Anal. Chem. 1999, 71, 2814-2820
Adjusting the Distance of Electrochemical Microsensors from Secreting Cell Monolayers on the Micrometer Scale Using Impedance Rohit Kashyap and Miklo´s Gratzl*
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106
There are emerging applications of electrochemical microsensors where the distance of the sensor from an insulating plane needs to be adjusted and/or accurately known. The plane may be merely an obstruction or the source of a species whose release rate needs to be measured. An example is in cell secretion studies where a monolayer of cells is stimulated to secrete ions and/or other biochemical species which then diffuse away from the cells while being measured by a microsensor. Sensor response will thus depend on both the rate of release and the distance of the sensor from the cells. To obtain accurate release rates, the precision of the scheme to control electrode distance from the monolayer needs to be on the micrometer scale for species with ionic diffusivities. Optical (stereomicroscope and microruler) and mechanical (precalibrated micrometer screw) methods to precisely position the electrode are difficult to use under realistic circumstances (due to opaqueness of the chamber, and/or the medium, or irreproducible chamber depth). In this work we propose to correlate electrochemical cell impedance with sensor distance. This scheme has been used to adjust the distance of a chloride (tip diameter ∼250 µm) and a potassium (tip diameter ∼1000 µm) ion-selective microelectrode in the 0-250- and 0-2500-µm range, respectively, from a planar obstruction as well as from a monolayer of cells with a best precision of (5 µm (n ) 6) for the chloride and about (20 µm for the potassium sensor. Larger electrodes have a broader range of distances over which they are sensitive, albeit with a poorer spatial resolution. This was verified by using Ag disk electrodes of 250 and 500 µm in diameters in AgNO3 solution. The response of an electrochemical microsensor when measuring secretion from a biological source depends on both the released amount of the secreted species and the distance of the sensor from the secreting source(s). Quantitative and dynamic characterization of the release process thus requires accurate knowledge of electrode distance from the substrate(s). A practical example in which distance from a cell layer is critical in the evaluation of results is electrochemical monitoring of drug efflux from a monolayer of cancer cells with a flatly placed cylindrical carbon fiber microelectrode.1 A similar problem exists in measur(1) Yi, C.; Gratzl, M. Biophys. J. 1998, 75, 2255-2261.
2814 Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
ing the secretion of ions from epithelial cell layers with ionselective microelectrodes (µISEs).2,3 In this latter scheme, which is used here as an example, secretion of ions from monolayers of a goblet cell line4 is monitored where the monolayer studied is placed inside a Ussing chamber, allowing for easy manipulation of the conditions on the basolateral and apical sides of the cells (Figure 1A). Net electrolyte flux from the cells is determined by passing a current in the direction opposite to the ion flow so that the potential measured across the cell monolayer is maintained constant. To estimate potential effects of electrode misplacement on µISE response to ion secretion, we used a simplified problem by assuming that net efflux from the cell monolayer is due only to secretion of chloride ions and then solved the corresponding diffusion problem to simulate the spatiotemporal distribution of the secreted ions for experimental efflux data.5 This model predicts that an error of placing the electrode at 200 µm instead of 100 µm from the monolayer could result in a difference of up to 50% in the sensed instantaneous local concentration, for the exact same secretion process. Thus, distance of the electrode must be accurately known to a precision of ∼10 µm if the secretion process and its dynamics are to be quantified from µISE measurements. Carbon fiber microdisk electrodes have been used to make quantitative electrochemical measurements with temporal information about release of neurotransmitters6,7 and insulin (a peptide hormone)8 from single cells during exocytosis. A similar scheme has been recently used to monitor efflux of anticancer drugs from sensitive and multidrug-resistant single cancer cells.9 In all of these measurements, the largest possible portion of secreted molecules needs to be detected for quantification. On the other hand, high temporal resolution is required to understand the process of (2) Kashyap, R.; Hopfer, U.; Gratzl, M. Dynamic measurement of Ca2+ release during mucin secretion from HT29-Cl.16E cells. To be submitted to J. Membr. Biol. (3) Kashyap, R.; Hopfer, U.; Gratzl, M. Cl- and K + secretion from HT29-Cl.16E, a mucin secreting cell line. To be submitted to J. Membr. Biol. (4) Merlin, D.; Guo, X.; Martin, K.; Laboisse, C.; Landis, D.; Dubyak, G.; Hopfer, U. Am. J. Physiol. 1996, 271, C612-C619. (5) Kashyap, R. Ph.D. Thesis, Case Western Reserve University, 1998-99. (Chapter 2: Development and testing of a mathematical model to study the spatial and temporal distribution of chloride ions from epithelial cells). (6) Wightman, R. M.; Schroeder, T. J.; Finnegan, J. M.; Ciolkowski, E. L.; Pihel, K. Biophys. J. 1995, 68, 383-390. (7) Chen, G.; Gavin, P. F.; Luo, G.; Ewing, A. G. J. Neurosci. 1995, 15, 77477755. (8) Paras, C. D.; Kennedy, R. T. Anal. Chem. 1995, 67, 3633-3637. (9) Lu, H.; Gratzl, M.: Monitoring drug efflux from sensitive and multidrug resistant single cancer cells. Anal. Chem., in print. 10.1021/ac9808876 CCC: $18.00
© 1999 American Chemical Society Published on Web 06/10/1999
Figure 1. Experimental setup for simultaneous transepithelial current measurement and potentiometric measurement of ionic concentrations in a modified Ussing chamber. (A) The standard four electrodes are used for voltage clamp measurements (Ia and Ib are platinum current passing electrodes; Ra and Rb are Ag|AgCl reference electrodes with junctions, placed in the apical and basolateral chambers). Cl- µISE is the chloride ion-selective electrode; RISE is the reference electrode for potentiometric measurements. (B) Schematic diagram (approximately to scale) showing the distance scheme with a Ag|AgCl microelectrode (250-µm tip chloride ISE) as used in this work. The reference (250-µm Ag|AgCl wire ring of 6-7 mm in diameter) is not shown.
release. It is therefore necessary that the electrode be placed almost touching the studied cell. These measurements are typically done in a transparent cell culture dish placed on the stage of a microscope, and thus the electrode is easily positioned against the cell using a micromanipulator while being observed under the microscope. When overall efflux, representative of an entire cell monolayer, is of interest, such as in this work, then different strategies must be adopted. If the electrode is a long cylindrical carbon fiber, then it can be positioned horizontally against the cell population since due to its microscopic cross section it will not interfere with the diffusion of secreted material. Such is the case of drug efflux measurements from monolayers of cancer cells where the 5-mm fiber length ensures effective averaging.1 If, however, the electrode is disk shaped (like with ISEs) and the information sought is still average efflux, the electrode needs to be placed at a distance far enough from the monolayer where the secretion from the individual cells is homogenized by radial diffusion, and also so that the electrode does not hinder this process due to its rather large size. Even though in such cases the electrode is required to be not very close to the cells, accurate determination of the distance of the electrode is necessary for a quantitative interpretation of the results, according to our simulations.5
The complete embodiment of a Ussing chamber and electrodes (see Figure 1A) cannot be mounted on a microscope stage, partly because of the size of the setup and partly because epithelial cells during secretion experiments need to be inside a CO2 incubator that typically cannot accommodate a microscope. Also, to control electrode placement, a (microscopic) vertical distance would have to be assessed. For all these reasons, only a stereomicroscope outside the incubator could be used in combination with a microruler. Obviously, this arrangement could not provide the ∼10-µm resolution shown as necessary by our simulation studies.5 All these difficulties with an optical approach are further compounded in practice by the opaqueness of the chamber, or medium, or both, and because the apical side is also quite crowded with different electrodes. Mechanical methods (precalibrated micrometer screw) cannot be used to adjust the effective distance either due to irreproducible depth of the chambers. Therefore, to conduct such measurements properly, first a technique other than optical or mechanical to measure and adjust sensor distance from cell monolayers accurately and precisely on the micrometer scale needs to be devised and tested. In this work, we propose an in situ scheme that correlates ac impedance of the electrochemical cell with “effective” sensor distance from a planar obstruction. The overall impedance includes solution resistance, Rs, which for a disk electrode is concentrated near the disk,10 and the Faradaic resistance, RF, which changes due to local depletion of the electroactive material and variations in current distribution along the electrode surface. The idea of this work is that when such a sensor is close to a cell monolayer, i.e., approximately at a distance in the order of its own tip diameter or less, current response to a small voltage excitation will change significantly as the distance from the cells varies. This can be used to position an electrode with the spatial resolution required as predicted by the model.5 The idea is tested here with three ion-selective electrodes of different electrochemical properties, thus proving feasibility for a broad spectrum of electrochemical sensors. The first is a Ag|AgCl microdisk electrode (d ∼250 µm), used to assess chloride secretions from epithelial cells.3 This electrode has a virtually nonpolarizable interphase and a relatively large surface area, resulting in a very low Faradaic resistance in the low-kiloohm range (∼20 kΩ/mm2). The second example chosen is a valinomycin-based potassium microelectrode (o.d. ∼1000 µm; ionselective membrane diameter ∼500 µm) significant in basic electrolyte secretion studies. This sensor has a highly polarizable electrochemical interphase resulting in Faradaic impedances in the low-medium megaohm range (∼15 MΩ/mm2). The third tested example is a silver microdisk electrode (to measure Ag + concentrations in a nonphysiological context) made in two different sizes (250 and 500 µm), to study the effect of sensor diameter on spatial resolution and dynamic range of the distance scheme. The proposed scheme works well for all three sensors including the potassium electrode where, in principle, variations in solution resistance induced by changing distance could be expected to get completely obscured by the high Faradaic and ISE membrane impedances. (10) Newman, J. J. Electrochem. Soc. 1966, May, 501-502.
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All these sensors are potentiometric electrodes normally operated at zero (net) current, and yet, the impedance-based distance scheme (obviously requiring net currents to pass) is feasible. Adaptation of the same idea to amperometric sensors (operated at nonzero currents) is then even more straightforward. EXPERIMENTAL SECTION Apparatus. A Ag|AgCl microdisk electrode for chloride sensing (Figure 1B) is fabricated by inserting a silver wire (d ∼250 µm) inside a polyethylene tubing (i.d ∼0.6 mm, Cole-Palmer) which is then pulled over an alcohol burner. Cutting it normal to its axis exposes a Ag disk surrounded by a thin plastic ring. The disk is chloridized in 0.1 M HCl solution. The potassium microelectrode has a body of Tygon tubing (o.d. ∼1000 µm, i.d. ∼500 µm), one end of which contains the potassium ion-selective membrane. The membrane is formed by immersing the tip of the electrode body in a K+ ion-selective cocktail (Fluka). The cocktail enters the Tygon tubing by capillary action and then forms a membrane (
