Ag/AgCl electrode

An Ag/AgCl electrode (silver/silver chloride electrode) is silver coated with a layer of silver chloride and immersed in a solution that contains chloride ions. It is the usual electrode in patch clamp and nanopore recording, because it passes current by a reversible reaction in which silver chloride is turned into silver and chloride, or the reverse, and its potential stays close to its resting value while it does so.

In a nanopore cell one such electrode sits in each of the two chambers on either side of the membrane. They apply a voltage of tens to hundreds of millivolts across the pore and carry the ionic current, which is picoamperes to nanoamperes for pores a few nanometers wide, to the transimpedance amplifier.

The electrode reaction is

so the potential follows the activity of chloride in the solution according to the Nernst equation, falling by 59.2 mV at 25 °C for each tenfold rise in activity. At unit chloride activity the standard potential is 0.22234 V on the hydrogen scale at 25 °C, the value Bates and Bower of the US National Bureau of Standards fixed in 1954 from measurements on cells of hydrogen, hydrochloric acid and silver chloride, 81 cells at 25 °C and at least 24 at every temperature between 0 and 95 °C.^[1]

Because silver chloride is a sparingly soluble solid, the electrode carries current without much change of potential for as long as some chloride is present in the solution and some silver chloride is left on the metal. At one electrode silver chloride is reduced and chloride enters the solution; at the other, silver is oxidized and chloride leaves it. A platinum electrode in the same salt has no such reaction available, charges the layer of ions at its surface like a capacitor, and at higher voltages splits water. The behavior of the silver electrode is the reason it is called non-polarizable, and it is why a pore can be held at a constant voltage for minutes or hours.^[2]

A chloride layer is grown on clean silver by making the metal the anode in a chloride solution, by soaking it in sodium hypochlorite (bleach), or by treating it with ferric chloride, in which iron(III) takes electrons from the silver while chloride deposits as silver chloride.^[2] Silver chloride darkens in light, as photographic film does, and the electrodes in many nanopore setups sit in a dark Faraday cage.

The electrode stays still only while its chloride supply holds up. The charge that a coating can deliver is set by its mass: a layer 10 µm thick over 1 mm² holds about 37 millicoulombs.^[a] At 1 nA that lasts more than a year. At 10 µA, about what a 10 µm pore carries at 100 mV in 1 M potassium chloride, it lasts about an hour, and the potential drifts as the layer wears away.

A second source of offset arises when the two chambers hold different salt concentrations. Each electrode then sits at its own Nernst potential, and the cell produces a voltage with nothing applied. Wanunu and colleagues, who put a 20-fold salt gradient across a 4 nm pore to raise the capture rate of DNA, found the offset between their homemade Ag/AgCl electrodes to be purely Nernstian, −0.058 V times the logarithm of the concentration ratio. For a 20-fold ratio that is 75 mV. They nulled it before every experiment with the voltage compensation of an Axopatch 200B amplifier or with an agarose bridge, and they worked in a dark Faraday cage.^[3]

Most nanopore cells use two electrodes, each far from the pore. The solution between them and the interfaces at their surfaces add a series resistance to the circuit, and the fraction of the applied voltage that is lost is R_s/(R_pore + R_s). A review of ionic phenomena in nanoscale pores puts the resistance of the bulk solution in experiments below 5 kΩ, against pore resistances of megohms to gigohms.^[4] Against 100 MΩ, 5 kΩ is 0.005% of the voltage, and the electrodes do not matter.

A four-electrode cell separates the electrodes that carry current from the electrodes that sense voltage, so that the potential drop at the current-carrying interfaces does not enter the measurement. The arrangement pays when the pore is a low-resistance one. In the simulations of the open MR1 Nanopore Reader design, the series resistance of two remote electrodes, about 8.8 kΩ, takes 25% to 40% of the applied voltage when the pore is 10 µm wide, with a resistance of 13 to 27 kΩ, and 82% to 88% when it is 100 µm wide.^[5]

The design draws four pre-chloridized silver ribbons, each 2.0 by 0.8 by 0.1 mm, cut from 99.9% silver foil that has been treated in 0.1 M ferric chloride. They sit in the fluid channels on the interposer board: a drive electrode and a sense electrode on the cis side, and a sense electrode and a current-return electrode on the trans side. Sense electrodes are drawn about 6.9 mm from the aperture, drive and return electrodes about 15.8 mm.^[6]

According to its specification, the arrangement leaves the amplifier measuring "the pure transmembrane ionic current with zero drift".^[6] Nothing has been built. The company's own status line for the project is "Designed. Simulated. Built and measured: not yet."^[7] A statement that the design can support is narrower than the specification's: the sense electrodes are placed to keep electrode polarization out of the voltage measurement.

• Four-electrode cell
• Patch clamp
• Potassium chloride
• Electric double layer