A local reverse electric field gated nanopore single molecule speed regulation system and method

Abstract

The present application relates to the technical field of nanometer hole precision instrument control, and particularly relates to a local reverse electric field gate nanometer hole single molecule speed regulation system and method. The system comprises a nanometer hole chip, a buffer pool, a main driving double electrode, a liquid microelectrode probe, a sub-micron three-axis micro-operation module, a high-frequency membrane patch amplifier and an upper computer; biological macromolecules are captured remotely by a global main driving electric field, a local reverse potential barrier is output by the liquid microelectrode probe to weaken the molecular net electrophoresis force, and the sub-micron three-axis micro-operation module is used to realize nanometer-level non-contact positioning of the probe and hardware emergency stop collision avoidance. The present application linearly extends the time of biological macromolecules passing through the hole under the premise that an exponential cliff attenuation of the molecular capture rate is not caused by spatial decoupling of the global electric field and the local reverse electric field, and fine current step signals corresponding to the dynamic conformation of proteins in the hole are completely retained.

Description

Technical Field

[0001] This invention relates to the field of nanopore precision instrument control technology, and in particular to a nanopore single-molecule speed regulation system and method with local reverse electric field gating. Background Technology

[0002] In existing solid-state or glass nanopore single-molecule detection techniques, it is typically necessary to apply a transmembrane bias voltage in the hundreds of millivolts (e.g., +100mV to +500mV) across an insulating film. This voltage creates a strong global electrophoretic driving force, prompting charged biomolecules to shuttle through the nanopores and generating a characteristic ion current blocking signal.

[0003] However, the loss of features caused by extremely fast through-hole penetration has been a long-standing pain point in this field. A core technical contradiction exists in conventional two-electrode systems: a higher main driving voltage is more conducive to overcoming the orifice entropy barrier to capture molecules and obtain a larger blocking current signal; however, an excessively high main driving electric field leads to excessively fast molecular through-hole speeds (often in the sub-millisecond range), causing high-frequency signals to be truncated by the patch-clamp's low-pass filter, resulting in the loss of waveform details at the bottom. Conversely, simply reducing the main driving voltage across the orifice, while slowing down the through-hole speed, leads to an exponential and precipitous decline in the molecular capture rate.

[0004] Existing technologies have attempted various solutions to address the aforementioned contradictions, but all have significant limitations in their applicability: 1. Limitations of solid-state integrated gated electrodes: Existing literature discloses the integration of gated electrodes inside or on the surface of solid nanopores to slow down molecular permeation. However, once the solid electrode is fabricated, the spatial distance between it and the pore opening is permanently fixed, making the fabrication extremely complex and not dynamically adjustable. At the same time, the extremely thin dielectric layer between the electrode and the conductive solution introduces huge parasitic capacitance, which severely compresses the high-frequency detection bandwidth.

[0005] 2. The destructive nature of mechanical contact manipulation: For example, the invention patent with publication number CN112941160B uses a three-dimensional platform to drive a solid nanoprobe to mechanically manipulate DNA molecules between two nanopores. In the detection stage, this technology is essentially still a two-electrode system, with no gating voltage applied to the third electrode. This hard-contact physical manipulation method easily alters the three-dimensional folding conformation of complex proteins, making it difficult to meet the requirements for non-destructive, high-resolution analysis at the single amino acid level.

[0006] 3. Spatial Positioning and Electrochemical Challenges of External Electrodes: Attempting to apply a localized reverse electric field using an external probe presents stringent spatial positioning challenges. If the probe is too far from the pore opening, the reverse electric field will diffuse or be shielded in the solution, making it difficult to effectively weaken the net electrophoretic force within the pore; if the probe is too close, it will cause nanopore blockage, impact damage, or background current drift. Furthermore, conventional microscopic positioning or mechanically preset coordinates for probes are simply insufficient to meet the requirements for non-destructive positioning at the nanoscale. Summary of the Invention

[0007] One objective of this invention is to propose a locally reverse electric field-gated nanopore single-molecule speed control system, overcoming the inherent defects of existing two-electrode nanopore systems, solid-state integrated gated electrodes, and mechanical contact probe manipulation schemes.

[0008] Another objective of this invention is to propose a locally reverse electric field-gated nanopore single-molecule speed regulation method, which is applied to the aforementioned speed regulation system. By decoupling the global capture electric field from the local reverse potential barrier spatial function, and combining it with ion current hardware direct-through tracking, hysteresis-free parallel micro-manipulation, and insulating liquid microelectrode probes, the passage time of biomacromolecules through the pore is linearly extended without causing an exponential drop in the molecular capture rate. This fully preserves the fine current step signal corresponding to the dynamic conformation within the protein pore, achieving fully automated, contactless, non-destructive, and high-resolution single-molecule detection.

[0009] To achieve this objective, the present invention adopts the following technical solution: A locally reverse electric field-gated nanopore single-molecule speed control system includes a nanopore chip, a buffer cell, a main driving dual electrode, a liquid microelectrode probe, a submicron-level triaxial micromanipulation module, a high-frequency patch-clamp amplifier, and a host computer. The nanoporous chip is disposed inside the buffer pool, which is filled with buffer solution; the nanoporous chip includes an insulating film and a tapered nanopore penetrating the insulating film. The main driving dual electrode includes a cis-driving electrode and an anti-driving electrode, which are respectively located on both sides of the nanopore; both the cis-driving electrode and the anti-driving electrode are electrically connected to the high-frequency patch-clamp amplifier, the cis-driving electrode is set as the system global reference ground, and the anti-driving electrode is connected to the main current detection input terminal of the high-frequency patch-clamp amplifier; The high-frequency patch-clamp amplifier is used to output a positive transmembrane bias voltage to the main driving dual electrodes to construct a global main driving electric field, to collect the ion current signal of the nanopore channel in real time and transmit it back to the host computer, and to receive digital commands issued by the host computer and output the detection voltage, main capture voltage and reverse gating voltage accordingly. The submicron-level triaxial micromanipulation module is equipped with a microelectrode holder, which mechanically holds the liquid microelectrode probe. The submicron-level triaxial micromanipulation module is used to drive the liquid microelectrode probe to complete three-dimensional displacement and servo locking actions within the buffer pool. When the liquid microelectrode probe is immersed in the buffer solution, it can output a reverse bias voltage and form a local reverse potential barrier superimposed on the global main driving electric field. The host computer is connected to the high-frequency patch-clamp amplifier and the submicron-level three-axis micro-manipulation module via a digital communication link. The host computer has a built-in closed-loop control algorithm, coordinate analysis program and data monitoring interface. The host computer is used to receive ion current signals, issue motion commands or emergency stop commands to the submicron-level three-axis micro-manipulation module, and schedule the high-frequency patch-clamp amplifier to perform smooth switching of multiple voltage levels.

[0010] Preferably, the submicron-level three-axis micro-manipulation module includes a micro-manipulation body, a drive component, and an end effector component; The micro-operation body is equipped with a main control board, which integrates a dual-core microcontroller. The dual-core microcontroller includes a first core and a second core. The first core is used to perform three-dimensional forward kinematics calculation and three-dimensional inverse kinematics calculation, and the second core is used to exclusively run the cascaded magnetic field orientation FOC three-loop control algorithm. The three sets of drive components are integrated on the outside of the micro-operation body, and the end effector is located at the output end of the three sets of drive components. The three sets of drive components drive the end effector to achieve independent movement of the X, Y and Z axes respectively. The single drive assembly includes a stepper motor, a sector rotor horn, a connecting rod, a miniature ball joint, an elastic preload element, and a magnetic gear encoder feedback unit; The end effector assembly includes an end connector block, a flexible pressure block, and a silicone sleeve, wherein the silicone sleeve is fitted onto the microelectrode holder. The output end of the stepper motor is fixedly connected to the center of the fan-shaped rotor corner. The edge of the fan-shaped rotor corner is arranged with a magnet array. The magnetic gear encoding feedback unit is arranged close to the magnet array. The magnetic gear encoding feedback unit feeds back the absolute rotation angle value of the stepper motor to the dual-core microcontroller in real time. The magnetic gear encoding feedback unit includes a magnetic encoder and a magnetic encoder chip. The connecting rod is hinged at both ends to the fan-shaped rotor horn and the end connecting block via the micro ball joint, and the elastic preload is provided at the micro ball joint to eliminate transmission backlash. The flexible pressure block is fixed to the end connector block, one end of the silicone sleeve is embedded in the flexible pressure block, and the other end of the silicone sleeve is sleeved and fixed to the microelectrode holder, which holds the liquid microelectrode probe.

[0011] Preferably, each group of stepper motors is equipped with a charge pump-free pure MOSFET H-bridge drive circuit, which receives the control signal output by the dual-core microcontroller to drive the stepper motor windings. The cascaded field-oriented FOC three-loop control includes an outer position loop, a middle speed loop, and an inner 30kHz current loop. It is used to force the excitation current component Id=0, adjust only the torque current component Iq, and directly generate a reverse braking current in the current loop to achieve rigid braking when receiving a hardware emergency stop signal.

[0012] After being mapped by the three-dimensional forward kinematics calculation and the three-dimensional inverse kinematics calculation, the magnetic gear encoding feedback unit enables the end effector to have a linear positioning resolution of 50nm.

[0013] Preferably, the liquid microelectrode probe includes a conical insulating shell, the interior of which is filled with an electrolyte and contains an Ag / AgCl electrode wire; The conical insulating shell is divided into a straight tube section and a pointed conical section. The pointed conical section has a pointed opening. The conical insulating shell radiates a local electric field outward only through the pointed opening, which is used to apply a reverse bias voltage to construct a local reverse electric barrier in the throat region of the nanopore. The Ag / AgCl electrode wire is embedded in the straight section of the conical insulating shell and does not extend into the pointed conical section; After the lead wire of the Ag / AgCl electrode wire passes through the reserved hole of the straight section of the conical insulating shell, it is sealed by secondary vacuum dripping and heating with low viscosity modified epoxy resin to prevent the electrolyte from leaking.

[0014] Preferably, the main control board is also provided with a high-speed analog comparator circuit, and the dual-core microcontroller is provided with a hardware external interrupt pin; The high-frequency patch-clamp amplifier is equipped with a BNC analog output channel, which maps the ion current into an analog voltage and connects it to the analog input terminal of the high-speed analog comparator circuit. The analog output terminal of the high-speed analog comparator circuit is connected to the hardware external interrupt pin of the dual-core microcontroller. When the ion current decays to a preset threshold, the high-speed analog comparator circuit undergoes a level flip and directly transmits the flip signal to the hardware external interrupt pin of the dual-core microcontroller. A hardware interrupt signal is generated in microseconds, bypassing software polling, and the cascaded magnetic field-oriented FOC three-loop control algorithm is directly activated to perform emergency braking.

[0015] Preferably, the host computer has built-in dual pre-alignment logic: The first pre-alignment logic is optical coarse alignment, which reduces the lateral deviation between the liquid microelectrode probe and the nanopore to the micrometer level by imaging with a long working distance objective lens; The second pre-alignment logic is an electrical fine pre-scan, which raises the liquid microelectrode probe to a safe height to perform a two-dimensional grating scan and locks the geometric center of the nanopore based on the extreme value of the ion current distribution.

[0016] A locally reverse electric field-gated nanopore single-molecule velocity modulation method, applied to a locally reverse electric field-gated nanopore single-molecule velocity modulation system as described above, includes the following steps: S1. Planar pre-alignment: The liquid microelectrode probe is locked above the nanopore coaxially through a dual process of optical coarse alignment and electrical grating fine scanning; S2. Construct an independent tracking and detection loop: Control the main driving dual electrodes to maintain zero potential silence, schedule the high-frequency patch clamp amplifier to output a detection voltage to the liquid microelectrode probe, and collect a stable initial baseline ion current I0. S3, Z-axis microstepping approximation: The submicron-level triaxial micro-manipulation module is controlled to drive the liquid microelectrode probe to move downward at a constant speed along the Z-axis, and the high-frequency patch-clamp amplifier transmits the instantaneous ion current It in real time; S4. Hardware Servo Locking: It and I0 are compared in real time. When It is lower than the current decay threshold of 0.85I0, the high-speed analog comparator circuit flips the level and sends the flip signal directly to the hardware external interrupt pin of the dual-core microcontroller. The hardware interrupt signal is generated in microseconds, bypassing software polling, and the cascaded magnetic field orientation FOC three-loop control algorithm is directly started to perform emergency stop braking and coordinate servo locking. The liquid microelectrode probe and the nanopore convergence range maintain a safe gap of 3~15nm. S5. Smooth mode switching: After locking, wait for the mechanical vibration to decay, and schedule the high-frequency diaphragm clamp amplifier to complete the ramp switching of the probe voltage returning to zero, the main drive voltage rising, and the reverse gate voltage falling with a fixed linear slope, so as to eliminate the step capacitive current spike. S6. Single-molecule detection speed regulation: The global main driving electric field captures biomacromolecules, the local reverse potential barrier weakens the net electrophoretic force of molecules, collects the waveform of the spread-type ion blocking current, and analyzes the secondary current fluctuations corresponding to the dynamic conformation in the molecular pore. S7. Quantitative linear speed regulation: Continuously adjust the reverse negative pressure output by the liquid microelectrode probe to achieve a continuously controllable extension of the biomolecule passage time, stable molecular blocking signal amplitude, and no exponential cliff attenuation of molecular capture rate.

[0017] Preferably, the gate voltage output by the liquid microelectrode probe can be selected as either DC mode or high-frequency pulse AC mode; The high-frequency pulse AC mode is limited to a frequency of 1~10kHz and a duty cycle of 20%~80%, and is used for long-term continuous detection to suppress the accumulation of microbubbles at the nozzle interface.

[0018] Preferably, the submicron-level three-axis micro-manipulation module uses an RP2350 dual-core microcontroller to execute the forward and inverse kinematic equations to convert the end coordinates to the stepper motor rotation angle. The inverse kinematic equations are as follows:

[0019] The length constraint equation for the connecting rod is as follows:

[0020] Substituting the inverse kinematic equations into the length constraint equations of the link, and rearranging, we obtain the trigonometric standard equations:

[0021] The set of equations for the positive kinematic spherical constraint:

[0022] After elimination, a linear mapping relationship is obtained:

[0023] Back substitution yields the quadratic characteristic equation:

[0024] in, , , These are the absolute coordinates of the X, Y, and Z axes of the miniature ball joint, which is hinged to the connecting rod of the i-th sector-shaped rotor corner of the submicron-level three-axis micro-manipulation module, after it has moved in three-dimensional space, in nm. , , These are the absolute coordinates of the i-th stepper motor rotation origin of the submicron-level three-axis micro-manipulation module in three-dimensional space along the X, Y, and Z axes, respectively, in nm; The radius of rotation of the angular fan-shaped rotor, in mm; Here, θ represents the actual rotation angle of the i-th stepper motor and the structural installation azimuth angle of the i-th stepper motor on the horizontal projection plane, respectively, in °. , , These correspond to the absolute coordinates of the liquid microelectrode probe tip on the horizontal, vertical, and axial axes in three-dimensional space, respectively, in nm. The unit is the uniform length of the connecting rod, in mm; , , These are the trigonometric substitution coefficients derived from known geometric quantities such as target coordinates, motor origin coordinates, and mechanical rod length when solving the inverse kinematics equation of the i-th drive component. , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the first drive assembly to the connecting rod; , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the second drive assembly to the connecting rod; , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the third drive assembly to the connecting rod; , , , , All of these are fitting coefficients for the characteristic equation, obtained by fitting measured motor rotation angle and end coordinate data; By solving the above set of equations, the three-dimensional absolute coordinates of the target coordinates are inverted to correspond to the stepper motor rotation angle, and the measured rotation angle of the stepper motor is inverted to correspond to the liquid microelectrode probe.

[0025] One of the above technical solutions has the following beneficial effects: 1. Resolving the core technical contradiction while balancing capture efficiency and pore resolution: This invention adopts a spatial decoupling architecture of global main driving electric field + local reverse electric potential barrier. The global high voltage ensures long-distance molecule capture and avoids an exponential drop in capture rate. The liquid microelectrode probe applies a reverse electric field only in the throat region of the nanopore, precisely weakening the net electrophoretic force of molecules, linearly extending the pore time, and completely preserving the fine current fluctuations corresponding to the protein conformation. This solves the inherent contradiction of traditional two-electrode systems that "increase voltage to speed up but lose details, and decrease voltage to slow down but lose molecules".

[0026] 2. Nanoscale fully automatic non-contact positioning, hardware anti-collision to prevent sample damage: Equipped with optical coarse alignment + electrical grating fine scanning dual coaxial calibration, the alignment accuracy is better than 10nm; a dual-core microcontroller combined with a high-speed analog comparator circuit realizes microsecond-level anti-collision interruption, bypassing the upper computer software delay, and triggering FOC rigid brake in real time to stably maintain a 3~15nm non-contact safety gap between the probe and the nanopore, without mechanical impact or irreversible damage to the nanopore, and the whole process is automated without manual fine adjustment.

[0027] 3. Low-noise electromagnetic isolation architecture, suitable for picoampere-level weak ion current detection: The micro-operation drive adopts a charge pump-free pure MOSFET H-bridge drive, eliminating the wideband EMI noise of the charge pump; it is equipped with a three-layer three-dimensional electromagnetic isolation structure, with electrical and physical dual isolation between the power drive circuit and the electrochemical detection circuit, and the system baseline noise can be controlled to within 2.5pA, ensuring distortion-free acquisition of high-frequency fine current signals.

[0028] 4. Backlash-free parallel three-axis micro-manipulation, 50nm-level high-precision closed-loop positioning: The three-axis parallel kinematic structure combined with the elastic pre-tightened micro ball joint completely eliminates transmission backlash; the magnetic gear non-contact absolute angle encoding feedback, combined with real-time forward / inverse kinematics calculation, enables the end effector to stably achieve 50nm linear positioning resolution, with no coordinate drift during long-term hovering, and is suitable for nanoscale precision approximation control.

[0029] 5. Dual-mode gated voltage, suitable for both short-term high-precision and long-term high-throughput experiments: The gated voltage supports both DC and 1~10kHz high-frequency pulsed AC modes: DC mode is used for short-term fine resolution of single-molecule conformations; the pulsed AC mode can suppress the accumulation of microbubbles at the tube opening, support continuous detection for more than 8 hours, with baseline drift amplitude less than 0.5pA, greatly improving experimental throughput.

[0030] 6. Smooth voltage switching throughout the process to eliminate capacitive current spikes: The hardware supports linear ramp voltage switching, and the probe voltage, main capture voltage, and reverse gate voltage rise and fall synchronously and smoothly, avoiding capacitive spikes caused by step voltage changes. The baseline remains stable throughout the process, preventing spike signals from masking weak protein conformation secondary current fluctuations. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of a locally reverse electric field-gated nanoporous single-molecule speed regulation system according to the present invention; Figure 2 This is a schematic diagram of the liquid microelectrode probe inside the buffer pool in a locally reverse electric field-gated nanopore single-molecule speed regulation system of the present invention. Figure 3 This is a schematic diagram of the submicron-scale triaxial micromanipulation module and liquid microelectrode probe in a locally reverse electric field-gated nanopore single-molecule accelerometer system according to the present invention. Figure 1 ; Figure 4 This is a schematic diagram of the submicron-scale triaxial micromanipulation module and liquid microelectrode probe in a locally reverse electric field-gated nanopore single-molecule accelerometer system according to the present invention. Figure 2 ; Figure 5 yes Figure 3 A magnified view of a section at point A in the middle; Figure 6 yes Figure 3A magnified view of a section at point B in the middle; Figure 7 This is a comparison chart of the results of BSA protein sequencing using the traditional two-electrode scheme and the present invention. In the attached diagram: 1. Nanopore chip; 11. Insulating film; 12. Nanopore; 2. Buffer pool; 3. Main driving dual electrodes; 31. Cis-driven electrode; 32. Reverse-driven electrode; 4. Liquid microelectrode probe; 41. Conical insulating shell; 42. Ag / AgCl electrode wire; 5. Submicron-level triaxial micro-manipulation module; 51. Micro-manipulation body; 52. Drive assembly; 52. Stepper motor; 521. Fan-shaped rotor horn; 522. Connecting rod; 523. Micro-ball joint; 524. Elastic preload; 525. Magnetic gear encoding feedback unit; 526. Magnetic encoder; 5261. Magnetic encoder chip; 5262. Magnet array; 527. End effector; 53. End connector; 531. Flexible pressure block; 532. Silicone sleeve; 533. High-frequency patch clamp amplifier; 6. Host computer; 7. Detailed Implementation

[0032] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0033] like Figure 1-6As shown, a locally reverse electric field-gated nanopore single-molecule speed control system includes a nanopore chip 1, a buffer pool 2, a main driving dual electrode 3, a liquid microelectrode probe 4, a submicron-level triaxial micromanipulation module 5, a high-frequency patch-clamp amplifier 6, and a host computer 7. First, the nanopore chip 1 is placed inside the sealed buffer pool 2, and a conical nanopore 12 is formed on the insulating film 11 of the nanopore chip 1. Second, the cis driving electrode 31 and the anti driving electrode 32 are respectively arranged on both sides of the nanopore chip 1. The two, together with the high-frequency patch-clamp amplifier 6, construct a global main driving electric field. The cis driving electrode 31 serves as a unified reference potential to ensure the uniformity of the potential reference of the entire system. The anti driving electrode 32 is connected to the high-frequency patch-clamp amplifier 6 as the ion current detection end. Applying a positive transmembrane bias voltage to both can construct a global main driving electric field for long-distance capture of biomacromolecules. After the entire buffer pool is assembled, it is directly placed inside a double-layer copper mesh Faraday shielded box and an air-suspended vibration isolation optical platform to simultaneously isolate external vibrations and electromagnetic interference, making it suitable for picoampere-level weak ion current detection scenarios. The high-frequency patch-clamp amplifier 6 has two core functions: first, it collects the picoampere-level ion current of the nanopore channel and uploads it to the host computer 7 in real time; second, it receives digital commands from the host computer 7 and outputs capture voltage to the main driving dual electrode 3 and detection voltage / reverse gating voltage to the liquid microelectrode probe 4, respectively. Finally, the submicron-level three-axis micro-manipulation module 5 acts as a three-dimensional actuator, flexibly clamping the liquid microelectrode probe 4 and driving it to complete a fully automated process of coaxial pre-alignment, Z-axis non-contact approximation, and hardware servo locking. The liquid microelectrode probe 4 is immersed in the buffer solution in the sealed buffer pool 2, independently generating a local reverse potential barrier, which is spatially superimposed with the global main driving electric field, weakening the net electrophoretic force on biomolecules only in the throat region of the nanopore. Most importantly, the host computer 7 communicates with the high-frequency patch-clamp amplifier 6 and the submicron-level three-axis micro-manipulation module 5 only through the USB / serial digital bus. There is no direct electrical path to the liquid microelectrode probe 4, thus avoiding analog signal crosstalk. The multi-layer electromagnetic isolation layer isolates the power drive circuit and the electrochemical detection circuit in layers, blocking the wideband electromagnetic noise generated by the stepper motor 521 from coupling into the weak ion current acquisition path.

[0034] This architecture independently separates the electrochemical acquisition, electric field control, and three-dimensional micro-motion modules to work collaboratively. The global electric field is responsible for capturing molecules at long distances, while the local reverse electric field only brakes molecules in the key area of ​​the via. This fundamentally solves the contradiction between the traditional two-electrode capture rate and the via speed. The host computer 7 only issues digital commands and does not connect to the analog detection circuit, which greatly reduces the baseline noise introduced by stray capacitance. The multi-layer isolation structure is suitable for picoampere-level ultra-weak ion current detection scenarios.

[0035] In a preferred embodiment, the nanochip is made of solid silicon nitride with a thickness of 20nm and a pore size of 30nm. The buffer solution ratio is 1mM KCl + 10mM Tris-HCl + 1mM EDTA, the pH value is adjusted to 8.0, the global main drive voltage is selected in the range of 100mV to 500mV, and the host computer 7 adopts USB 2.0 high-speed serial communication with a communication refresh rate set to 1kHz.

[0036] Further explanation: The submicron-level three-axis micro-manipulation module 5 includes a micro-manipulation body 51, drive components 52, and end effector components 53. The micro-manipulation body 51 houses a main control board, which integrates a dual-core microcontroller, specifically the RP2350 model. This dual-core microcontroller includes a first core and a second core. The first core performs three-dimensional forward kinematics calculations and three-dimensional inverse kinematics calculations, while the second core exclusively runs the cascaded magnetic field-oriented FOC three-loop control algorithm. Three sets of drive components 52 are integrated externally into the micro-manipulation body 51. The end effector components 53 are located at the output ends of the three sets of drive components 52, each driving the end effector components 53 to achieve independent X, Y, and Z axis movements. Each drive component 52 includes a stepper motor 521, a sector-shaped rotor horn 522, a connecting rod 523, a miniature ball joint 524, an elastic preload element 525, and a magnetic gear encoder feedback unit 526. The end effector components 53 include an end-connecting block 531 and a flexible pressure block 526. 32 and silicone sleeve 533, silicone sleeve 533 is sleeved with microelectrode holder; the output end of stepper motor 521 is fixedly connected to the center of sector rotor horn 522, magnet array 527 is arranged on the edge of sector rotor horn 522, magnetic gear encoder feedback unit 526 is arranged close to magnet array 527, magnetic gear encoder feedback unit 526 feeds back the absolute rotation angle value of stepper motor 521 to dual-core microcontroller in real time, magnetic gear encoder feedback unit 526 includes magnetic encoder 5261 The magnetic encoder chip 5262; the two ends of the connecting rod 523 are respectively hinged to the fan-shaped rotor horn 522 and the end connecting block 531 through the micro ball joint 524; the elastic pre-tightening member 525 is provided at the micro ball joint 524 to eliminate the transmission backlash; the flexible pressure block 532 is fixed to the end connecting block 531; one end of the silicone sleeve 533 is embedded in the flexible pressure block 532; the other end of the silicone sleeve 533 is sleeved and fixed to the microelectrode holder; the microelectrode holder holds the liquid microelectrode probe 4.

[0037] To further explain, each stepper motor 521 is equipped with a charge pump-free pure MOSFET H-bridge drive circuit. The pure MOSFET H-bridge drive circuit receives the control signal output by the dual-core microcontroller to drive the windings of the stepper motor 521. The cascaded field-oriented three-loop control includes an outer position loop, a middle speed loop, and an inner 30kHz current loop, which is used to force the excitation current component Id=0 and only adjust the torque current component Iq. When receiving a hardware emergency stop signal, it directly generates a reverse braking current in the current loop to achieve rigid braking. After the magnetic gear encoding feedback unit 526 is mapped by three-dimensional forward kinematics calculation and three-dimensional inverse kinematics calculation, the end effector 53 has a linear positioning resolution of 50nm.

[0038] The submicron-level three-axis micro-manipulation module 5 adopts a three-axis parallel kinematic architecture. The dual-core microcontroller is integrated into the main control board inside the module as the top-level computing unit. Three sets of identical drive components 52 are integrated into the overall base of the micro-manipulator. Each set of independent drive end effector components 53 completes single-axis linear motion. The transmission link of a single drive unit is a stepper motor 521 rigidly connected to a fan-shaped rotor horn 522. The edge of the fan-shaped rotor horn 522 is arranged with a magnetic array 527. The two ends of the connecting rod 523 are hinged to the fan-shaped rotor horn 522 and the end connecting plate respectively by micro ball joints 524. The elastic preload 525 continuously applies centripetal tension to the ball joint to eliminate the bidirectional backlash of the transmission. The flexible pressure block 532 and the silicone sleeve 533 form a flexible clamping structure to avoid the rigid stress cracking of the brittle conical insulating shell 41. Position feedback relies on the magnetic gear encoding feedback unit 526, preferably using a magnet array 527 composed of 17 sets of N / S alternating permanent magnets, and is paired with the MT6835 magnetic encoding chip to achieve non-contact absolute angle acquisition. The single magnetic field period has a chordal span of 6mm. The encoding chip adopts a 21-bit subdivision design, and after eliminating electrical noise, the effective chordal resolution is 45.7nm. After kinematic mapping, the end effector 53 stably achieves a linear positioning accuracy of 50nm.

[0039] Taking any drive component 52 as an example, the stepper motor 521 rotates, causing the motor rotor horn to swing in an arc. Ends A and B of the connecting rod 523 are permanently hinged to the rotor horn and end effector respectively via miniature ball joints 524. Simultaneously, the elastic preload 525 located at the ball joint provides continuous centripetal tension, ensuring the ball joint fits tightly into the socket. This design ensures that the connecting rod 523 transmits force strictly along its axis during both push and pull movements, completely eliminating mechanical backlash. Therefore, the three-dimensional spatial translation of the liquid microelectrode probe 4 is determined by the spatial geometric constraints formed by the combined push and pull of the three connecting rods 523.

[0040] At the sensing feedback and calculation level: When the rotor horn 522 of the motor swings with the stepper motor 521, the magnet array 527 fixed on it tangentially sweeps across the magnetic encoding chip 5262. Utilizing the non-contact magnetic gear amplification effect, the magnetic encoding chip 5262 provides real-time, high-resolution feedback of the absolute rotation angle value of the stepper motor 521. The dual-core microcontroller, by acquiring the absolute rotation angles of the three stepper motors 521 in real time, can accurately calculate the current absolute coordinates of the liquid microelectrode probe 4 in the XYZ space using the forward kinematic equations. Conversely, by inputting the desired XYZ target coordinates, the inverse kinematic equations can convert them into the target rotation angles of the three motors and execute the calculations.

[0041] The hardware driver hierarchy is clearly defined. The RP2350 dual-core microcontroller outputs PWM control signals to each stepper motor. The 521 chip is equipped with a TB6612 pure MOSFET H-bridge driver circuit, eliminating the built-in charge pump oscillation circuit and completely eliminating wideband electromagnetic radiation interference caused by the charge pump. The two core computing powers of the RP2350 dual-core microcontroller are completely isolated. The first core is dedicated to executing the three-dimensional forward and inverse kinematic equations, while the second core exclusively uses a 30kHz cascaded field-oriented three-loop FOC control loop. The loop is divided into an outer position loop, a middle speed loop, and an inner 30kHz current loop. Through Clarke and Park transformations, the alternating stator current of the motor is decoupled into orthogonal Id and Iq components. The control logic forces the excitation component Id=0, eliminating permanent magnet demagnetization and useless heat loss. All drive power is used to adjust the torque component Iq. When an emergency stop signal is triggered, it can directly output a reverse instantaneous braking torque to achieve sub-millisecond rigid braking. Compared to traditional orthogonal slides, the parallel motion structure is more rigid and smaller. The elastic preload ball joint completely eliminates long-term hovering coordinate drift. Dual-core computing power isolation ensures that trajectory planning and high-speed braking do not compete for hardware resources. The charge-free pump drive chip can be paired with multi-layer electromagnetic isolation to control the system baseline noise to within 2.5pA. 50nm-level closed-loop positioning can stably maintain a safe insulation gap of 3~15nm between the probe and the nanopore. In the preferred implementation, the single connecting rod 523 has a uniform length of 72mm, the micro ball joint 524 has an arrangement radius of 0.5mm, the RP2350 main frequency is 150MHz, the TB6612 power supply voltage is 12V, the FOC inner loop update frequency is fixed at 30kHz, and the emergency stop reverse torque is 1.2 times the rated torque of the motor.

[0042] In a single-molecule detection system, the high-frequency patch-clamp amplifier 6 needs to record picoampere levels (10). -12A) The weak ion current means that any external high-frequency electromagnetic radiation will cause signal distortion. Conventional motor driver chips usually integrate charge pump circuits, whose high-frequency charging and discharging radiates broadband spurious electromagnetic noise. This invention uses a TB6612 pure MOSFET H-bridge driver with the charge pump removed, and a P-channel MOSFET for the high-side switch, eliminating charge pump EMI interference from the bottom layer; at the same time, it can be equipped with three-layer three-dimensional electromagnetic isolation: solid copper shielding on the driver PCB, single-end grounding of the motor shielded twisted pair cable, and physical grounding between the power circuit and the detection pool, completely blocking the coupling of drive noise to the detection channel.

[0043] To further explain, the liquid microelectrode probe 4, serving as the carrier for generating a localized reverse electric field, includes a conical insulating shell 41. The interior of the conical insulating shell 41 is filled with electrolyte and contains an Ag / AgCl electrode wire 42. The conical insulating shell 41 is divided into a straight tube section and a pointed conical section. The pointed conical section has a pointed opening, and the conical insulating shell 41 radiates the localized electric field outward only through the pointed opening to apply a reverse bias voltage to construct a localized reverse potential barrier in the throat region of the nanopore. The Ag / AgCl electrode wire 42 is embedded in the straight tube section of the conical insulating shell 41 and does not extend into the pointed conical section. After the lead wire of the Ag / AgCl electrode wire 42 passes through the reserved hole in the straight tube section of the conical insulating shell 41, it is sealed by secondary vacuum dripping and heat curing with low-viscosity modified epoxy resin to prevent electrolyte leakage.

[0044] In a preferred embodiment, the liquid microelectrode probe 4 is made of a high-purity quartz conical insulating shell 41 with an overall length of 45-50 mm, an outer diameter of 1.0 mm, an inner diameter of 0.5 mm, a tapered section of 3-5 mm at the tip, and a taper controlled at 1.6°-2.0°. The inner diameter of the tip opening can be selected from 100 nm to 500 nm. The shell is filled with 1 mM HCl electrolyte that has been ultrasonically degassed. An Ag / AgCl electrode wire 42 with a diameter of 0.25 mm is built in. The electrode wire is pre-chlorinated in a 0.1 mM HCl solution with a constant current density of 1 mA / cm² for 1500 seconds. The end of the electrode wire 42 terminates in a straight tube area 5-8 mm above the tapered section, without extending into the tapered section or out of the tube opening. This allows it to be kept away from the electrochemical reaction zone at the tip, significantly reducing electrochemical polarization baseline drift. The liquid microelectrode probe 4 features a double-sealed structure, relying on a medical-grade silicone sealing ring to radially compress the sleeve, combined with vacuum drip curing of EPON828 modified epoxy resin to prevent capillary creep leakage of the electrolyte. Electrolyte infusion is achieved by slowly injecting it from the end of the straight tube section using a 0.16mm ultrafine quartz needle. After infusion, the tube is centrifuged at 3500rpm for 5 minutes to completely remove any tiny air pockets. A trace amount of 0.01% Triton X-100 is added to the electrolyte to reduce the solid-liquid interfacial tension and inhibit the adhesion and nucleation of microbubbles generated by the electrochemical reaction at the tube opening. The insulating quartz tube wall confines the electric field to radiate only from the narrow tip opening, preventing the large-scale diffusion of the reverse electric field from interfering with the global main electric field captured by peripheral molecules. A local reverse potential barrier is precisely constructed only in the throat region of the nanopore. The entire structure has no metal entities extending into the reaction zone, thus avoiding steric hindrance that could interfere with fluid and electric field distribution. Long-term continuous detection does not result in bubble blockage or baseline drift, making it suitable for uninterrupted protein sequencing experiments lasting tens of hours.

[0045] Furthermore, the main control board also includes a high-speed analog comparator circuit, and the dual-core microcontroller has a hardware external interrupt pin. In a preferred embodiment of the invention, the independently configured high-speed analog comparator circuit on the main control board uses a comparator chip with a nanosecond-level response time, such as the TLV3501 high-speed comparator chip manufactured by Texas Instruments (TI) or the ADCMP601 ultrafast comparator chip manufactured by Analog Devices (ADI). The high-frequency patch-clamp amplifier is equipped with a BNC analog output channel, which maps the ion current to an analog voltage and connects it to the analog input terminal of the high-speed analog comparator circuit. The analog output terminal of the high-speed analog comparator circuit is connected to the hardware external interrupt pin of the dual-core microcontroller. When the ion current decays to a preset threshold, the high-speed analog comparator circuit undergoes a level flip and directly transmits the flip signal to the hardware external interrupt pin of the dual-core microcontroller, generating a hardware interrupt signal in microseconds. This bypasses software polling and directly initiates the cascaded magnetic field-oriented FOC three-loop control algorithm to perform emergency braking.

[0046] Because the high-frequency patch-clamp amplifier 6 is equipped with a dedicated BNC analog output channel, it can linearly map the real-time acquired nanopore ion current into a continuous analog voltage. This voltage is then directly connected to the high-speed analog comparator circuit via a coaxial cable without attenuation. The system only presets a 15% current attenuation threshold on the host computer 7, i.e., It < 0.85I0. The hardware comparison relies on pure hardware logic to perform level discrimination, without the need for software sampling or polling. Once the analog voltage is detected to be below the threshold, a highest-priority hardware interrupt signal can be generated directly within 2 to 5 microseconds, bypassing the tens of millisecond delays caused by the host computer 7's operating system and serial communication. This directly wakes up the second core to run the 30kHz FOC emergency stop algorithm and output reverse braking torque, preventing irreversible damage caused by the probe's high-speed descent and impact on the nanopore from a hardware perspective. At the same time, it does not occupy the host computer 7's data processing and trajectory planning computing power. High-volume signal acquisition and anti-collision protection can operate synchronously and stably. The response delay of the high-speed analog comparator circuit is preferably controlled within 3 microseconds, and the interrupt priority is set to the highest on the chip, enabling it to preempt all regular computing processes. Because it uses an external, independent high-speed analog comparator chip, the analog voltage of the patch clamp is directly compared with its threshold voltage (corresponding to 0.85 times the initial current reference voltage) using pure hardware circuitry. Once triggered, the digital high / low level output of the comparator directly triggers the RP2350's GPIO external hardware interrupt (EXTI).

[0047] To further explain, the host computer 7 has a built-in dual pre-alignment logic, which is the core pre-step of the entire automated positioning process. The first pre-alignment logic is optical coarse alignment, which reduces the lateral deviation between the liquid microelectrode probe 4 and the nanopore to the micrometer level through long working distance objective lens imaging. The second pre-alignment logic is electrical fine pre-scan, which raises the liquid microelectrode probe 4 to a safe height to perform two-dimensional grating scanning and locks the geometric center of the nanopore based on the extreme value of ion current distribution.

[0048] The host computer 7 incorporates a two-stage fully automatic coaxial calibration algorithm to resolve the probe-nanopore 12XY plane misalignment issue. The first stage is optical coarse alignment. The system can be equipped with 40x to 100x long working distance microscope objectives, achieving a total magnification of 400x to 1000x when combined with a CCD imaging system. The operator or image algorithm uses the complete overlap between the diffraction spot at the probe tip and the image of the transparent target area of ​​the nanopore as the alignment criterion, which can quickly reduce the XY lateral deviation to the range of 1-2 micrometers. After coarse alignment, the micro-manipulation module raises the probe to a safe height of 500nm above the chip to perform the second stage of electrical alignment. The system employs a fine pre-scan with a scanning range of 2μm×2μm rectangular grating and a scanning step size of 0.1μm. It simultaneously acquires the ion current signal corresponding to each coordinate to generate a two-dimensional current distribution map. Since the nanopore is the only ion conduction channel of the insulating film 11, the extreme point of current intensity in the distribution map is the nanogeometric center. The system automatically locks the coordinate with a servo, and the final coaxial alignment accuracy is better than 10nm. This electrical scanning method is not affected by solution turbidity or poor optical transmittance, breaks through the Abbe diffraction limit of optical microscopes, and is fully automated without repeated manual fine-tuning, which greatly reduces the operation threshold and human alignment error.

[0049] A locally reverse electric field-gated nanopore single-molecule velocity modulation method, applied to a locally reverse electric field-gated nanopore single-molecule velocity modulation system as described above, includes the following steps: S1. Planar Pre-alignment: Through a dual process of optical coarse alignment and electrical grating fine scanning, the liquid microelectrode probe 4 is locked coaxially above the nanopore. S2. Construction of an Independent Tracking Detection Loop: The main driving dual electrodes 3 are controlled to remain at zero potential, and the high-frequency patch-clamp amplifier 6 is scheduled to output a detection voltage to the liquid microelectrode probe 4, acquiring a stable initial baseline ion current I0. S3. Z-axis Microstep Approach: The submicron-level three-axis micro-manipulation module 5 is controlled to drive the liquid microelectrode probe 4 to move uniformly downwards along the Z-axis, while the high-frequency patch-clamp amplifier 6 transmits the instantaneous ion current It in real time. S4. Hardware Servo Locking: It and I0 are compared in real time. When It is lower than the 0.85I0 current decay threshold, the high-speed analog comparator circuit undergoes a level flip, and the flip signal is directly connected to the hardware external interrupt pin of the dual-core microcontroller, generating a hardware interrupt signal in microseconds, bypassing software polling. The cascaded magnetic field-oriented FOC three-loop control algorithm is directly activated to perform emergency stop braking and coordinate servo locking, maintaining a safe gap of 3~15nm between the liquid microelectrode probe and the nanopore convergence range; S5, Smooth mode switching: After locking, wait for mechanical vibration to decay, and schedule the high-frequency patch-clamp amplifier 6 to complete the ramp switching of zeroing the detection voltage, raising the main driving voltage, and lowering the reverse gate voltage with a fixed linear slope, eliminating the step capacitive current spike; S6, Single molecule detection speed regulation: The global main driving electric field captures biomacromolecules, and the local reverse potential barrier weakens the net electrophoretic force of molecules, collecting the waveform of the extended ion blocking current and analyzing the secondary current fluctuations corresponding to the dynamic conformation within the molecular pore; S7, Quantitative linear speed regulation: Continuously adjust the reverse negative voltage output by the liquid microelectrode probe 4 to achieve a continuously controllable extension of the biomacromolecule passage time, stable molecular blocking signal amplitude, and no exponential cliff decay of the molecular capture rate.

[0050] To further explain, the gate voltage output by the liquid microelectrode probe 4 can be selected as either DC mode or high-frequency pulse AC mode; the high-frequency pulse AC mode is limited to a frequency of 1~10kHz and a duty cycle of 20%~80%, and is used for long-term continuous detection to suppress the accumulation of microbubbles at the tube interface.

[0051] To further explain, the submicron-level three-axis micro-manipulation module 5 uses the RP2350 dual-core microcontroller to execute the forward and inverse kinematic equations to complete the conversion between the end coordinates and the rotation angle of the stepper motor 521; The inverse kinematic equations are as follows:

[0052] The length constraint equation for the connecting rod 523 is as follows:

[0053] Substituting the inverse kinematic equations into the length constraint equations of link 523, we obtain the trigonometric standard equations:

[0054] The set of equations for the positive kinematic spherical constraint:

[0055] After elimination, a linear mapping relationship is obtained:

[0056] Back substitution yields the quadratic characteristic equation:

[0057] in, , , The absolute coordinates of the X-axis, Y-axis, and Z-axis of the micro-ball joint 524, which is hinged to the i-th sector-shaped rotor 522 and the connecting rod 523 of the submicron-level three-axis micro-manipulation module, after moving in three-dimensional space, are in nm. , , These are the absolute coordinates of the rotation origin of the i-th stepper motor 521 of the submicron-level three-axis micro-manipulation module on the X, Y, and Z axes in three-dimensional space, respectively, in nm; The radius of rotation of the angular fan-shaped rotor, in mm; These are the actual rotation angle of the i-th stepper motor 521 and the structural installation azimuth angle of the i-th stepper motor 521 on the horizontal projection plane, respectively, in °; , , These correspond to the absolute coordinates of the liquid microelectrode probe tip on the horizontal, vertical, and axial axes in three-dimensional space, respectively, in nm. The unit is the uniform length of the connecting rod, in mm; , , These are the trigonometric substitution coefficients (intermediate transition parameters) derived from known geometric quantities such as target coordinates, motor origin coordinates, and mechanical rod length when solving the inverse kinematics equation of the i-th drive component. , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the first drive assembly to the connecting rod; , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the second drive assembly to the connecting rod; , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the third drive assembly to the connecting rod; , , , , All of these are fitting coefficients for the characteristic equation, obtained by fitting the measured motor rotation angle and end coordinate data. By solving the above set of equations, the three-dimensional absolute coordinates of the target coordinates are inverted to the corresponding rotation angle of the stepper motor 521 and the measured rotation angle of the stepper motor 521 is inverted to the liquid microelectrode probe 4.

[0058] Applied to the aforementioned locally reverse electric field-gated nanopore single-molecule accelerometer system, this method consists of seven closed-loop steps. Each step relies on the aforementioned hardware structure, control circuit, and kinematic algorithm to operate collaboratively. The first step executes a dual pre-alignment process to lock the probe and nanopore coaxial position, corresponding to the two-stage coaxial calibration logic built into the host computer. The second step controls the cis and trans main driving electrodes to maintain a 0V quiescent state, schedules the patch clamp to output a +50mV detection voltage to the probe, and waits for the baseline to stabilize before acquiring the initial baseline ion current I0. The independent detection loop is not affected by stray interference from the main trapping electric field, avoiding premature molecule aspiration and pore blockage before positioning. The third step involves controlling the micro-manipulation module to drive the probe to move uniformly downwards along the Z-axis with a tiny step size of 0.1 μm. The patch clamp continuously transmits the real-time instantaneous ion current It. During the movement, the RP2350 uses the forward and inverse kinematic equations to calculate the target coordinates and motor rotation angles in real time. The half-angle substitution is used to solve the target rotation angles of each motor without singularities, and the calculation is then executed. During the hardware locking phase, the actual rotation angles of the three motors are read, and the quadratic terms are eliminated by substituting them into the forward kinematic spherical constraint equations to obtain a linear mapping relationship. The quadratic characteristic equation is obtained by substituting back and solving it. Then, the effective real roots are selected by combining the geometric constraints of the mechanism, and the current three-dimensional absolute coordinates of the probe are rapidly inverted.

[0059] The system continuously compares It with the 0.85I0 baseline threshold in real time. When the ion current decay reaches the 15% judgment condition, the high-speed analog comparator circuit inside the RP2350 generates the highest priority hardware interrupt within 2.8μs. The second core immediately initiates a 30kHz cascaded FOC reverse braking and PId servo lock, completing the S4 hardware servo lock step. After locking, the probe tip and the nanopore convergence range stably maintain an insulation safety gap of about 10nm. The corresponding law of this gap is calibrated based on the Poisson-Nernst-Planck equation, the Navier-Stokes equation coupled finite element model, and SICM approximation curve. The entire threshold is applicable to solid nanopore diameters of 20~50nm, probe openings of 100~300nm, and 1M For KCl high ionic strength buffer, the larger the probe opening size, the larger the safety gap of the pore corresponding to the same decay threshold. Subsequent control experiments were conducted by assembling 100nm and 300nm opening probes to verify this rule. The measured gap was 3nm when the 100nm opening probe triggered 15% decay, and the gap was 15nm when the 300nm opening probe triggered decay, which is completely consistent with the model calculation results.

[0060] After the hardware servo lock is completed, the system maintains a 10ms mechanical silent buffer period to absorb residual micro-vibrations of the linkage 523 and the end effector 53. After the buffer is completed, the high-frequency patch-clamp amplifier 6 performs a smooth ramp voltage switching with a uniform linear slope of 10mV / ms. First, the probe +50mV detection voltage is smoothly dropped back to 0V, and the reverse drive electrode 32 is simultaneously ramped up to the standard +300mV main capture voltage. At the same time, the probe voltage is synchronously dropped to the set reverse gate negative voltage. There are no step voltage changes throughout the process, avoiding capacitive peak current and electroosmotic drift, and ensuring that the baseline is stable and undisturbed throughout the process. This smooth switching function is realized by the high-frequency patch-clamp amplifier's built-in multi-level voltage linear output hardware module.

[0061] After voltage switching, pretreated BSA samples were injected into the cis-chamber of sealed buffer tank 2. BSA lyophilized powder was dissolved in standard buffer to prepare a working concentration of 50 nM. After preparation, the sample was centrifuged at 14000 rpm for 10 minutes to remove micron-sized aggregates, followed by secondary filtration through a 0.22 μm filter membrane. The patch clamp was set to a 100 kHz signal acquisition bandwidth and a 10 kHz fourth-order Bessel low-pass filter to output the ion current. The measured baseline RMS noise of the system was 2.3 pA. To quantitatively verify the linear speed regulation performance of this invention, under a fixed main drive voltage of +300 mV, the reverse gate negative pressure of the liquid microelectrode probe 4 was adjusted sequentially, and the median characteristic peak data of each group of experiments were collected and the standard deviation was recorded. Sequencing results were referenced. Figure 7 As shown, the complete quantitative comparison data table is as follows:

[0062] As shown in the table, the BSA molecule pore passage time exhibits a highly linear positive correlation with the gated negative pressure within the negative pressure range of -10mV to -200mV. The single-molecule blocking rate remains at around 33.9% throughout the range with only minor fluctuations. The molecule capture rate decreases gradually with increasing negative pressure, without the exponential drop seen in traditional low-pressure solutions. When the gated voltage is set to -100mV, the typical BSA pore passage time is extended from 0.80ms in the traditional dual-electrode system to 1.61ms, with a speed adjustment factor exceeding 2 times. The current waveform is fully extended on the time axis, and the step-like secondary current fluctuations of multiple corresponding protein pore dynamic conformations can be clearly distinguished at the bottom of the waveform, solving the pain point of losing fine signals during rapid pore passage in existing technologies. During the experiment, the liquid microelectrode probe 4 can switch between two gating modes. The DC mode is suitable for short-term, high-precision protein conformation analysis. If long-term, high-throughput detection of more than eight hours is to be carried out, it can be switched to a high-frequency pulse AC mode of 1~10kHz with a duty cycle of 20%~80%. The preferred mode is 5kHz with a 50% duty cycle. The alternating electric field continuously breaks the gas nucleation equilibrium at the tube opening interface, inhibiting the adhesion and accumulation of microbubbles. After eight hours of continuous acquisition, there is no obvious bubble accumulation at the tube opening, and the overall baseline drift is less than 0.5pA, which greatly extends the continuous detection time.

[0063] Furthermore, the functional units in the various embodiments of the present invention can be integrated into a processing module, or each unit can exist physically separately, or two or more units can be integrated into a module. The integrated module can be implemented in hardware or as a software functional module. If the integrated module is implemented as a software functional module and sold or used as an independent product, it can also be stored in a computer-readable storage medium.

[0064] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A locally reverse electric field-gated nanoporous single-molecule speed regulation system, characterized in that, It includes a nanopore chip (1), a buffer pool (2), a main driving dual electrode (3), a liquid microelectrode probe (4), a submicron-level triaxial micromanipulation module (5), a high-frequency patch clamp amplifier (6), and a host computer (7). The nanoporous chip (1) is disposed inside the buffer pool (2), which is filled with buffer solution; the nanoporous chip (1) includes an insulating film (11) and nanopores (12) penetrating the insulating film (11). The main driving dual electrode (3) includes a cis-driving electrode (31) and an anti-driving electrode (32), which are respectively placed on both sides of the nanopore; both the cis-driving electrode (31) and the anti-driving electrode (32) are electrically connected to the high-frequency patch-clamp amplifier (6), the cis-driving electrode (31) is set as the system global reference ground, and the anti-driving electrode (32) is connected to the main current detection input terminal of the high-frequency patch-clamp amplifier (6); The high-frequency patch-clamp amplifier (6) is used to output a positive transmembrane bias voltage to the main driving dual electrode (3) to construct a global main driving electric field, to collect the ion current signal of the nanopore channel in real time and transmit it back to the host computer (7), to receive the digital instructions issued by the host computer (7) and output the detection voltage, main capture voltage and reverse gating voltage accordingly. The submicron-level triaxial micro-manipulation module (5) is equipped with a microelectrode holder, which mechanically holds the liquid microelectrode probe (4). The submicron-level triaxial micro-manipulation module (5) is used to drive the liquid microelectrode probe (4) to complete three-dimensional displacement and servo locking actions within the buffer pool (2). When the liquid microelectrode probe (4) is immersed in the buffer solution, the liquid microelectrode probe (4) can output a reverse bias voltage and form a local reverse potential barrier superimposed on the global main driving electric field. The host computer (7) is connected to the high-frequency patch clamp amplifier (6) and the submicron-level three-axis micro-operation module (5) respectively through a digital communication link. The host computer (7) has a built-in closed-loop control algorithm, coordinate analysis program and data monitoring interface. The host computer (7) is used to receive ion current signals, send motion commands or emergency stop commands to the submicron-level three-axis micro-operation module (5), and schedule the high-frequency patch clamp amplifier (6) to perform smooth switching of multi-level voltage.

2. The locally reverse electric field-gated nanoporous single-molecule speed regulation system according to claim 1, characterized in that, The submicron-level three-axis micro-manipulation module (5) includes a micro-manipulation body (51), a drive component (52), and an end effector component (53). The micro-operation body (51) is equipped with a main control board, which integrates a dual-core microcontroller. The dual-core microcontroller includes a first core and a second core. The first core is used to perform three-dimensional forward kinematics calculation and three-dimensional inverse kinematics calculation, and the second core is used to exclusively run the cascaded magnetic field orientation FOC three-loop control algorithm. The three sets of drive components (52) are integrated on the outside of the micro-operation body (51), and the end effector (53) is located at the output end of the three sets of drive components (52). The three sets of drive components (52) drive the end effector (53) to achieve independent movement of the X, Y and Z axes respectively. The single drive assembly (52) includes a stepper motor (521), a sector rotor horn (522), a connecting rod (523), a miniature ball joint (524), an elastic preload (525), and a magnetic gear encoder feedback unit (526). The end effector assembly (53) includes an end connector (531), a flexible pressure block (532), and a silicone sleeve (533), the silicone sleeve (533) being fitted onto the microelectrode holder; The output end of the stepper motor (521) is fixedly connected to the center of the fan-shaped rotor corner (522). The edge of the fan-shaped rotor corner (522) is arranged with a magnet array (527). The magnetic gear encoding feedback unit (526) is arranged close to the magnet array (527). The magnetic gear encoding feedback unit (526) feeds back the absolute rotation angle value of the stepper motor (521) to the dual-core microcontroller in real time. The magnetic gear encoding feedback unit (526) includes a magnetic encoder (5261) and a magnetic encoder chip (5262). The connecting rod (523) is hinged at both ends to the fan-shaped rotor horn (522) and the end connecting block (531) respectively through the micro ball joint (524). The elastic preload (525) is provided at the micro ball joint (524) to eliminate transmission backlash. The flexible pressure block (532) is fixed to the end connector (531), one end of the silicone sleeve (533) is embedded in the flexible pressure block (532), and the other end of the silicone sleeve (533) is sleeved and fixed to the microelectrode holder, which holds the liquid microelectrode probe (4).

3. The locally reverse electric field-gated nanoporous single-molecule speed regulation system according to claim 2, characterized in that, Each set of stepper motors (521) is equipped with a charge pump-free pure MOSFET H-bridge drive circuit. The pure MOSFET H-bridge drive circuit receives the control signal output by the dual-core microcontroller to drive the windings of the stepper motor (521). The cascaded magnetic field oriented FOC three-loop control includes an outer loop position loop, a middle loop speed loop, and an inner loop 30kHz current loop. It is used to force the excitation current component Id=0, only adjust the torque current component Iq, and directly generate a reverse braking current in the current loop to achieve rigid braking when receiving a hardware emergency stop signal. After the magnetic gear encoding feedback unit (526) is mapped by the three-dimensional forward kinematics calculation and the three-dimensional inverse kinematics calculation, the end effector component (53) is equipped with a linear positioning resolution of 50nm.

4. The locally reverse electric field-gated nanoporous single-molecule speed regulation system according to claim 2, characterized in that, The liquid microelectrode probe (4) includes a conical insulating shell (41), the interior of which is filled with electrolyte and contains an Ag / AgCl electrode wire (42). The conical insulating shell (41) is divided into a straight tube section and a pointed conical section. The pointed conical section has a pointed opening. The conical insulating shell (41) radiates a local electric field outward only through the pointed opening, which is used to apply a reverse bias voltage to construct a local reverse electric barrier in the throat region of the nanopore. The Ag / AgCl electrode wire (42) is embedded in the straight section of the conical insulating shell (41) and does not extend into the pointed conical section; After the lead wire of the Ag / AgCl electrode wire (42) passes through the reserved hole of the straight section of the tapered insulating shell (41), it is sealed by secondary vacuum dripping and heating with low viscosity modified epoxy resin to prevent the electrolyte from leaking.

5. The locally reverse electric field-gated nanoporous single-molecule speed regulation system according to claim 2, characterized in that, The main control board is also equipped with a high-speed analog comparator circuit, and the dual-core microcontroller is equipped with a hardware external interrupt pin. The high-frequency patch-clamp amplifier is equipped with a BNC analog output channel, which maps the ion current into an analog voltage and connects it to the analog input terminal of the high-speed analog comparator circuit. The analog output terminal of the high-speed analog comparator circuit is connected to the hardware external interrupt pin of the dual-core microcontroller. When the ion current decays to a preset threshold, the high-speed analog comparator circuit undergoes a level flip and directly transmits the flip signal to the hardware external interrupt pin of the dual-core microcontroller. A hardware interrupt signal is generated in microseconds, bypassing software polling, and the cascaded magnetic field orientation FOC three-loop control algorithm is directly started to perform emergency braking and coordinate servo locking.

6. The locally reverse electric field-gated nanoporous single-molecule speed regulation system according to claim 1, characterized in that, The host computer (7) has built-in dual pre-alignment logic: The first pre-alignment logic is optical coarse alignment, which reduces the lateral deviation between the liquid microelectrode probe (4) and the nanopore to the micrometer level by imaging with a long working distance objective lens; The second pre-alignment logic is an electrical fine pre-scan, which raises the liquid microelectrode probe (4) to a safe height to perform a two-dimensional grating scan and locks the geometric center of the nanopore based on the extreme value of the ion current distribution.

7. A method for regulating the velocity of single molecules in nanopores under locally reverse electric field gating, characterized in that, The method applied to a locally reverse electric field-gated nanoporous single-molecule speed regulation system as described in any one of claims 1-6 includes the following steps: S1. Planar pre-alignment: The liquid microelectrode probe (4) is locked above the nanopore coaxially through a dual process of optical coarse alignment and electrical grating fine scanning. S2. Construct an independent tracking and detection loop: Control the main driving dual electrode (3) to maintain zero potential silence, schedule the high-frequency patch clamp amplifier (6) to output detection voltage to the liquid microelectrode probe (4), and collect a stable initial baseline ion current I0; S3, Z-axis microstep approximation: The submicron-level triaxial micro-manipulation module (5) is controlled to drive the liquid microelectrode probe (4) to move down at a constant speed along the Z-axis, and the high-frequency patch clamp amplifier (6) transmits the instantaneous ion current It back in real time; S4. Hardware Servo Locking: It and I0 are compared in real time. When It is lower than the current decay threshold of 0.85I0, the high-speed analog comparator circuit flips the level and sends the flip signal directly to the hardware external interrupt pin of the dual-core microcontroller. The hardware interrupt signal is generated in microseconds, bypassing software polling, and the cascaded magnetic field orientation FOC three-loop control algorithm is directly started to perform emergency stop braking and coordinate servo locking. The liquid microelectrode probe and the nanopore convergence range maintain a safe gap of 3~15nm. S5. Smooth mode switching: After locking, wait for the mechanical vibration to decay, and schedule the high-frequency diaphragm clamp amplifier (6) to complete the ramp switching of the probe voltage returning to zero, the main drive voltage rising, and the reverse gate voltage falling with a fixed linear slope, so as to eliminate the step capacitive current spike. S6. Single-molecule detection speed regulation: The global main driving electric field captures biomacromolecules, the local reverse potential barrier weakens the net electrophoretic force of molecules, collects the waveform of the spread-type ion blocking current, and analyzes the secondary current fluctuations corresponding to the dynamic conformation in the molecular pore. S7. Quantitative linear speed regulation: Continuously adjust the reverse negative pressure output by the liquid microelectrode probe (4) to achieve a continuous and controllable extension of the biomolecule pore passage time, stable molecular blocking signal amplitude, and no exponential cliff decay of molecular capture rate.

8. The method for localized reverse electric field gating of nanopore single-molecule speed regulation according to claim 7, characterized in that, The gate voltage output by the liquid microelectrode probe (4) can be selected in DC mode or high-frequency pulse AC mode; The high-frequency pulse AC mode is limited to a frequency of 1~10kHz and a duty cycle of 20%~80%, and is used for long-term continuous detection to suppress the accumulation of microbubbles at the nozzle interface.

9. The method for regulating the speed of a single molecule in a nanopore under locally reverse electric field gating according to claim 1, characterized in that, The submicron-level three-axis micro-manipulation module (5) executes the forward kinematics equations and the inverse kinematics equations through the RP2350 dual-core microcontroller to complete the conversion between the end coordinates and the rotation angle of the stepper motor (521); The inverse kinematic equations are as follows: The length constraint equation for the connecting rod (523) is as follows: Substituting the inverse kinematic equations into the length constraint equations of the link (523), we obtain the trigonometric standard equations: The set of equations for the positive kinematic spherical constraint: After elimination, a linear mapping relationship is obtained: Back substitution yields the quadratic characteristic equation: in, , , The absolute coordinates of the X-axis, Y-axis, and Z-axis of the micro-ball joint (524) that is hinged to the i-th sector rotor corner (522) and the connecting rod (523) of the submicron-level three-axis micro-manipulation module after moving in three-dimensional space, in nm; , , These are the absolute coordinates of the rotation origin of the i-th stepper motor (521) of the submicron-level three-axis micro-manipulation module in three-dimensional space along the X, Y, and Z axes, respectively, in nm; The radius of rotation of the angular fan-shaped rotor, in mm; These are the actual rotation angle of the i-th stepper motor (521) and the structural installation azimuth angle of the i-th stepper motor (521) on the horizontal projection plane, respectively, in °; , , These correspond to the absolute coordinates of the liquid microelectrode probe tip on the horizontal, vertical, and axial axes in three-dimensional space, respectively, in nm. The unit is the uniform length of the connecting rod, in mm; , , These are the trigonometric substitution coefficients derived from known geometric quantities such as target coordinates, motor origin coordinates, and mechanical rod length when solving the inverse kinematics equation of the i-th drive component. , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the first drive assembly to the connecting rod; , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the second drive assembly to the connecting rod; , , These are the absolute coordinates of the X, Y, and Z axes in three-dimensional space of the micro-ball joint that connects the fan-shaped rotor corner of the third drive assembly to the connecting rod; , , , , All of these are fitting coefficients for the characteristic equation, obtained by fitting the measured rotation angle and end coordinate data of the stepper motor. By solving the above set of equations, the three-dimensional absolute coordinates of the target coordinates are inverted to the corresponding rotation angle of the stepper motor (521) and the measured rotation angle of the stepper motor (521) is inverted to the liquid microelectrode probe (4).

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