Fig. 1. The steps in fabricating a silicon substrate for use as a planar patch-clamp. (A) Circles (2 μm diameter) were defined in a photoresist (white layer) using optical lithography and then etched to a depth of 20–40 μm using deep reactive ion-etching (DRIE). The SU-8 resist was then stripped off with an acid bath and the wafer was inverted for back-side processing. A 1 mm diameter well, centered on the 2 μm pore, was defined using optical lithography. DRIE was used to etch the 1 mm well to a depth such that a small pore was formed connecting the base of the well to the other side of the wafer. A 0.5–1 μm thick SiO₂ film was then deposited over the surface of the entire wafer (using plasma enhanced chemical vapor deposition). This film electrically isolates the front side of the wafer from the back and reduced the pore diameter to 1.5–1.8 μm. The deposition conditions could be varied to deposit thicker oxides and thereby reduce pore diameters as small as 0.7 μm. (B) A scanning electron micrograph (SEM) of a 1.7 μm diameter pore micromachined in the silicon chip. (C) SEM image of a “nanopore”.

Fig. 2. Assembly of the patch-clamp device. (A) Aligning and sealing the processed silicon chip (grey wafer) between an upper and lower chamber (yellow and blue) electrically isolates one side of the chip from the other (accept through the pore in Si), and enables independent experimental manipulation of each side. The top and bottom chambers are composed of cured PDMS (yellow) coupled to machined polycarbonate (light blue) supports. The bottom polycarbonate support interfaces the lower PDMS vertical channel to a syringe which is used to apply negative pressure. (B) Except for the area immediately surrounding the pore on the topside and the 1 mm well on the underside of the chip, the entire top and bottom surfaces of the silicon wafer were stamped with SU-8 photoresist (green) to enable sealing to the PDMS layers and reduce the chip capacitance to as low as 15 pF. (C). A single RIN m5F cell is drawn onto the pore (black spot) following suction. A few seconds after landing on the pore, an individual cell tends to center itself over the pore. This image is of a chip that was recycled after being previously used to patch-clamp a cell.

Fig. 3. This figure represents a summary of 63 wafer-based patching experiments using RIN m5F cells in which a wafer/cell seal resistance of at least 40 MΩ (10×the maximum access resistance, R_a) was achieved. Sixty percent of all RIN m5F experiments are represented in this figure. The others did not lead to at least a 40 MΩ seal. (A). Characterization of the dimensions of all 63 chip micropores by access resistance (R_a) measurements. Access resistances >10 MΩ are most likely due to debris contamination. (B) Distribution of RIN m5F cell membrane sealing statistics indexed by the measured micropore R_a values for all of the chips with R_a<10 MΩ (55 chips). A total of five gigaseals were achieved (three for R_a<3.0 and two for R_a>4.0). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 4. Schematic representations of CHO-K1 cells forming a gigaseal in various configurations and the accompanying recorded currents. All currents are in response to a −10 mV pulse from a holding potential of 0 mV. (a) The leak current measured through the chip pore without a cell present. This current was used to calculate the access resistance (R_a) of the pore, which in this case was 8.36 MΩ. (b) The recorded leak current decreased as a CHO cell was drawn to the pore by applying suction to the bottom side of the chip. (c) After prolonged application of gentle suction, a gigaohm seal between the cell and chip was formed, and the leak current became minimal giving a resistance of 3.96 GΩ. This patch is considered to be in the cell-attached mode. (d) The application of additional pressure leads to rupture of the membrane that spans the pore, and the patch goes to the whole-cell mode, and the seal resistance falls to about 250 MΩ. The vertical scale on the bottom plot is reduced relative to the top plot to emphasize the capacity transient that occurs upon voltage-clamping the whole-cell rather than a patch of the cell.

Fig. 5. Recorded Maxi K currents from voltage-clamped HIT-T15 cells in whole-cell configuration. The microchip access resistance (R_a) was 5.1 MΩ. Prior to transition to whole-cell mode, the cell-attached seal resistance was 150 MΩ. (A) The voltage pulse protocol for whole-cell mode measurements of K⁺ currents. (B) The K⁺ current recordings in whole-cell mode. (C) Recorded K⁺ currents from the same cell following the addition of 12.5 mM TEA to the top chamber. The capacity transients were manually removed from the current traces in order to focus on the ion channel activities.

Fig. 6. K⁺ currents from delayed rectifier K⁺ channels recorded from a voltage-clamped RAW 264.7 macrophage in whole-cell configuration. The microchip access resistance (R_a) was 1.7 MΩ. Before transitioning to whole-cell mode, the cell-attached seal resistance was 80 MΩ. K⁺ currents were measured in response to the indicated voltage pulse protocol. The capacity transient was manually removed to focus on the current measurements.

Fig. 7. A microfluidic channel designed to focus the cells over the region of the pore for patching experiments. (A) The design of the PDMS flow-through, focusing channel is illustrated with input and output channels (labeled) for the introduction and extraction of cells and fluids. The dot on the right side of the channel represents the pore that was micromachined into the underlying silicon substrate. (B) Two optical micrographs of the same PDMS microchannel. The left image shows the flow direction (arrow) as it passes through the 100 μm wide focusing channel into the 500 μm wide patching chamber that contains the micropore. The right image shows the region of the chamber that contains the micropore. The inset reveals a HIT-T15 cell that was localized to the micropore using laminar flow. Following suction, all excess cells were washed away using external buffer. (C) Whole-cell mode recordings of the same localized HIT-T15 cell, revealing the electrophysiological signatures of non-inactivating Maxi K-ion channels.