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US-12625129-B2 - Systems and methods for inertial-kinetic capture and sensing of single molecules

US12625129B2US 12625129 B2US12625129 B2US 12625129B2US-12625129-B2

Abstract

The subject invention pertains to a nanopore sensing device for inertial-kinetic translocation and sensing of single molecules. Some embodiments comprise a centrifuge rotor; a centrifuge tube; single or multiple flow cell modules; a nanopore module consisting of single or multiple nanopores; a signal detection module; a signal amplifier module; a control module; and a wireless communication module. Through the kinetic regulation of a centrifugal force field while maintaining a counter-balanced state of electrophoretic and electroosmotic forces in the nanopore by adjusting the pH value of the electrolyte in nanopore or the surface charge excited on the silicon nanopore using visible light, the precise regulation of molecular translocation parameters, such as speed, direction, and molecular selectivity, is provided for optimizing the temporal and spatial resolutions of molecular sensing with high S/N ratio signal readout.

Inventors

  • Ho-Pui HO
  • Jianxin Yang
  • Wu Yuan

Assignees

  • THE CHINESE UNIVERSITY OF HONG KONG

Dates

Publication Date
20260512
Application Date
20230929

Claims (17)

  1. 1 . A nanopore sensing device, comprising: a centrifuge rotor ( 1 ); a centrifuge tube ( 2 ); and a nanopore module comprising single or multiple nanopores ( 4 ) located within the centrifuge tube ( 2 ), wherein each nanopore ( 4 ) has radial asymmetry about a central axis.
  2. 2 . A nanopore sensing device, comprising: a centrifuge rotor ( 1 ); a centrifuge tube ( 2 ); a nanopore module comprising single or multiple nanopores ( 4 ) located within the centrifuge tube ( 2 ); single or multiple flow cell modules ( 3 ) and ( 5 ) separated by the nanopores ( 4 ); and a signal detection module ( 6 ) spanning the nanopore ( 4 ).
  3. 3 . The nanopore sensing device of claim 2 , comprising: a signal amplifier module ( 7 ); a control module ( 8 ); and a wireless communication module ( 9 ).
  4. 4 . The nanopore sensing device of claim 3 , wherein the signal amplifier module ( 7 ), the control module ( 8 ), and the wireless communication module ( 9 ) are each, respectively, mounted within and configured and adapted to rotate with the centrifuge tube ( 2 ).
  5. 5 . The nanopore sensing device of claim 4 , wherein the centrifuge rotor ( 1 ) is configured and adapted to provide different rotation speed and direction to the centrifuge tube ( 2 ).
  6. 6 . The nanopore sensing device of claim 4 , wherein the signal detection module ( 6 ) spanning the nanopore module ( 4 ) is configured and adapted to provide different voltage potential.
  7. 7 . The nanopore sensing device of claim 6 , wherein the signal amplifier module ( 7 ) is configured and adapted to detect a sensing signal flowing over time across the detection module ( 6 ) spanning the nanopore module ( 4 ).
  8. 8 . The nanopore sensing device of claim 7 , wherein the control module ( 8 ) is configured and adapted to form a digital representation of the signal detected by the signal amplifier module ( 7 ).
  9. 9 . A method for inertial-kinetic translocation and sensing of single molecules, the method comprising: providing a nanopore sensing device, that comprises a nanopore, a driving circuit, a sensing circuit, and a communications device, each respectively located within a centrifuge tube; applying, via the driving circuit, an electrical potential across the nanopore; applying, via operation of the centrifuge tube, a centrifugal force across the nanopore; driving through the nanopore, via inertial-kinetic translocation, a molecule to be measured; and capturing, via the sensing circuit, a digital representation of a sensing signal across the nanopore over time.
  10. 10 . The method according to claim 9 , comprising: transmitting, via the communications device, the digital representation of the sensing signal; and receiving, at a location outside the centrifuge tube, the digital representation of the sensing signal.
  11. 11 . The method according to claim 9 , comprising: extracting, from the digital representation of the sensing signal, a characteristic feature.
  12. 12 . The method according to claim 11 , wherein the characteristic feature is selected from the group consisting of an amplitude of a signal pulse amplitude; a dwell time of a signal pulse; a localized peak value in one of the foregoing; a mean, median, or standard deviation of a series of any of the foregoing; a decay time in any of the foregoing; a change in one of the foregoing; a difference between two of the foregoing; and a count of any of the foregoing.
  13. 13 . The method according to claim 11 , wherein the characteristic feature comprises a dwell time or a pulse amplitude.
  14. 14 . The method according to claim 11 , wherein the characteristic feature comprises a ratio of a second-peak amplitude to a first-peak amplitude.
  15. 15 . The method according to claim 11 , wherein the characteristic feature comprises a count of the number of peaks per molecule translocation.
  16. 16 . A nanopore sensing device, comprising: a centrifuge rotor ( 1 ); a centrifuge tube ( 2 ); a nanopore module consisting of single or multiple nanopores ( 4 ) located within the centrifuge tube ( 2 ); single or multiple flow cell modules ( 3 ) and ( 5 ) separated by nanopore module ( 4 ); a signal detection module ( 6 ) spanning the nanopore ( 4 ); a signal amplifier module ( 7 ); a control module ( 8 ); and a wireless communication module ( 9 ); wherein the signal amplifier module ( 7 ), the control module ( 8 ), and the wireless communication module ( 9 ) are each, respectively, mounted within and configured and adapted to rotate with the centrifuge tube ( 2 ); wherein the centrifuge rotor ( 1 ) is configured and adapted to provide a rotation speed between 1,000 and 4,000 revolutions per minute (rpm) to the centrifuge tube ( 2 ); wherein the nanopore structure is wide applicable; wherein the nanopore has an adjustable pore size; and wherein adjustable pore size is adjustable within a range of nanometers.
  17. 17 . The nanopore sensing device of claim 16 , wherein the signal detection module ( 6 ) spanning the nanopore module ( 4 ) is configured and adapted to provide a voltage potential between 0.3 volts (V) and 0.6 V; wherein the preamplifier circuit board ( 7 ) is configured and adapted to detect a sensing signal flowing over time across the signal detection module ( 6 ) spanning the nanopore module ( 4 ); and wherein the microcontroller with the control module ( 8 ) is configured and adapted to form a digital representation of the sensing signal detected by the signal amplifier module ( 7 ).

Description

TECHNICAL FIELD OF THE INVENTION The invention relates to high sensitivity measurement devices. More particularly the invention relates to improved systems and methods of nanopore sensing. BACKGROUND OF THE INVENTION When a stream of molecules drift through a nanometer-size pore (also known as nanopore) driven by a potential difference imposed across the nanopore, the ionic channel is temporally blocked and resultant sensing signals (in terms of current, voltage, resistance, conductance) are generated [ACS Chem. Biol. 2012, 7, 1935-1949; Phys. Chem. Chem. Phys., 2022, 24, 19948-19955]. Nanopore sensing is a technique realised by measuring the sensing signals with electrodes across the nanopore [Nat. Nanotech. 12, 360-367 (2017); Nat. Nanotech. 17, 708-713 (2022); Nat. Nanotech. 17, 976-983 (2022); Nature Reviews Materials 2020, 5 (12), 931-951]. This technique can be integrated into portable sensing devices with electronics [K. Chuah, et al. Nature Communications 2019, 10, 2109]. Indeed, the so-called nanopore sequencing technique has made significant contributions in many branches of life sciences in the last two decades [N. S. Galenkamp, et al. Nature Communications, 9, 4085 (2018); Bayley, H. Nanopore sequencing: from imagination to reality. Clin. Chem. 61, 25-31 (2015).] In principle, a nanopore of appropriate structural dimension can resolve the sizes and configurations of the molecules in question [Phys. Life rev. 9, 125-158 (2012).]. Electrokinetic translocation of a single molecule is commonly utilized in nanopore, and the speed of translocation is determined by the electrophoretic force and the viscous drag of the molecules in the solution and the pore. However, the translocation speed of molecules is challenging to control in nanopore, leading to the sensing signals of short dwell time and low conformation sensitivity. High translocation speed and low conformation sensitivity on signal readings have limited the accuracy of nanopore in molecular discrimination [Adv. Mater. 2018, 30, 1704680; Venkatesan, B. M. & Bashir, R. Nanopore sensors for nucleic acid analysis. Nat. Nanotechnol. 6, 615-624 (2011)]. Although speed control with protein motors has been successfully demonstrated with biological nanopores, it remains challenging to achieve a stable feed rate of the protein motor and a high conductance drop as in solid-state nanopores [Fragasso, ACS nano, 2020, Brinkerhoff, Science 2021]. On the other hand, a nanopositioner has been utilized to achieve controlled translocation in glass nanopore. However, this method requires tethered molecules, inhibiting them from fully translocating [Leitao Nat Nanotec 2023]. BRIEF SUMMARY OF THE INVENTION Embodiments of the subject invention provide systems and methods to drive nano-sized objects through a nanopore by centrifugation, so that the molecules under investigation experience inertial-kinetic controlled translocation and regulated dwell time in nanopore with high conformation sensitive signal readouts. The use of inertial forces generated by centrifugation has effectively decoupled the single-molecule translocation process from experimental parameters (such as ionic strength and bias voltage) and signal detection process which use the same pair of electrodes applying bias voltage crossing the nanopore. In certain embodiments the electrophoretic and electroosmotic forces are effectively counter balanced by adjusting the pH value of the electrolyte in nanopore or the surface charge excited on the silicon nanopore with using light, while the electric field still covers the nanopore as an independent sensing method. While electrokinetic translocation commonly results in high and uncontrolled translocation speeds of single molecules in nanopore and non-uniform conductance signals of low conformation sensitivity and short dwell time from micro- to milli-seconds [Tang, L. et al. Nat. Commun. 12, 913 (2021).], the inertia-kinetic translocation can effectively control the speed and direction of single-molecule translocation, leading to an unform sensing readouts of high conformation sensitivity and long dwell time up to hundreds of milli-seconds and a capability of programmable and selective sensing of single molecules from the complex made of multiple molecules. In addition, the inertia-kinetic translocation can help realize the reversible sensing and selective translocation of single molecules, achieving repetitive and addressable sensing of molecules with high spatial and temporal resolution. Therefore, a sensing signal, including its signal-to-noise ratio and dwell time, can be optimized by independently controlling the centrifugal force in nanopore to achieve a highly distinguishable molecular fingerprint of single molecule with improved detection limit. Embodiments provide a centrifuge tube like in-tube nanopore sensing device, which can be conveniently placed in the centrifuge machine to achieve an inertial-kinetic nanopore sensing system. Such in-tube nan