Timothy M. Kubow, Karen C. Cheung, Loren F. Bentley, and Luke P. Lee ... advantages. Traditionally, the pipette is lowered at an askew angle, making it difficult ...
MICROFABRICATED PATCH-CLAMP ARRAY FOR NEURAL MEMS APPLICATIONS Timothy M. Kubow, Karen C. Cheung, Loren F. Bentley, and Luke P. Lee Joint Graduate Group in Bioengineering University of California San Francisco / Berkeley Department of Bioengineering, University of California, Berkeley Berkeley Sensor and Actuator Center, University of California, Berkeley Berkeley, CA 94720, U.S.A. Abstract Microfabricated patch clamping devices comprising planar arrays of individually addressable nozzles, fluidic channels and electrodes have been developed. Patch clamp based electrophysiological techniques are among the most widespread methods in neurophysiology and are used to address a broad range of cellular physiology and quantitative biological questions. Among the limitations of the technique are the difficulty of obtaining multiple patches on connected cells or on the same cell, limited stability of patches, and constraints on chemical and optical access to the patched membrane. The parallel array device will enable the formation of multiple seals simultaneously. The structure facilitates visualization of the interior of the patched membrane during electrical recording, as well as delivery of chemicals. The microfabrication technique gives precise control over the capacitive and resistive characteristics of the electrode channels, as well as the flow resistance, which are important factors in patch clamp recording. The device is fabricated using an SOI wafer and Deep Reactive Ion Etching to create an array of cylindrical nozzles, each of which has a core of silicon dioxide and interior walls of silicon nitride. Vertical channel segments and plumbing holes are fabricated by deep reactive ion etching through the wafer. Important electrical properties of the device were characterized, and patch clamping was attempted. Introduction Patch clamp electrophysiology is one of the most important techniques in modern biology. The awarding of the Nobel Prize to its inventors Neher and Sakmann in 1991 highlighted its significance to the field. Its continued relevance to biology is illustrated by the over 1500 citations with it as a subject in 2001. The original technique [1] allowed the first direct recording of electrical currents through a single membrane channel. Since then the technique has branched out to include many variations, including whole-cell patch, inside-out patch, and outside-out patch. For a thorough discussion of the technique see [2]. Its use in the study of membrane channels has led to many important findings about the fundamentals of biology as well as to the discovery and understanding of innumerable pharmaceutical products [3]. Despite the fact that it is an extremely important technique and is now commonplace, patch clamping still requires a skilled researcher to perform the procedure, thus leading to a recent surge in interest in automating the technique. Many researchers have now begun to make patch clamp devices in novel ways [4, 5, 6]. Many companies are now selling or are developing automated patch clamp devices. One approach to automation is to fabricate the equivalent of a patch pipette on a silicon chip. The equivalent to the pipette could be as simple as a micron sized hole or, in this study, a three-dimensional microfabricated nozzle. In addition to automation, putting a patch pipette on chip has other advantages. Traditionally, the pipette is lowered at an askew angle, making it difficult
to find the plane of the membrane. Thus, a membrane patch in the horizontal plane would allow for improved optical microscopy. This could include advanced techniques such a FRET microscopy. Moreover, fabrication on chip allows for the possibility of more complex fluidic control on both sides of the patched membrane. Wire to Amplifier Pipette Tip Membrane Patch
CELL
Figure 1. Illustration of traditional patch clamp technique. At first glance, the technique is deceptively simple. A glass pipette filled with an electrolyte solution is lowered to the surface of a cell. The pipette is pushed into the cell creating tension, and then a brief pulse of suction is given to seal the cell membrane to the pipette. An illustration of the technique can be seen in Fig. 1 above. The challenge of the technique in the scales involved. The pipette typically has a diameter on the order of microns. To accomplish this, a thin glass capillary tube is pulled under heat from a hot wire or laser. By varying the speed of the pull and heat, the researcher can adjust the diameter and taper of the pipette. Pipettes are often fire polished in order to get a smooth tip that will attach more readily to the cell. Variations on this are still being developed [7]. In addition to the shape of the its mouth, the pipette resistance and capacitance as an electrode, as well as the dielectric properties of the pipette wall, are important [8].
Figure 2. Illustrations of the patch-clamp nozzle design.
In this paper, we present the design, fabrication, and characterization of a silicon-based patch clamp nozzle. Its design incorporates a novel three-dimensional structure as seen in Fig. 2 above. The design also allows it to be arrayed, allowing for many parallel patches in future devices. We characterized its electrical properties and found that it is similar to traditional patch pipettes. We attempted to perform a patch-clamp experiment on a frog (Xenopus) oocyte, but as yet we have been unsuccessful in achieving a seal to the cell membrane. Experimental Details The patch device was fabricated as follows. The process starts with a 75 micron SOI wafer. First, the bottom fluidic channel is formed by a deep reactive ion etch (DRIE) on the backside which is stopped by the buried oxide layer. Next, the nozzle channel is formed by a second DRIE from the front side that again is stopped by the buried oxide. Third, an oxidation step both defines the nozzle shape and provides some electrical insulation. This oxidation step forms the nozzle because it is followed by a brief reactive ion etch that selectively attacks the silicon. Thus, only the oxide wall remains, forming the nozzle structure and the surrounding well. A final nitride deposition step provides more insulation and further shapes the nozzle structure. The basic flow of fabrication can be seen in Fig. 3 below.
Step 1 Step 4
Step 2
Step 5 Step 3 Figure 3. Device fabrication steps: Step 1. Back side DRIE of SOI wafer to form fluidic connection. Step 2. Front side DRIE to form nozzle channel. Step 3. Oxidation step. Step 4. RIE to form nozzle and well. Step 5. Nitride deposition everywhere to insulate device. Silicon is gray, oxide gold, and nitride blue.
The final structure can be seen in the scanning electron micrograph in Fig. 4 below. The nozzle and the surrounding well are clearly illustrated. The thickness of the nozzle wall can also be seen; however, the connecting channel below can not be seen. The nozzle is slightly off center due to slight misalignments in the fabrication.
10 µm
Figure 4. Scanning electron micrograph of nozzle and surrounding well. Electrical measurements and patch-clamp experiments were carried out on a standard physiology rig. The microfabricated chip is glued to a cell culture dish with silicon spacers. A fluidic connection to a glass capillary tube is made for easy interface to a standard patch clamp amplifier and data acquisition system (Axon Instruments, Union City, CA). The device is filled up to the nozzle through the glass capillary with an electrolyte solution.
Figure 5. Current measured through a 3 micron nozzle. A 1500ms long step of 10mV was applied starting at t=200ms.
The device is interfaced to the electronics through an Ag Ag/Cl wire placed in the capillary. The dish is filled with a “bath” electrolyte solution and a reference electrode is placed in this bath. Both electrolyte solutions are varied to match the intracellular and extracellular solutions of the cell under study. The electrical measurements are taken from this setup. Micromanipulators are also used to control the electrodes and maneuver a cell over the nozzle. We characterized the basic electrical properties of the device by applying 10 millivolt steps across the electrodes and measure the resulting currents. The trace above in Fig. 5 is for a 3 micron nozzle. Based on the voltage step and the time course of the current, we calculated the resistance and capacitance as 3.08 M ohm and 50 n F, respectively. We attempted to patch a cell by rolling it on top of the nozzle and applying suction. Normally, one sees the resistance increase rapidly as the pipette seals with the cell membrane. Typically the resistance reaches around a G ohm forming a “giga seal.” In our case, we saw the resistance increase slightly and stop. It remained the same even after the cell was removed. This indicates the nozzle became clogged with debris and was rendered useless for patching. Summary In this study, we have demonstrated a novel three-dimensional nozzle array for patch clamp physiology. We have described its fabrication as well as electrical testing. Its resistance closely matches traditional patch pipettes. However, its capacitance is much higher than normal. This could present problems in measuring some membrane channel currents that are extremely fast acting. It is unclear what is creating this large capacitance at this moment; however, it may be remedied by simply changing the experimental setup. If not, our design and fabrication allows for future fabrications to adjust both the capacitance and resistance values. Although we have been unable to patch cells, we believe that practice and improvements in manipulation of the cell can overcome this problem. Future devices may also incorporate dielectrophoretic trapping to improve performance. Similarly, the design allows for incorporating biological based activation such as growth factors or other signaling molecules. Finally, integrating fluidics on chip will be important for making parallel patching and automation a reality. Acknowledgments We would like to thank Pau Gorostiza, Elena Molokanova, John Long, and Rob Froemke for their assistance. TMK was supported by a Howard Hughes Predoctoral Fellowship. KCK was supported by a Whitaker Fellowship. LFB was supported by a NIH Traineeship. References 1.E. Neher, B. Sakmann, Nature 260, 5554 (1976). 2.B. Sakmann, E. Neher, Eds., Single-Channel Recording, (Plenum Press, New York, 1983). 3.J. Connolly, C. Kennedy, Journal of Receptor and Signal Transduction Research 21, 21 (2001). 4.N. Fertig, A. Tilke, R. H. Blick, J. P. Kotthaus, J. C. Behrends, and G. ten Bruggencate, Applied Physics Letters 77, 8 (2000). 5.C. Schmidt, M. Mayer, and H. Vogel, Angewandte Chemie International Edition 39,17 (2000).
6.N. Fertig, Ch. Meger, R. H. Blick, Ch. Trautmann, and J. C. Behrends, Physical Review E 64, 4 7.M. B. Goodman, S. R. Lockery, Journal of Neuroscience Methods 100, 1-2 (2000). 8.R. A. Levis, J. L. Rae, Biophysical Journal 65, 4 (1993).
