compartments. A very well-established one is the patch clamp technique awarded the Nobel Prize for Medicine in 1991.1 Locally, highly resolved ion currents over ion channels have been traced using this technique, although it does not allow ion-selective measurements. However, the patch clamp method is not a destruction-free technique. Ion-selective electrodes have increased the possibility of resolving local specific ion exchange in the micrometer range. In the best case, working with a tip size in the range of 100 nm has been found feasible.2 The most successful technique so far was found by Fan and Bard,3 who were able to identify a single ion exchange by electrochemical detection of single molecules based on redox systems and amperometric microtips combined with scanning electrochemical microscopy. However, the high noise registered affected the resolution. In contrast to these techniques, AFM allows a high resolution of the ion exchange combined with high selectivity and sensitivity. The modification of the AFM tip with an ion-selective coating may allow use to be made of the boundary potential in order to map ion concentrations or specific ion channels on living cell membranes. By measuring force vs distance curves with AFM, it should be feasible to map the specific ion exchange over boundaries, since electrostatic forces are more intensive by a †

Swiss Federal Institute of Technology. University of Zu ¨ rich. (1) Sakmann, B.; Neher, E. Angew. Chem. 1992, 104, 837-843. (2) Lantner, F.; Steiner, R. A.; Ammann, D.; Simon, W. Anal. Chim. Acta 1982, 135, 51. (3) Fan, F.-R. F.; Bard, A. J. Science 1995, 267, 871. ‡

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factor of 20 than other forces traced by AFM. A boundary potential resulting from an ion exchange is generated at the AFM tip, which must exceed the double-layer force (dipole on the tip side vs induced dipole on the substrate side) between AFM tip and substrate in an electrolyte or buffer solution. As a consequence of the boundary potential, the gap between the ion-releasing surface and the ion-selective cantilever tip is polarized, which contributes to the electrostatic forces exerted onto the cantilever. The method presented in this paper aims at showing the preparation of an ion-selective AFM tip similar to the preparation of ion-selective electrodes. The tips were coated with a thin layer of a PVC solution, which incorporates ionophores and which, in addition, allowed the chemical composition of the surface to be controlled. To show the feasibility of using these tips to trace local ion activities selectively, the modified tips were compared when used on plasticized ion-releasing PVC substrates and on a blank PVC substrate. The factors that influence the force-distance curves of the AFM cantilever, as well as the force selectivity coefficients, Kfij, were investigated. EXPERIMENTAL SECTION Materials. PVC (high molecular, 100 000) was obtained from Fluka (Buchs, Switzerland). The ionophore valinomycin, potassium tetrakis(4-chlorophenyl) borate (KTpClPB), o-nitrophenyl octyl ether (o-NPOE), bis(1-butylpentyl) adipate (BBPA), tetrahydrofuran (THF), and cyclohexanone came from Fluka. DD16C5 came from Keio University, (Yokohama, Japan). Alkanthiol HS(CH2)15COOH was synthesized according to ref 4. Substrates. The potassium-releasing substrate was as follows: 1.3 (2 wt %, 2.31 × 0.10-2 mol kg-1) and 6.5 mg (10 wt %, 10.9 × 0.10-2 mol kg-1) of KTpClPB, 56 mg of PVC, 56 mg of o-NPOE (1-nitrophenyloctyl ether), and 2 mL of THF. The sodium-releasing substrate was as follows: 1.3 (2 wt %, 2.31 × 0.10-2 mol kg-1) and 6.5 mg (10 wt %, 10.9 × 0.10-2 mol kg-1) of sodium tetrakis[3,5-bis(trifluorophenyl)] borate (NaR), 56 mg of PVC, 56 mg of o-NPOE, and 2 mL of THF. Tip Coating. The potassium-selective membrane consisted of the following: 2.3 mg of valinomycin (1.16 × 0.10-2 mol kg-1), 54.7 mg of PVC, and 122.9 mg of BBPA, dissolved in 3 mL of cyclohexanone. The blank membrane consisted of the following: 56 mg of PVC, and 120 mg of BBPA, dissolved in 3 mL of cyclohexanone. The sodium-selective membrane was as follows: 2.3 mg of DD16C5 (1.16 × 0.10-2 mol kg-1), 54.7 mg of PVC, and 122.9 mg of BBPA, dissolved in 3 mL of cyclohexanone. Coating Technique for AFM Tips. Initially, the chip that carries the cantilevers was immersed in a 3 mM hydrophilic alkanethiol (HS(CH2)15COOH) THF solution for 10 min. This procedure serves to protect the gold-coated surface, which reflects the laser beam of the AFM, from the coating polymer film. Subsequently the chip was washed with ethanol and then with 1,4-dioxane. A membrane solution, composed of 33 wt % PVC, 65 wt % BBPA, and 1 wt % valinomycin was dissolved in cyclohexanone. The cantilever and tip were then immersed in 10 µL of the homogeneous membrane solution and centrifuged at 160g. To centrifuge the probe, the chip was glued onto a ceramic holder and positioned into an Eppendorf tube. From experiments with (4) Zaugg, F. G. Thesis 13182, Swiss Federal Institute of Technology, 1999.

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Figure 1. (a) Normal AFM tip of the type Ultralevers. The image is taken by transmission electron microscope (TEM). (b) Shows the same tip after coating with a polymer layer. The black arrow shows an accumulation of PVC on the cantilever. In (c), a confocal laser scanning microscopy image of the coated cantilever (image size 35 µm) is shown. Right-hand side and bottom section, cross sections through the cantilever and the tip apex. The polymer film incorporates a fluorescent dye (4′,5′-dibromofluorescein octadecyl ester) (bright areas). The effect of the surface modification by alkanethiols is shown in both cross sections. The reflecting side of the cantilever, which controls the position of the tip, is protected by alkanethiols (white arrow).

optical membranes, it is well known that the polymer films are optically homogeneous and transparent.5 All components dissolved in the polymer layer are mobile, and the mass balances are diffusion-controlled (see section on Diffusion of the Ions). The thickness of the film was measured applying TEM (Philips CM100 operated at 80 kV). First, a bare AFM tip was imaged (Figure 1a). Second the tip was coated with the polymer film and imaged once more under identical conditions (Figure 1b). In a third step, a gold film of 5-nm thickness was sputtered onto the polymercoated tip in order to confirm the measurements in the second experiment and to respect uncontrolled reflection. The diameters of the tips as measured in the three experiments were compared (Figure 1a and b). The adhesion of the polymer coating after AFM measurements was tested and confirmed with a confocal laser scanning microscope (CLSM) by substituting valinomycin with a fluorescent chromoionophore (see legend for Figure 1c). Atomic Force Microscope. For the AFM experiments, an Autoprobe LS (Park Scientific Instruments, Sunnyvale, CA) (5) Spichiger, U. E.; Bott, M.; Citterio, D. Proc. SPIE-Int. Soc. Opt. Eng. 1995, 2508, 179. (6) Ha¨mmerle, S. Thesis 12290, Swiss Federal Institute of Technology, 1997. (7) Watson, G. S.; Gibson, C. T.; Myhra, S. Nanotechnology 1996, 7, 259-262.

modified with pulsed laser6 or a Bioscope (Digital Instruments, Santa Barbara, CA) was used. The dried samples were glued to magnetic punches for the Autoprobe LS and on glass slides for the Bioscope. Ultralevers are silicon cantilevers, with a nominal spring constant of 0.06 N/m (Park Scientific Instruments), where the cantilever length is 200 µm and the tip length is 3.2 µm, were used in most experiments. The remaining experiments were performed with Nanoprobes, with a spring constant of 0.06 N/m (Digital Instruments), where the cantilever length is 200 µm and the tip length is 1.15 µm. The second type is less sharp and consists of silicon nitride (Si3N4). Verification of the spring constants of the cantilever was done according to the method developed by Watson et al.7 The spring constants thus measured were not significantly different from those given by the manufacturers. The spring constant of the coated cantilever was measured and evaluated on a statistical basis with the same method. A constant of 0.9 ( 0.3 N/m was obtained for 20 different tips of each manufactured type. The force vs distance curves were taken at a scan rate of 10 Hz. Net Force Calculation. The net force, Fnet, is calculated as adhesion force measured on a PVC layer containing a lipophilic potassium or sodium salt, Fsalt in PVC, minus the adhesion force on a bare PVC layer, FPVC, blank,:

Fnet ) Fsalt in PVC - FPVC, blank

(1)

Elastic Behavior Measured with an AFM (Nanoindentation).8 The elastic behavior of a material can be measured macroscopically with the strain force analysis (SFA) or microscopically by nanoindentation. With the first method, a sample of material with a well-defined size is stretched with a constant speed. For the calculation of the nanoindentation ∆z, it is necessary to measure force vs distance curves. To interpret the force vs distance curve, according to ref 8, the curve that describes the contact mode is considered, which coincides with the negative part of the x-values. This part of the curve is fitted with a polynom such as

force ) a∆zb

(2)

The term a gives information about the elastic modulus and b characterizes the tip shape. We assumed a parabolic tip shape, where R is the radius and E* is sample elasticity.

force (∆z) ) (4/3R1/2E*∆z1.5)

(3)

The relationship between the Young’s modulus and the sample elasticity is given by the next equation (where Esample , Etip):

1/E* ) (1 - µsample2)/Esample

(4)

For the Poisson ratio (µ), several approximations have to be done. The Poisson ratio of the sample, µsample, was supposed to be 0.17 as for cells8 and the one of the substrate 0.4 (as for pure PVC). RESULTS AND DISCUSSION Hypothesis. The prerequisites of the work presented here are as follows: an AFM tip modified with a K+- and a Na+(8) Vinckier, A.; Semenza, G. FEBS Lett. 1998, 430, 12-16.

selectivefilm, a K+- or, respectively, a Na+-releasing substrate, and an electrolyte solution (NaCl, KCl). The AFM tip is coated with a permselective layer containing valinomycin, a potassium-selective ionophor, and DD16C5, a sodium-selective ionophor. The substrate is based on plasticized PVC, which contains lipophilic potassium and a sodium-releasing salt. If sodium and potassium are mobile in both phases, i.e., both within the PVC substrate and within the electrolyte solution, an exchange of the potassium ion in the substrate with the sodium ion of the electrolyte will occur. Theoretically, the exchange will be stopped when equilibrium conditions are installed. The equilibrium conditions are defined by the solubility of the ions in each phase. As a matter of fact, K+ complexes to valinomycin are more stable by a factor of 104 than those of the sodium ion.9 At the tip coating in NaCl solution, the ionophore will primarily extract Na+. However, if K+ is leaching from the substrate and the AFM tip coated with the potassium-selective film approaches the substrate, Na+ will immediately be replaced by K+ 10 and K+ will preferably be extracted into the tip coating. This phase change of K+ raises the electrical boundary potential at the tip, which compensates for the change in the chemical potential. Simultaneously, the interface between the tip and the substrate will be polarized, and double-layer forces as described in ref 11 will add to the boundary potential forces. Therefore, a change in the adhesion forces exerted on the AFM tip is expected on a potassium-containing substrate in NaCl and LiCl solutions compared to those on the tip on a blank substrate. The adhesion forces are influenced by the ionic strength of the environment solution.12 The ionic strength directly correlates with the thickness of the Debye layer. The distance at which two neighboring charges influence each other and therefore at which an interaction can be registered13 defines the Debye layer. These forces can be approximated by estimates of the electrostatic forces and should be in the range of nanonewtons, which is measurable with AFM. The main contribution has to be referred to the electrostatic forces given by the Coulombic force term, and therefore, an approximation with equations that contain the dielectric constant of the environment solution as a parameter is feasible. Coating Technique. To generate ion-selective AFM tips, a cantilever was coated with a potassium-selective plasticized PVC layer incorporating valinomycin or DD16C5. The tip coated with a film containing valinomycin is assumed to be potassium selective, whereas that containing DD16C5 is selective to sodium ion. The thickness of the polymer film measured with TEM was 7.5 ( 2.5 nm (Figure 1a and b). The total tip apex diameter was measured as 25 ( 5 nm, from which the contact surface can be calculated if the tip apex can be considered as a circular section of a parabolic tip. The estimated contact area is ∼490 ( 216 nm2. The increase in the spring constant by a factor of ∼15 times is given by the distribution of the polymer coating due to centripetal (9) Fluka Chemie AG, Buchs, Switzerland, Catalogue Selectophore. (10) The first-order rate constant of the exchange of water against other ligands of an alkaline metal ion in an octahedral complexes is maximum and in the order of 108 s-1: Huheey, J.; Keller, E.; Keiter, R. Anorganische Chemie; Walter de Gruyter: Berlin, New York, 1995. (11) Takano, H.; Kenseth, J. R.; Wong, S.; O′Brien, J.; Porter, M. D. Chem. Rev. 1999, 99, 2845-2890. (12) Butt, H.-J. Nanotechnology 1992, 3, 60-68. (13) Israelachvili, J. Intermolecular & surface forces; Academic Press: London,

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force caused by the centrifugation of the AFM chip. Centrifugation of tips causes a very thin polymer layer at the very apex of the tip, but the cantilever is coated with a layer of ∼100 nm of ionselective polymer film. AFM Experiments. The difference between the substrate and the coating membrane is that the substrate consists of the same type of PVC layer but exhibits increased viscosity. In addition, the substrate contains the ion-exchanger KTpClPB for the K+/ Na+ exchange and NaR for the Na+/K+ exchange. The substrate and the tip are immersed in an aqueous buffered solution of sodium chloride (0.15 M NaCl, pH 6.5), potassium chloride (0.15 M KCl, pH 6.5), or lithium chloride (0.15 M LiCl, pH 6.5). Experience with optode membranes5 and other analytical techniques, such as potentiometry, suggests that the mobile cations of the substrate equilibrate with those in the aqueous phase, whereas the anions are fixed within the organic substrate owing to their lipophilicity. The ion-exchange equilibrium is determined by the free energy of hydration of the cations (lyotopic or Hofmeister series). Force Curves Taken with a Modified Tip with Valinomycin. The following describes the ion exchange between an AFM cantilever coated with a potassium-selective PVC membrane, M, and a substrate, S, from where potassium ions were released, in NaCl solution, pH 6.5 (Figures 2 and 3). It was assumed that a new equilibrium state is established between the tip coating and the substrate if the cantilever approaches the surface of the substrate. The situation is shown schematically in Figure 2. In the first step, the membrane contains the neutral ionophore valinomycin, L, and a small fraction of complexed sodium ions while working in 0.15 M aqueous NaCl solution (aq). In microelectrodes, the extraction of potassium compared to that of sodium into an organic membrane phase containing valinomycin is favored by a factor of 3.2 × 104.14 The polymeric substrate contains the lipophilic ion-exchanger KTpClPB (R-K+), which allows K+ to be exchanged with Na+ in the NaCl solution (aq) as shown by the mass balances below (eq 5). In close proximity to the substrate surface, the K+ released from the substrate (s) will be extracted into the membrane (m), which coats the tip, as described by the mass balance of the extraction equilibrium in (eq 6).

substrate: R-(s) + K+(s) + Cl-(aq) + Na+(aq) T R-(s) +

Figure 2. Schematic drawing of the force vs distance curve during retraction of the tip. The tip apex is coated with a K+-selective membrane, which contains the ionophore L, on a K+-releasing substrate (K+R-) in 0.15 M NaCl. The trace of the tip is associated with different stages in the selective ion-exchange process (insets A-D). If no exchangeable ions are offered in the aqueous phase (Na+), the ion exchange does not work, and the typical force vs distance curves cannot be observed. (A) The tip is in contact with the substrate in a position where attractive and repulsive forces compensate each other. (B) The electrostatic repulsion progressively weakens the attractive forces and the tip changes from the contact to the noncontact regime. As discussed in the section Diffusion of the Ions, a steady-state situation of ion exchange can be assumed for a short moment. In point C, the resting position is finally restored and the K+ exchange will be partially reversed.

Na+(s) + Cl-(aq) + K+(aq) (5) Tip membrane

in NaCl solution: L(m) + Na+(aq) + Cl-(aq) T NaL+(m) + Cl-(aq) (6) close to the K+-releasing substrate:

NaL+(m) + K+(aq) + Cl-(aq) T KL+(m) + Na+(aq) + Cl-(aq) (7) The binding constants of valinomycin to potassium are in the 3692 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

range of > 8 × 103 kg mol-1.14 The relative binding constants in water and membrane are presented in ref 15. According to Meers and Feigenson,16 the association constant measured in methanol is of the same order of magnitude as that in a PVC membrane. In eqs 6 and 7, the mass balance describing the charge separation at the boundary is given. The changes in the chemical potentials of the ions involving the tip coating and the electrolyte are compensated for by a varying surface potential, whereas the boundary potential of the substrate may not vary relevantly at constant concentration of the electrolyte and of KTpClPB in the (14) Ammann, D.; Chao, P.; Simon, W. Neurosci. Lett. 1987, 74, 221. (15) (a) Spichiger-Keller, U. E. Chemical Sensors and Biosensors for medical and biological application; Wiley-VCH Verlag GmbH: Weinheim, 1998. (b) Wipf, H. K.; Pioda, L. A. R.; Stefanac, Z.; Simon, W. Helv. Chim. Acta 1968, 51, 377-381. (16) Meers, P.; Feigenson, G. N. Biochim. Biophys. Acta 1988, 938, 469-482.

Figure 3. Experimental force vs distance curves in an intermittentcontact regime taken on a plasticized PVC substrate with two different concentrations of the lipophilic potassium salt KTpClPB (2 and 10 wt %) and the reference PVC substrate without KTpClPB. During the retraction phase, a typical shape was observed where the indentation is reduced and the adhesion forces increase with an increasing potassium concentration of the substrate.

substrate. The force curve was collected by monitoring the cantilever deflection vs separation distance as the tip and substrate are brought into contact and subsequently separated in 150 mM NaCl solution at pH 6.5. The cantilever deflection was converted to applied force. The force curve in Figure 2 was divided into several regions: In (A), the tip is in contact with the substrate in a position where attractive and repulsive forces compensate each other. The adhesive contact was referred to electrostatic interactions. The electrostatic interactions are caused by the electrically polarized interface between aqueous electrolyte and polymer film. In (B), the electrostatic repulsion progressively weakens the attractive forces and the tip changes from the contact to the noncontact regime. As discussed in the section Diffusion of the Ions (below), a steady-state situation of ion exchange can be assumed for a short moment. In point C, the resting position is finally restored and the K+ exchange will be partially reversed. The force vs distance curves for both phases of the cantilever motion, contact and retraction, varied with the concentration of KTpClPB within the polymer layer of the substrate and with the potassium activity at the tip apex (Figure 2). The trace of the cantilever motion between (A) and (C) represents the pull-off forces. In (D), the situation of the coated film in the resting position with ∼1 µm distance from the substrate is depicted. The adhesion forces in the retraction phase measured with a valinomycin-coated tip on a blank PVC substrate were 9.8 ( 3.3 nN, but the ones measured on 2 wt % KTpClPB were 100 ( 33 nN. The forces increased for the substrate containing 10 wt % KTpClPB to 340 ( 113 nN (see Figure 3). Although, there is no linear dependence between the salt concentration of the substrate and the forces registered, there is a significant increase in the adhesion forces when concentrations of the ion exchanger in the substrate are compared. In the following, the retraction phases were analyzed in more detail in order to evaluate the adhesion forces and the variable elastic behavior of the tip at the substrate boundary. In Figure 3, the interpretation of the force vs distance curve during retraction

is shown. Initially, similar experiments were performed in water rather than NaCl solution. During these experiments, no modification of the cantilever motion could be observed. The reason is that, due to the absence of NaCl in solution, no ion exchange between valinomycin and potassium ions could occur. Diffusion of the Ions. The kinetics of the ion complexation process shown here is assumed to be higher than the diffusion kinetics and will therefore not limit the ion-exchange process much.13 The ion exchange will be diffusion limited, at least at the substrate boundary. However, the short path lengths of the diffusion processes that are involved in increasing the attractive and repulsive forces at the AFM tip means that the ion exchange at the boundaries can be equilibrated very quickly. When the cantilever approaches the substrate, the two processes, namely, ion exchange at the substrate boundary and at the AFM tip, will equilibrate for a very short moment when the cantilever changes its direction. At this moment, the motion of the cantilever will be governed by the electrostatic interactions and polarization of the solvent in the gap between the surfaces, which can be easily understood with Debye length theory (see below). It has to be assumed that a charge separation occurs owing to the extraction of K+ to form KL+ at the boundary of the tip membrane. Clcompensates for this surface charge in the aqueous boundary phase. The change in the chemical potential of these species is compensated for by the boundary potential measured in potentiometry. At the same time, the release of K+ in exchange for Na+ at the substrate/NaCl boundary polarizes the interface between the substrate (membrane) and the environment (solution). R- and Cl- compensate for the charge number of the cations. In close proximity to the surface, the electrostatic interactions and the polarization of the boundaries will determine the motion of the cantilever and will allow a close approximation of the tip corresponding to the Debye lengths of Na+, K+, and KL+ relative to the anions in each phase. The Debye length is related to the ionic strength of the solution, to the local ionic strength, and to the local permittivity. Since the ionic strength is relatively high, the van der Waals forces will cooperate with electrostatic forces, whereas capillary forces will be negligible. The influence of van der Waals forces increases with the ionic strength. To evaluate the rate-limiting step of the ion exchange, several estimates were done. The time required for an ion to pass through the gap between the tip and the substrate is thought to be primarily diffusion controlled. The diffusion time of an ion in the aqueous phase is of the order of d2/2Daq, where Daq is the diffusion coefficient, DK+, of K+ or DNa+, of Na+ in water (DK+ ) 1.96 × 10-9 m2 s-1, DNa+ ) 1.33 × 10-9 m2 s-1).11,17 This means, a potassium ion should be able to cross a gap of 1 nm in the aqueous phase within approximately 2.5 × 0.10-8 s or 0.25 ns (3.7. 10-8 s for Na+). Since cNaCl in the aqueous solution is higher than the molality of the ligand and the ion exchanger within the substrate or, respectively the film y, the Na+ concentration should not limit the ion exchange. An overall apparent diffusion coefficient, Ds, which was related to Ca2+/H+ exchange within the same highly viscousPVC substrate (DCa2+ ) (1.1-1.8) × 10-13 m2 s-1), was determined for optode membranes.5 The diffusion coefficient for alkaline ions in the same medium is twice that of Ca2+, in the (17) Cussler, E. L., Ed. Diffusion, Mass Transfer in Fluid Systems; Cambridge University Press: New York, 1984.

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Table 1. Adhesion Forces (Mean ( 2SD) Calculated from the n Repetitions of Force vs Distance Curves, Accounting for the Spring Constant of the Cantilevera

K+ concn, substrate

total adhesion force (nN) (n ) 50)

E modulus nanoindentation (MPa)13

E modulus wet (MPa)

reference, PVC layer 2 wt % KTpClPB 10 wt % KTpClPB

9.826 ( 3.275 100.855 ( 33.615 340.110 ( 113.37

0.6066 ( 0.1213 59.5 ( 8.9 65.4 ( 13.1

4.08 ( 0.64 5.07 ( 0.15 6.84 ( 0.77

a The tip coatings incorporated 55 mmol kg-1 valinomycin. The E modulus measured by nanoindentation (mean ( 2SE) with the AFM is described by Vinckier8 and was performed with Park LS from Park Scientific.6 The E modulus of the conditioned, wet and isolated membranes were determined via strain force analysis (Mecmensin M 1000E, mean ( 2SD).

Figure 4. An error signal image, which results from the motion of the cantilever, coated with a solvent polymeric membrane of 55 mol kg-1 valinomycin, on two neighboring substrates. The substrate is scanned in contact mode. On the left is a substrate without lipophilic potassium salt; on the right is a substrate containing 10 wt % KTpClPB. The small capillary forces, which induce the apparent roughness of the surface, are smoothed as a consequence of the interaction forces arising at the boundary potential.

range of 3 × 10-13 m2 s-1.5 Therefore, the limiting rate should be the diffusion of the ions within the substrate where a potassium ion is able to transit a gap of 1 nm within 1.7 × 10-6 s. For comparison, the cantilever moves with a speed of 20 µm s-1 or is displaced by 1 nm within 5. 10-5 s, 10 times slower than the diffusion of Ca2+ within the polymer membrane and 105 times slower than the diffusion of Na+ ions in water. The force-distance curve shows the typical deflection motion of an AFM tip on soft substrates in contact due to indentation. However, the electrostatic interactions seem to cause an increase in hardness during the retraction phase, where from calculations about the diffusion speed of ions a steady state can be assumed. To explain these preliminary results, it was concluded that the electrostatic interactions considerably influence the force vs distance curves of the cantilever coated with an ion-selective membrane layer (see Table 1). The Elastic Behavior. Further investigations (Figures 3 and 4) were aimed at characterizing the interactions in more detail. Two main topics were investigated: first, the changes in the elastic modulation of the cantilever motion on PVC substrates due to the ion-selective interactions; second, the forces that are exerted onto the cantilever, in competition with and in addition to its own weight. Tips coated with potassium-selective membrane layers 3694

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Table 2. Net Forces (Mean ( 2SD) Calculated from the Results in Table 1, Column 2, Referred to the Experimental Results of a Blank Membrane (Not Shown Here) According to Eq 4a K+ concn, substrate

total measd net forces (nN) (n ) 50)

mean distance measd (nm) (n ) 50)

reference, PVC layer 2 wt % KTpClPB 10 wt % KTpClPB

90.65 ( 33.5 330.15 ( 113.0

76 ( 15 150 ( 25 409 ( 74

a Mean minimum distance between the cantilever and the surface of the substrate in the same experiments for two different concentrations of the ion exchanger in the substrate and a concentration of 55 mmol kg-1 valinomycin in the tip coating. The measurements were achieved with Park LS from Park Scientific.6

incorporating valinomycin (55.5 mol kg-1) showed an increasing stiffness or less elastic behavior (Figure 4, right-hand side) vs the same substrate (10 wt % KTpClPB) with increasing concentrations of valinomycin. These effects were not observed with a blank PVC coated and an uncoated Si3N4 tip (Figure 4, left-hand side). Nevertheless AFM imaging of the surfaces of both substrates, that with and that without KTPClPB, shows an identical mechanical roughness. Generally, the following behavior was observed: In contact mode, the tip jumped into attraction and moved below the original surface (negative distance) because of the high elasticity of the substrate (see Figure 3). In addition, typical elastic behavior was noticed upon retraction of the AFM tip that inversely correlated with the concentration of the ion exchanger of both the coating and the substrate and with the conditioning period of the coated tip in NaCl solution. The elastic behavior was completely compensated for by the electrostatic interactions for more concentrated substrates and coatings. The concentration of the ion exchanger in the substrate in these experiments clearly dominates the response behavior and, therefore, the closest distance between the tip apex and the substrate (see Figure 2A-C). The influence of van der Waals and electrostatic forces cannot be distinguished in these experiments. In the following, the cantilever deflection in the retraction phase was transformed into the quantity (N) of adhesion forces in order to quantify the results,16,18 as summarized in Table 1. The net adhesion forces induced by the interaction of the coated tip with the ion-exchanging substrate were calculated by subtracting the nonspecific electrostatic and van der Waals forces referred to a blank PVC substrate from the experimentally determined total forces (see eq 1). The results are shown in Table 2. Table 2 shows a clear influence of the KTpClPB concentration of the substrate. The increasing concentration goes along with stronger adhesion forces in the retraction phase and an increasing closest distance between the substrate and the tip. These differences cannot be explained by the variation in the Young’s moduli (Table 1) measured with strain force analysis or nanoindentation by AFM.8 The E modulus measured with strain force analysis was 2 times lower than the one measured with nanoindentation for 2 wt % and 5 times lower for 10 wt % KtPClPB substrate. This means that the difference in elasticity cannot be explained by the elasticity of the substrate, but has rather be referred to the electrostatic forces exerted onto the AFM tip. (18) Lechner, M. D., Gehrke, K., Nordmeier, E. H., Eds. Makromolekulare Chemie; Birkha¨user: Basel, 1993.

The lifetime of the cantilever coating was restricted to less than 3 days storage in water. Leaching of the ionophore valinomycin, which exhibits a relatively low lipophilicity of log PDSC ) 8.6,19 from the very thin coating was observed, and went along with a loss in adhesion force from 360 ( 120 to 27 ( 9 nN within 8 days. Selectivity. The selectivity factor describes the discrimination of interfering ions relative to the target ion. This parameter is important, therefore, to see which factors influence the efficient functioning of the AFM tip. Sodium ions are discriminated from potassium ions by a factor of -3.2 × 104 by valinomycin in microelectrodes.14 It was postulated that the forces measured are specific for the ionophore and, therefore, give a direct indication of its selectivity. The selectivity coefficient describes the affinity of the ligand to a competing compound relative to a target compound. In this case, the adhesion forces of the coated AFM tip in the retraction phase measured on a sodium-releasing substrate were compared to those on a potassium-releasing substrate for both ionophores, valinomycin and DD16C5. The selectivity can be calculated in the following way:

selectivity ) log Kfi,j ) log[forcesubstrate with interfering ion/force substrate with target ion] (8) A negative sign indicates discrimination of the interfering ion relative to the target ion as in potentiometry. As described previously, a competing electrolyte in solution is necessary in order to obtain an ion exchange at the substrate/solution boundary for selectivity measurements. For example, if the tip is coated with a polymeric membrane containing valinomycin, the substrate will be based on KTPClPB, and sodium ions will be present in the solution. The results obtained with a tip, coated with a PVC film containing valinomycin as measured with the microscope Bioscope are shown in Figure 5. The range at which the forces were measured with AFM is between 10 mM and 1 M. In this range, the ionic strength of the solution increases. The ionic strength of the solution influences the Debye length, on which the type of forces measured is dependent. The range between 10 and 100 mM is dominated by electrostatic forces (Table 3, -2.4 ( 0.2), but for concentrations higher than 150 mM van der Waals forces become more important (Table 3, -2.8 ( 0.4). The overall selectivity of valinomycin obtained with the AFM is -2.5 ( 0.5, which is comparable with the one given by Fluka9 (-1.9 to -3.6 with comparable membrane composition). In this case, the overall selectivity can be considered because, for the value -2.5 ( 2SD, 2SD is smaller than the margin of error with AFM measurements. Selectivity was measured additionally with a tip coated with a polymeric film containing DD16C5, a sodium sensor (Figure 5). The overall selectivity was -4.0 ( 0.5. The value is comparable with that obtained with ion-selective electrodes from the manufacturer Dojindo (Tokyo, Japan). In the electrostatic forces range, the selectivity is -4.40 ( 0.02, but in the van der Waals forces domain, it is smaller, -4.0 ( 0.2. In both cases, the selectivity measured with the AFM method is comparable to the one given by the manufacturer. Hence, the (19) Weisenhorn A. L.; Khorsandi M.; Kasas S.; Gotzos V.; Butt H. J. Nanotechnology 1993, 4, 106-113.

Figure 5. (9) Selectivity of the valinomycin measured with an AFMcoated tip with a permselective polymer layer containing the ionophore at 55 mol kg-1. Substrate with lipophilic potassium salt (10 wt % KTpClPB) or lipophilic sodium salt (10 wt % NaR) were used. The salt solutions had a pH of 6.5. The measurements were performed at room temperature in a salt solution environment. The target ion for valinomycin is potassium. ([) Selectivity of the DD16C5 measured with an AFM-coated tip with a permselective polymer layer containing the ionophore at 55 mol kg-1. Substrates with lipophilic potassium salt (10 wt % KTpClPB) or lipophilic sodium salt (10 wt % NaR) were used. The salt solutions had a pH of 6.5. The measurements were performed at room temperature in a salt solution environment and were the same as for valinomycin, but in this case the target ion was sodium. Table 3. Selectivity Measured with AFM Coated Tips for DD16C5, a Sodium Ion Sensor, and Valinomycin, a Potassium Ion Sensor selectivity (AFM) concn range (mM)

valinomycin

DD16C5

0-100 300-1000

-2.4 ( 0.2 -2.8 ( 0.4

-4.40 ( 0.03 -4.0 ( 0.2

method can be considered as an alternative for measuring the selectivity of an ionophore incorporated into a polymer membrane at the molecular level. In this case, diffusion processes may show a negligible influence on the value of the selectivity coefficient.20 The results obtained with the macroscopic method of ISE are comparable to the microscopic one, which was determined with the new technique, using AFM. ACKNOWLEDGMENT This work was sponsored by the Swiss National Research Foundation, SNF 36. We are grateful to Prof. K. Suzuki and Dr. D. Citterio for delivering the sodium ionophore DD16C5. Dr. A. Vinckier (Institute of Anatomy, University of Zurich, Switzerland) carried out the TEM experiment. We gratefully acknowledge the cooperation of the Institute of Biomedical Engineering, ETH Zurich, Switzerland. We thank Dr. U. Ziegler for CLSM investigations, M. Colussi for the strain force analysis investigations, and Prof. Dr. P. Grosscurth, Prof. Dr. G. Folkers, and Prof. U. W. Suter for the helpful discussion and the material support. Received for review January 31, 2000. Accepted May 4, 2000. AC000084U (20) Dinten, O.; Spichiger, U. E.; Chaniotakis, N.; Gehrig, P.; Rusterholz, B.; Morf, W. E.; Simon, W. Anal. Chem. 1991, 63, 596.

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