Patch clamp tips

Fig. 4.24. Gold plated patch clamp tips (corteousy E. Neher). An oscillating field of 12 V at 100 Hz was applied (a) and the fluctuation of TMR labeled DNA molecules and/or nucleotide was measured (b) [50]...

The patch-clamp technique is based on the formation of a high resistance seal (109-10lon) between the tip of a glass micropipette and the cell membrane it touches (gigaohm-seal). This technique allows recordings of ionic currents through single ion channels in the intact cell membrane and in isolated membrane patches at a... 

In the whole cell model, a gigaseal is formed as the pipette is attached to the cell, and then a more dynamic suction is applied, which causes the interior of the cell to be sucked into the pipette tip (Fig. 3a). This action allows current and conductance of the entire cell to be measured. Therefore, the whole cell model measures changes caused by many ion channels on the entire cell membrane. Additionally, the liquid content of the cell will mix and equilibrate with the solution in the pipette, which allows pharmacological agents to be administered into the cell. Of the patch clamp techniques, the whole cell method is the most common and can be used to determine how pharmacological agents affect the total conductance of neurons. 

In the outside-out model, the pipette is attached to the entire cell as in the whole cell model, followed by a sharp pull that causes the cell membrane to break and reseal with the pipette tip (Fig. 3b). With the extracellular region exposed, channel activity as a response to different external stimuli can be probed. This configuration is less common than the inside-out method. Using an outside-out method, single-channel opening activity has been recorded while various neurotransmitters were released. For example, this patch clamp method was used as a detector for capillary electrophoresis separations of GABA, glutamate, and NMDA (7). 

Lepple-Wienhues A, Ferlinz K, Seeger A, Schafer A (2003) Flip the tip an automated, high quality, cost-effective patch clamp screen. Recept Channels 9(1) 13-17... 

The potential probes employed so far have all consisted of glass tubes that were pulled out to a capillary at the tip and contained a reference electrode inside the tube. As mentioned earlier, the diameter of the tip restricts the spatial resolution in the second setup. With the patch clamp technique, possible to produce capillaries with openings as small... 

This technique allows the study of single-ion channels as well as whole-cell ion channel currents. Essentially, the patch-clamp technique is an improved and refined version of the voltage-clamp technique. It requires a low electrical noise borosiUcate glass electrode, also known as a patch electrode or patch pipette, with a relatively large tip (>1 pm) that has a smooth surface rather than a sharp tip as with the conventional microelectrodes. This is a major difference between the patch electrode and the sharp electrode used to impale cells directly through the cell membrane (Figure 16.20). 

Figure 16.20 Comparison between the conventional (sharp) microelectrode and patch-clamp technique. The sharp microelectrode Is less than 1pm at the tip and Impales the cell, whereas the patch-clamp electrode Is over 1pm at the tip and forms a gigaseal (>10 G l) with the cell membrane.

The patch-clamp electrode is pressed against a cell membrane and suction is applied to pull the cell membrane inside the electrode tip. The suction causes the cell to form a tight, high-resistance seal with the rim of the electrode, usually greater than 10 giga Ohms, which is called a gigaseal (Figures 16.20 and 16.21). 

The sequence of open and closed states of an individual ion channel can be revealed by the patch clamp technique developed by Neher, Sakmann, Sigworth, Hamill, and others (I, 2). A small piece of cell membrane, which may contain one ion channel, is sealed in the tip of a micropipette. The picoampere currents when the channel is open can be resolved. Thus, as shown in Figure 1, the durations of the times spent in the open and closed states can be measured. Because this measurement is performed on a single protein molecule, it is a much more sensitive probe of protein kinetics than other biochemical or biophysical techniques that average their signals over many molecules in different states. 

In order to avoid the effect of membrane tension when pressing an AFM tip against the membrane, we can use the method of confocal microscopy, although with a lower sensitivity. Consider a spherical cell of radius R and area Aq that is whole-cell patch clamped. Apply a negative holding potential AC/ to the patch pipette, i.e. to the cell interior. In this case... 

Patch Clamp Measurements On-Chip, Fig. 3 Multipipette tip on-chip fabrication... 

Measrirements with bilayers formed on the tip of a glass micropipette ( dip-stick bilayers ) using dialysis liposomes were accomplished as previously described (4). For patch clamp measurements, giant liposomes were formed from the dialysis liposomes using the del dration/rehydration technique (5). 

Measuring the electrode resistance The filled patch pipette is mounted on the holder. At this point, a positive pressure is applied to the pipette, to avoid the tip collecting dust when it crosses the air-water interface when it is introduced into the bath, or collecting debris from the preparation when it is already in the bath. Meanwhile, a small voltage signal, such as 2 mV and 2 ms pulses, is input to the patch-clamp amplifier command circuit. The recorded current signal will mainly correspond to the holder and pipette associated capacity, and no resistive current is observed (Fig. 5A). 

K ionophore sensor (61). To make -selective SECM tips, borosilicate capillaries (o.d./i.d. 1.0/0.58 mm) are cleaned in a 1 1 (v/v) mixture of concentrated sulfuric acid and 30% hydrogen peroxide overnight, washed and dried at 120 °C for 30 min. Using a laser-based pipette puller, patch clamp type pipettes are produced (orifice radius = 0.7-20 pm). The inner wall of each pipette is silanized with a toluene solution of trimethylchlorosilane (5-100% v/v). The solution is removed from the pipette after 5-30 min, emptied using a syringe, and then connected to a vacuum pump to remove any residual silanizing vapor. The experimental system can be represented by the following cell ... 

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