Dual pore—control and sensor device
Two-pore devices and method for sequencing are described. A two-pore device can include first chamber, a second chamber, and a third chamber, wherein the first chamber is in communication with the second chamber through a first nanopore, and wherein the second chamber is in communication with the third chamber through a second nanopore. The device can also include sensing circuitry for measuring electrical signals associated with a target at a nanopore, and control circuitry for controlling motion of the target at a nanopore. The device can include and/or switch between sensing and control modes for each of the first nanopore and the second nanopore. Sequencing methods can implement a two-pore device in relation to translocation of a target through one or more nanopores, switching between sensing and control modes as appropriate, and measuring aspects of the target using in sensing modes.
1. A two-pore device comprising:
an upper chamber, a middle chamber and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first nanopore, and wherein the middle chamber is in communication with the lower chamber through a second nanopore,
a sensing circuitry, a control circuitry, and a switch controlling connectivity to the sensing circuitry and the control circuitry for each of the first nanopore and second nanopore,
each sensing circuitry configured to, when connected to the corresponding switch, apply a constant voltage across the corresponding nanopore and further configured to measure sensing current through the corresponding nanopore,
each control circuitry configured to, when connected to the corresponding switch, apply a dynamic voltage across the corresponding nanopore, the applied dynamic voltage determining a controlled motion of a charged polymer across the first nanopore and the second nanopore, wherein the applied dynamic voltage is applied using a direct current-biased alternating current signal source, and
wherein the sensing current is derived from the charged polymer during the controlled motion.
2. The two-pore device of claim 1 , wherein each sensor circuitry comprises a transimpedance amplifier.
3. The two-pore device of claim 2 , wherein the transimpedance amplifier is one of a patch clamp or voltage clamp amplifier.
4. The two-pore device of any one of claim 1 , wherein each control circuitry comprises a phase lock loop (PLL).
5. The two-pore device of claim 4 , wherein the control circuitry of the first nanopore is configured to generate an oscillatory voltage output based feedback from the sensor circuitry of the second nanopore.
6. The two-pore device of claim 5 , wherein a phase difference between a frequency of the oscillatory voltage output and the frequency of the sensing current is fixed over time.
7. The two-pore device of claim 5 , wherein the oscillatory voltage output is provided to a voltage-controlled amplifier (VCA) that applies the dynamic voltage across the second nanopore.
8. The two-pore device of claim 1 , wherein the middle chamber is electrically coupled as an electrical return path for both the sensor circuitry and the control circuitry of at least one of the first nanopore or second nanopore.
9. The system of claim 1 , comprising a first operation mode in which the switch of the first nanopore is connected to the sensing circuitry of the first nanopore and the switch of the second nanopore is connected to the sensing circuitry of the second nanopore.
10. A two-pore device comprising:
an upper chamber and a lower chamber, wherein the upper chamber is in communication with the lower chamber through a first nanopore and a second nanopore,
a sensing circuitry a control circuitry, and a switch controlling connectivity to the sensing circuitry and the control circuitry for each of the first nanopore and second nanopore,
each sensing circuitry, when connected to the corresponding switch associated with the corresponding nanopore, configured to apply a constant voltage across the corresponding nanopore and further configured to measure sensing current through the corresponding nanopore,
each control circuitry, when connected to the corresponding switch associated with the corresponding nanopore, configured to apply a dynamic voltage across the corresponding nanopore, the applied dynamic voltage determining a controlled motion of a charged polymer across the first nanopore and the second nanopore, wherein the applied dynamic voltage is applied using a direct current-biased alternating current signal source, and
wherein the sensing current is derived from the charged polymer during the controlled motion.
11. The two-pore device of claim 10 , wherein the device further comprises a first membrane layer that includes the first nanopore, a second membrane layer that includes the second nanopore, and a conductive middle layer between the first membrane and second membrane layer.
12. The two-pore device of claim 11 , wherein the conductive middle layer is electrically coupled as an electrical return path for both the sensor circuitry and the control circuitry of at least one of the first nanopore or second nanopore.
13. A method for sequencing a polynucleotide, the method comprising:
loading a sample comprising the polynucleotide in a first chamber of a nanopore device;
translocating the polynucleotide through a first nanopore by using a sensing circuit of the first nanopore to apply a first voltage across the first nanopore;
translocating the polynucleotide through a second nanopore by using a sensing circuit of the second nanopore to apply a second voltage across the second nanopore;
adjusting the first voltage applied across the first nanopore by reversing a polarity of the first voltage applied by the sensing circuit of the first nanopore;
adjusting the second voltage across the second nanopore by disconnecting a switch associated with the second nanopore from the sensing circuit of the second nanopore and connecting the switch to a control circuit of the second nanopore, wherein the adjusted second voltage generates an electrical force that opposes an electrical force generated by the adjusted first voltage, and wherein the adjusted second voltage is applied using a direct current-biased alternating current signal source; and
identifying nucleotides of the polynucleotide.
14. The method of claim 13 , wherein the adjusted second voltage is an oscillating control circuit voltage and wherein identifying nucleotides of the polynucleotide comprises:
for an initial oscillation of the applied oscillating voltage, detecting a first change in ionic current across the first nanopore corresponding to a first translocation of a nucleotide of the polynucleotide; and
for a subsequent oscillation of the applied oscillating voltage, detecting a second change in ionic current across the first nanopore corresponding to a second translocation of the nucleotide of the polynucleotide.
15. The method of claim 14 , wherein the adjusted second voltage is applied based on a feedback signal captured by the sensing circuit of the first nanopore.
16. The method of claim 15 , wherein the feedback signal is a current measured across the first nanopore, the current being a measure of polynucleotide movement across the first nanopore.
17. The method of claim 16 , wherein the measure of polynucleotide movement is a measure of one of polynucleotide position, velocity, or acceleration.
18. The method of claim 15 , wherein the feedback signal is designed in either a frequency domain or a time domain by using one of feedforward or feedback.
19. The method of claim 15 , wherein the feedback signal is designed using an estimator and filter that are co-designed to estimate molecule-induced changes in current.
20. The method of claim 13 , wherein the adjusted second voltage is applied with a frequency range between 0.001 Hz and 100 MHz and an amplitude range between 0.001 mV and 10 V.