Diamond & Related Materials 14 (2005) 2139 – 2142 www.elsevier.com/locate/diamond
A diamond-on-silicon patch-clamp-system J. Kusterer a,*, A. Alekov b, A. Pasquarelli a, R. Mu¨ller a, W. Ebert a, F. Lehmann-Horn b, E. Kohn a a
Department of Electron Circuits and Devices, University of Ulm, Germany b Department of Applied Physiology, University of Ulm, Germany Available online 27 July 2005
Abstract Here, we present a planar multi-patch-clamp-system based on a nano-crystalline diamond patch-membrane used for the investigation of ion channels in cell-membranes. In such a system the cell-membrane is electrically stimulated through a potential difference in the electrolyte across the patch-membrane. Usually, the patch-membrane material is a highly insulating dielectric. The use of diamond allows to add highly localized probes in close proximity to the clamping area for a localized analysis of the stimulation mechanism. The technological concept, realization and first electrical characteristics of the principle structure are discussed. D 2005 Elsevier B.V. All rights reserved. Keywords: Diamond film; Nano-crystalline; Microstructure; Biomedical application
1. Introduction The inside of a living cell is separated from the outside by a membrane. Information to the exterior is mediated by ion-channels, which have the ability to regulate the flow of ions like Na+, K+, Cl , etc. through the cell membrane. To analyze such transport phenomena, the patch-clamp technique using capillaries with small inner diameter (approx. 1 Am) has been pioneered by E. Neher and B. Sakmann [1]. However, the capillaries prevent miniaturization and may imply high parasitic impedances. Cells are individually contacted electrically by an electrolyte of intra- and extracellular solution. The technique does not allow local differentiation, is time-consuming and requires skilful handling of the fragile pipette tool. Signal conditioning and acquisition are performed by (bulky) external equipment. For these reasons planar structures, which can be arranged in arrays, have been developed [2 –4]. The heart of such a planar system is a dielectric patch-membrane at the
* Corresponding author. Universita¨t Ulm, Abt. EBS, Albert-EinsteinAllee 45, 89081 Ulm, Germany. Tel.: +49 731 5026187; fax: +49 731 5026155. E-mail address:
[email protected] (J. Kusterer). 0925-9635/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.diamond.2005.06.011
bottom of a compartment, containing a centre hole of approx. 1 Am in diameter. The dielectric patch-membrane needs to have high fracture strength to withstand the pressure difference needed for the tight contact between the cell-membrane and the patch-membrane, which in turn is needed to suppress any bypass current (GV-seal). Usually such dielectric membrane materials are Si3N4 or SiO2 [2,3]. The use of semiconductor patch-membranes would allow to integrate local probes for stimulation, potential recording or heating. It would therefore allow a high local resolution in the dynamic analysis of the ion channel activation mechaelectrodes
silicon
cell
I
diamond (insulating) electrolyte
Fig. 1. Schematic cross section of a single patch-clamp-compartment and principle of measurement.
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a) standing wave ripples of photoresist
b) copper cylinder
Fig. 2. a) Sacrificial Cu cylinder on diamond nucleation layer for selective diamond growth. b) Patch-hole after removing the sacrificial Cu cylinder.
nisms. Such an approach is pursued in the following using diamond as membrane material.
2. Technology The principle cross section of such a planar system is shown in Fig. 1, already showing the use of diamond. The materials approach may best be described as a diamond-on-Si technology. In this case a nano-crystalline diamond film has been deposited onto a Si base substrate by a two-step process, containing a first BEN and outgrowth step by HFCVD [5] and a second outgrowth step to approx. 2 Am thickness by MPCVD. Such a two-step process has proved very effective in reducing the residual pin-hole density, generating micro short circuits across the membrane submerged into the electrolyte. Into the Si base plate an array of compartments is etched down to the diamond interface by wet chemical etching. As a result, the bottom represents the diamond patch-membrane and the compartment the cell-chamber.
Thus, the cell is clamped against the diamond membrane surface, which had been the interface to the Si base material (Fig. 1). It is therefore as smooth as the polished Si surface used for nucleation and not related to the topography of the nano-crystalline membrane surface. Each membrane includes one hole in its centre with a diameter of approx. 1 –2 Am. The hole is fabricated by a specific selective diamond growth technique, using electroplated Cu cylinders as sacrificial material (Fig. 2a and b). The process is based on previous work, where a Cu sacrificial layer technology had been developed for the fabrication of high aspect ratio capillaries [6]. The patchmembrane is positioned and sealed onto a bath chamber, the reduced pressure in the bath chamber generating suction of the cell membrane into the patch-hole. A smooth membrane surface is essential to prevent any ionic bypass current and enable a GV-isolation between the top and bottom electrolyte. Both, the top cell-chamber and the bottom bath-chamber contain Pt electrodes to contact the electrolyte. Pt does not require chemical treatment and does not wear out in the electrolyte. The chip contains an
upper electrode
lower electrode
clamp hole
1.5 µm
Fig. 3. Array of compartments etched into the Si base material. The bottom of the compartments represents the diamond membrane. At the acceleration voltage used (10 kV) the membrane is transparent and top and bottom Pt-electrodes can be identified. The insert shows the patch-hole in detail (diameter 1.5 Am).
J. Kusterer et al. / Diamond & Related Materials 14 (2005) 2139 – 2142
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copper 100000 measured data fitted curve
nano-diamond
R [kΩ]
silicon
selective diamond growth
10000
1000
nano-diamond silicon 100 0,1
1
silicon etching in KOH
hole diameter [µm]
nano-diamond silicon
Fig. 6. Dependence of hole series resistance to hole diameter measured in extracellular solution. The fitted curve estimates the resistance for submicron-sized holes.
testing. The pressure difference between the cell-compartments and the bath-chamber is obtained by closing the electrolyte inlet to the cell compartments, while applying a low pressure transient to electrolyte bath-chamber.
Pt deposition of electrodes Fig. 4. Schematic fabrication routine of the chip fabrication.
array of cell compartments, which can be addressed individually. The chip, chamber array and centre patchhole are shown in Fig. 3. In the SEM picture taken at 30 kV acceleration voltage, where the diamond membrane is already transparent, the Pt-electrodes are also seen. The chip fabrication routine is described in Fig. 4. An assembled system consisting of the chip, sealing, PMMA bath-chamber and an elastomer to extend the volume of the cell-compartments is shown in Fig. 5. The extension of the cell chambers is needed to prevent drying during
3. Systems characteristics First individual elements of the system have been analysed. These were: – The resistance of the diamond membrane itself measured between the two platinum electrodes first in dry environment and then in contact with electrolyte at top and bottom: In both cases the resistance is very high with values in the range of several hundred GV.
outlet of electrolyte elastomer mould
inlet of electrolyte
bath chamber
patch-clampchip
Fig. 5. Assembly of patch-clamp-chip, elastomer extension and PMMA bath chamber.
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– The resistance in case of no membrane: This short circuit resistance is in the order of kV, representing the electrolyte system itself and thus depending on the electrolyte used. – The resistance of the system with patch-hole: Here patchholes of various diameters have been fabricated within the array with a diameter of 5 to 1.5 Am. The impedance measurement was carried out using an extracellular solution containing the following substances (in mMol/ l): NaCl (150), KCl (2), CaCl2 (1.5), MgCl2 (1), HEPES (10). Fig. 6 shows that the dependence of resistance on the patch-hole area is linear. Its value is typically between 100 kV and 1 MV for micron sized holes. In the specific case the membrane resistance itself (without clamp hole) was determined to approx. 240 GV. The isolation of the membrane is therefore sufficiently high to form a GV-seal with a contacted cell. The series resistance of the electrolyte is in the same order of magnitude compared to other planar systems [7,8]. In the next generation of technology, an active layer of doped diamond will be added for the monolithic integration of micron size electrodes. Such electrodes, when based on boron doped diamond may be electrochemically inactive, causing no parasitic effects. Together with a further reduction in patch-hole diameter it is expected that this will enable to characterize the dynamics of a single ion channel with high spatial resolution. However, reducing the patchhole diameter will also increase the hole resistance. With the presently used membrane thickness (2 Am) a 100 nm hole is expected to provide a series resistance of approx. 100 MV and an associated parasitic potential drop, leading to a quite challenging signal-to-noise ratio. It seems therefore essential to be able to engineer the shape of the through-hole by tailoring the sacrificial layer technique.
4. Conclusion A novel planar patch-clamp-system has been fabricated based on a nano-crystalline diamond patch-membrane. Into
this membrane micron sized patch-holes were integrated by a selective diamond growth and sacrificial layer technology as developed previously. The diamond membrane shows indeed high insulation in the range of several hundred GV and a smooth surface topography, sufficient to allow the realization of a GV-seal needed during in-vitro testing. The employed diamond technology offers the possibility to extend the system to highly localized ion-cannel characterization through the integration of electrochemically inert micron-sized probes. Acknowledgements Many thanks are to C. Janischowsky for stimulating discussions on nano-crystalline film growth, to Y. Men for assistance in the patterning and e-beam lithography development. Financial help was in part provided by the DFG.
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