Electronic Machine Intelligence Lab (EMIL)

Priority: Feb. 20, 2007

Discusses a compartment (“cell”) structure wherein biological pores can be organized into a sensing structure. A means of interfacing this with an electronic reader is described. The patent claims are entirely made with regards to the cell.

• [0020] The inventors have also shown that a lipid bilayer will form across an aperture following the rehydration of dried lipids.
• [0023] As lipid bilayers are preferably formed from dried lipid, this allows the lipid to be stably stored in cell until it is needed to form a lipid bilayer.
• [0042] Cells having lipids deposited on an internal surface may be fabricated using methods including, but not limited to, drop coating, various printing techniques, spin coating,painting,dip coating and aerosol application.
• [0043] The lipids are preferably dried.
• [0048] The lipid bilayer is formed by introducing an aqueous solution into the chamber.
• [0052] By way of example, in one implementation, the aqueous solutions may be 10 mM PBS containing 1.0M sodium chloride (NaCl) and having a pH of 6.9.
• [0053] The lipid bilayer is formed as the interface moves past the aperture.
• [0054] The interface may move past the aperture as many times as is necessary to form the lipid bilayer.
• [0060] The invention preferably also involves inserting membrane proteins into the lipid bilayer once it has been formed.
• [0063] The lipids, the aperture and the membrane proteins may be covered by the aqueous solution in any order, although as already discussed the aqueous solution preferably covers the lipids first.
• [0065] The membrane proteins are preferably dried.

• [0162] Refers to Fig. 14 where the “cell” and “electrical reader” are shown (items 2 and 3 respectively).

• [0166] A thin membrane 10 (0.1–10-um thick) with an aperture 11 that is < 25 um holds the lipid bilayer.
• [0168] The membrane 10 is supported by two sheets 12 placed on either side of the membrane which leave a portion (a “window”13) of the membrane around the aperture exposed.

• [0169] The sheets 12 should be < 0.5-mm thick.
• [0170] Hollow chambers 16-1 and 16-2 are formed above and below the sheet/membrane complex (the “septum” 17).
• [0171] The chambers could be 1–3000-um high and each have a volume of 0.1–250 uL.
• [0188] You want to make the chamber low enough such that surface tension prevents the liquid from spreading throughout the cell (Fig. 20 below).

• [0189] Moving this liquid wall over the aperture in the direction of the arrow A creates the lipid bilayer.

• [0217] …, in one type of cell 2, the secondary chamber 16-2 may contain a gel 50 as shown for example in the cell 2 of Fig. 20.

• [0228-0233] Describe a Faraday cage addition to the apparatus.
• [0236] 63 is at virtual ground and is called the “working contact”, 62 sets the bias and is called the “reference contact”. …the reference contact 62 provides a bias voltage potential relative to the working contact 63, whilst the working contact 63 is at virtual ground potential and supplies the current signal to the circuit 90.
• [0240] The first amplifier stage 93 takes the input which could be 10s-100s pA and may (FOR EXAMPLE) use a 500-MΩ feedback resistance.
• [0241] With a 100-pA current this could produce a voltage of 50-mV. The second stage 94 should boost this by say 50 times to 2.5 volts such that it is appropriate for an ensuing microcontroller.
• [0241] If your 1st stage's bandwidth is not big enough to capture the whole signal use the second stage to boost signals in this frequency range. This is essentially a very simplistic equalization method and effectively adds more noise than other techniques. At these signal levels it may be ok, but still an opportunity for innovation. Is something like a decision feedback equalizer (DFE) possible? These may only be good for data not measurements.
• [0242] To save power, PNP BJTs are used to switch power rails ON/OFF to the analog circuitry.
• [0244] The A-to-D may be a SAR or a V-to-F type with a sampling rate at least twice the bandwidth of 94.

• [0250] The reference voltage of 62 is set by a LPF'd PWM waveform. This is a convenient way of getting digital signals to set an analog level.
• [0252] If you want to array it is suggested that you just use an analog multiplexer. That is multiple analog circuits 93 and 94 would be used and multiplexed into a single microcontroller.

• [0264] The state of the cell may be detected on the basis of predetermined thresholds or adaptive thresholds.
• [0266] Also store the time duration of a state.
• [0266] This could be indicative of the number of binding events. For example what if you are sensing multiple pores (with one channel) the multiple binding events may combine to appear as signals with variable time durations.
• This processing strategy is referenced in Dec. 01, 2009 patent.

• [0270] With the reader empty (and essentially capacitive since there is no cell placed between the reader's electrodes) apply a 20-mV amplitude 50-Hz triangular waveform (centred at 100-mV) to make sure that you measure the appropriate (20-pA) square wave current. This is state S1.

• [0271] After inserting a dry cell a similar (albeit with changed amplitude) current should be measured. Expect say a 25% increase in the measured current.
• [0273] With the ionic fluid introduced into the cell a large current should flow thus saturating the amplifier current output and therefore indicating that the cell now contains ionic fluid.

• [0274] When the lipid bilayer forms you should again see a drop in the current (you have re-established a capacitance). Typically 250-pA amplitude centred at 0 pA. Typical DC resistance is 10 GΩ.

• [0277] Protein insertion into the bilayer can be sensed. So you know how many pores you have in your bilayer as they interface with it.