Current compensation and frequency following method for nanopore single-molecule sequencing platform

Abstract

The invention discloses a current compensation and frequency following method for a nanopore single molecule sequencing platform. A nanopore is placed in a left cavity and a right cavity which are different in concentration, command voltage is applied to the nanopore, the command voltage enables biological single molecules to move and penetrate through the nanopore, and the movement of the biological single molecules enables the number of ions in the left cavity and the number of ions in the right cavity to change so as to generate ion current. The stray capacitance and the parasitic capacitance existing in the nanopore can also generate disturbance current, meanwhile, the movement speed of the biological single molecule driven by the command voltage can also enable a traditional circuit not to follow to cause signal loss, and according to the compensation method and design, the disturbance current and frequency are compensated by changing the change of the access resistance value in the adjusting circuit. According to the invention, the disturbance current during nanopore single molecule sequencing signal detection can be effectively adjusted, the signal change is timely followed, the phenomenon of signal loss is not generated, the accurate control of the movement of biological single molecules is realized, and the repeated measurement of a single basic group in DNA sequencing is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of electronic information technology, and specifically relates to a current compensation method and a frequency following method for a nanopore single-molecule sequencing platform, and a compensation circuit for implementing the method and design. Background Art

[0002] Since the concept of nanopore was proposed, it has attracted widespread attention. The concept of nanopore sequencing was first proposed in the 1980s. It is a technology for sequencing DNA based on nanopore sensors, involving multiple disciplines such as molecular biology, synthetic biology, and micro-nano processing technology. Nanopore sequencing technology is mainly divided into two categories: biological nanopores and solid-state nanopores.

[0003] A general nanopore is a natural nanopore material composed of a unique conical cavity structure with a diameter ranging from 1.4nm to 4.6nm. It can effectively identify and transmit single-stranded DNA molecules and analyze single nucleotides when double-stranded DNA is temporarily fixed in the narrow pore. The protein molecules are embedded in the phospholipid membrane, or on two-dimensional materials such as tungsten disulfide (WS2), molybdenum disulfide (MoS2), silicon nitride (SiNx), etc., a nanopore film with uniform and controllable pore size is prepared by using focused ion beam, focused electron beam, and chemical vapor deposition (LPCVD). Usually, a small amount of atomic layer of film material, such as graphene, boron nitride, molybdenum sulfide, etc., is transferred to a silicon substrate with a pyramid window to make a suspended membrane. Subsequently, a focused electron beam is used to punch a hole of several nanometers in the suspended membrane. Such a chip with a nanopore is generally cut into a small chip of about 3mm square. The nanopore part is generally added to a storage tank with two left and right chambers. The storage tank generally contains a conductive ion solution, such as potassium chloride (KC1) solution, etc. It is connected to the signal acquisition device through the silver / silver chloride (Ag / AgC1) electrode to collect information and obtain data, thereby recording the ion current of the nanopore. The silver / silver chloride (Ag / AgC1) electrode will cause the negatively charged DNA molecules in the ion solution to move and pass through the nanopore through the applied voltage. When a positive voltage is applied, a baseline current through the pore will be generated when the DNA molecules have not moved yet. When the negatively charged DNA molecules move through the nanopore under the influence of the positive voltage, the blocking effect of the molecules on the pore will affect the number of ions passing through the pore, causing current fluctuations. At this time, we will see spike fluctuations in the ion current. Expanding these signals, we can see that the signals have many different forms, representing the different molecular structures of the pores. Currently, the corresponding base information can be judged and identified by statistically observing the size of the ionic current and the degree of change in the current size when passing through the pore, as well as a large number of perforation events, to obtain the diffusion coefficient, mobility, conformation and other characteristics of the molecule.

[0004] The silver / silver chloride (Ag / AgCl) electrode can cause the negatively charged DNA molecules in the ionic solution to move and pass through the nanopores by applying an external voltage. Without any treatment, the DNA molecules can move at a speed of up to 10 per second. 6 The pore size can be scaled up to five orders of magnitude by adding proteins to the ends of the DNA molecules to specifically bind to the molecules on the nanopore or by using optical or magnetic tweezers to control the movement of the DNA. Improved modulation of the pore can also be scaled up to two orders of magnitude.

[0005] The main reasons for inaccurate signals are: first, the average time for a single base to pass through the hole is too short, resulting in the acquisition circuit system bandwidth being unable to meet the requirements and difficult to achieve accurate measurement; second, the difference in ion current caused by different bases is very small and is submerged in the recorded ion current noise signal.

[0006] In order to achieve the purpose of sequencing and solve the problem of insufficient resolution of nanopores, a concept is proposed, that is, after the DNA molecule passes through the nanopore, a reverse driving voltage is immediately applied, the purpose is to move the DNA molecule back through the nanopore, so as to achieve the purpose of multiple measurements of the same DNA molecule. If it is possible to reduce the delayed reaction time of the DNA molecule or even eliminate the delay so that the driving voltage stops before the DNA molecule completely passes through the nanopore, the DNA molecule can stay in the nanopore.

[0007] The advantages of this idea are that, first, it can effectively confine the molecule near the nanopore, and multiple measurements of the same DNA molecule can improve the resolution of the DNA sequence, especially when the nanopore resolution is not enough, this method can provide more data to make up for the lack of resolution. Second, it can capture more accurate sequence information through multiple measurements, thereby improving the signal-to-noise ratio.

[0008] However, this idea encountered a serious problem in actual experiments: the nanopore itself has corresponding parasitic capacitance and stray capacitance. Their existence will inevitably cause a transient capacitor charging and discharging process when the driving voltage is reversed, which will generate a disturbance current. The corresponding disturbance current generally lasts for tens to hundreds of microseconds. When the disturbance current has not completely disappeared, it will affect the DNA molecule. Different from the expected result at the beginning, the electric field force on the DNA molecule in the ionic solution will be greater than the theoretical positive, which will shorten the time for the DNA molecule to pass back to the nanopore. At the same time, the amplitude peak of this disturbance current is very large, which usually saturates the operational amplifier of the information acquisition system. When the information acquisition system is saturated, first, the output will be distorted and can no longer change according to the change of the input signal. Second, the gain of the operational amplifier is actually zero, and it can no longer amplify the input signal. Third, additional noise will be introduced, affecting the clarity of the signal. If the signal generated by the DNA molecule passing back to the nanopore happens to fall into the package and area of ​​the information acquisition system at this time, its signal will not be recorded, and thus the next change in the driving voltage cannot be triggered. Although there have been reports of thousands of DNA ping-pong motion experiments, no one has yet been able to shorten the delay time to less than milliseconds and achieve continuous ping-pong motion control. This shows that in order to achieve rapid and accurate motion control of the same DNA molecule and achieve sequencing purposes, the problem of perturbed current in the nanopore must be solved. At the same time, the situation of information loss caused by the DNA molecule passing through the pore too quickly must also be dealt with. Summary of the invention

[0009] The technical solution of the present invention is to overcome the shortcomings of the prior art and provide a current compensation method and frequency following method and circuit structure for a nanopore single molecule sequencing platform, which can fully compensate for the disturbance current in the nanopore and expand the circuit bandwidth so that the signal acquisition system meets the required frequency. Ultimately, the precise control of the movement of biological single molecules is achieved, which is helpful for the accurate measurement of single bases in DNA sequencing.

[0010] The technical solution provided by the present invention is a method for current compensation and frequency following of a nanopore single-molecule sequencing platform, which comprises the following steps:

[0011] A command voltage is applied to the left and right chambers of the nanopore with different concentrations. The command voltage will cause the biological single molecule to move and pass through the nanopore. The movement of the biological single molecule will change the number of ions in the left and right chambers and generate ion current. The stray capacitance and parasitic capacitance of the nanopore itself will also generate disturbance current. At the same time, the rapid movement of the biological single molecule driven by the command voltage also puts forward requirements for the frequency of the detection circuit. Among them, the disturbance current and frequency are compensated by changing the resistance value connected to the detection circuit until the frequency completely follows and the disturbance current is reduced.

[0012] The technical solution principle of the present invention is: the current compensation method and frequency following design of this nanopore single-molecule sequencing platform, the driving voltage and the ground terminal are respectively connected at both ends of the nanopore electrode to generate a voltage difference, the DNA molecule moves through the nanopore under the action of the voltage difference and generates changes in ion current, and the parasitic capacitance and stray capacitance of the nanopore itself will also generate disturbance current. At the same time, under the action of the driving voltage, the DNA molecule passes through the nanopore quickly, which also puts forward high bandwidth requirements for the information acquisition system. The compensation method realizes the compensation of disturbance current and the adjustment of bandwidth by connecting the compensation circuit until the disturbance current compensation and bandwidth expansion are completed.

[0013] Since the compensation method compensates for the parasitic capacitance and stray capacitance generated by the nanopore by designing the corresponding current compensation circuits 1 and 2, the time constant is adjusted by adjusting the values ​​of the variable resistor and the capacitor to achieve the purpose of compensating the disturbance current. The bandwidth of the signal acquisition system is expanded by the frequency compensation circuit to meet the required requirements. The driving voltage makes the current change generated by the movement of the DNA molecules consistent with the expected process, and the ion current in the solution itself does not change due to the access of the compensation circuit. Under the action of the compensation circuit, the signal-to-noise ratio of the signal is improved, and the information loss caused by the disturbance current and insufficient bandwidth is reduced. It is helpful for the accurate measurement of single bases in DNA sequencing. Both the current compensation circuits 1 and 2 perform current compensation, and their functions are to compensate for the current generated by the fast capacitor and the current generated by the slow capacitor respectively. The parasitic capacitance existing in the nanopore equivalent circuit can be divided into two types: fast capacitor and slow capacitor according to the size of the time constant. The phospholipid membrane capacitor Cm is a slow capacitor, and the partition capacitor Cg is a fast capacitor, so the two capacitors should compensate for the current generated by them respectively.

[0014] The present invention provides a circuit structure for realizing a current compensation method and frequency following of a nanopore single-molecule sequencing platform, comprising an equivalent circuit, current compensation circuits one and two, and a frequency compensation circuit, wherein the current compensation circuits one and two are connected in front of a signal amplification part, and the frequency compensation circuit is connected behind the signal amplification part; wherein the current compensation circuits one and two compensate for the current generated by a fast capacitor and the current generated by a slow capacitor respectively; the frequency compensation circuit is used to change the frequency in the circuit to prevent signal loss;

[0015] Specifically, current compensation circuit 1 and current compensation circuit 2 are respectively connected to the solution resistance and series resistance of the equivalent circuit, the equivalent circuit is connected to the signal detection amplifier circuit to detect and amplify the signal, and the signal detection amplifier circuit is then connected to the frequency compensation circuit to realize frequency compensation.

[0016] More specifically, the equivalent circuit in the nanopore detection device is: solution resistance Rr, nanopore membrane resistance Rm, phospholipid membrane capacitance Cm, series resistance Rs, partition capacitance Cg, partition capacitance Cg series solution resistance Rr, nanopore membrane resistance Rm in parallel with phospholipid membrane capacitance Cm and then in series with series resistance Rs, and finally in parallel with partition capacitance Cg in series with solution resistance Rr. Current compensation circuit 1 includes variable resistors Rp1, Ri, Rp2, Rx capacitor Cp and operational amplifiers A1 and A2, variable resistor Rp1 and one end of capacitor Cp are connected to the inverting input terminal of operational amplifier A1, Rp2 is connected to the non-inverting input terminal of A1 and connected to the inverting input terminal of operational amplifier A2, Ri is connected in parallel between Rp1 and Rp2, and Rx is connected in parallel between the inverting input terminal and the output terminal of A2. The second current compensation circuit includes Rm1 connected to the reverse input terminal of the operational amplifier A3, Rm2, Rm3, and Cm1 are respectively connected across the reverse input terminal and output terminal of the operational amplifiers A3, A4, and A5, Rp3 and Rm4 are respectively connected to the reverse input terminal and output terminal of A3, A4 and A4, A5, Rp4 is respectively connected to the reverse input terminal of A3 and A6, and the A6 in-phase input terminal is connected to the command voltage. The disturbance current generated by the nanopore can be compensated by the compensation circuit. The frequency compensation circuit includes a sliding rheostat Rp6 connected in parallel with the capacitor C, and the two ends are respectively connected to the reverse input terminal of the operational amplifier A7 and one end of the resistor R, the other end of the resistor R is connected in series with C4 and connected to the in-phase input terminal of A7, and the two ends of the sliding rheostat Rp5 are connected to the reverse input terminal and output terminal of A7. One end of Rp6 of the compensation circuit is connected to the detection device circuit to compensate the frequency.

[0017] The present invention can effectively adjust the disturbance current during nanopore single-molecule sequencing signal detection, timely follow the signal changes, and will not cause signal loss, thereby achieving precise control of the movement of biological single molecules and facilitating repeated measurement of single bases in DNA sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the structure of a nanopore sequencing platform detection device is shown;

[0019] Figure 2 Equivalent electrical diagram of the nanopore signal generating device;

[0020] Figure 3 Nanopore disturbance current compensation circuit design 1;

[0021] Figure 4 Nanopore disturbance current compensation circuit design 2;

[0022] Figure 5 Nanopore frequency compensation circuit design;

[0023] Figure 6A current comparison diagram before and after compensating for disturbance current according to the present invention is shown;

[0024] Figure 7 A frequency comparison diagram before and after frequency compensation according to the present invention is shown;

[0025] Figure 8 Shows the overall connection diagram. DETAILED DESCRIPTION

[0026] The present invention provides a current compensation method and frequency following design for a nanopore single molecule sequencing platform to accurately measure the current signal generated by the movement of a single biological molecule. The present invention is suitable for measuring the signal generated by the movement of a single chain molecule such as DNA.

[0027] Example 1: Current compensation method and frequency following design of nanopore single molecule sequencing platform

[0028] Figure 1 The schematic diagram of the structure of the nanopore sequencing platform device is shown. Figure 1 It can be seen that the general nanopore sequencing platform places the nanopore device in two chambers filled with electrolyte (such as KCl or Ag / AgCl system). Under the action of external voltage, when the single-stranded nucleotide sequence passes through the nanopore through the membrane, the base molecules will hinder the flow of ions, causing the current signal characteristics passing through the nanopore to change. Since different bases have different obstacles to the flow of ions, analyzing the current fluctuation signal can identify the base passing through the nanopore and complete the reading of the base sequence.

[0029] Figure 2 The equivalent electrical diagram of the nanopore signal generating device is shown. When an external driving voltage is applied, due to the presence of the partition capacitance Cg and the phospholipid membrane capacitance Cm, it will form multiple groups of RC circuits with its own solution resistance Rr, the nanopore membrane resistance Rm or other dielectric loss series resistance Rs, so there is an obvious disturbance current phenomenon. The disturbance current generally lasts for hundreds of microseconds or even tens of milliseconds. When the disturbance current exists, it will cause the data acquisition equipment to saturate and blank areas will appear in the record. This means that the device cannot record data normally, resulting in information loss.

[0030] At the same time, the perturbation current will also hinder the re-capture of the returning molecules, so that the continuous DNA ping-pong movement cannot continue for a long time. This will affect the continuity and efficiency of sequencing. During the time period when the perturbation current appears, a strong electric field will be generated. This strong electric field effectively pulls back the molecules that have just passed through the nanopore, affecting the movement of DNA molecules and causing the DNA to return to the nanopore earlier than theoretically predicted, affecting the accuracy of sequencing. Due to the influence of the perturbation current, the system signal-to-noise ratio may be reduced and the current measurement accuracy may be reduced, which is crucial for accurately identifying DNA sequences. In some cases, the perturbation current may also limit the maximum sampling frequency of the sequencing circuit.

[0031] The present invention provides such a method: a compensation method for perturbation current and frequency following in a nanopore, wherein a driving voltage interface and a grounding point are set at both ends of the nanopore to generate a driving voltage, the driving voltage is used to drive the movement of biological single molecules passing through the nanopore to generate ion current changes, and the nanopore itself has a capacitance to generate perturbation current. The compensation method compensates for the perturbation current by connecting a compensation circuit to the signal generation part of the nanopore and gradually changing the time constant of the compensation circuit until all perturbation currents are compensated. At the same time, a frequency compensation circuit is connected after the signal detection amplifier circuit, and the circuit frequency is compensated by adjusting the time constant of the compensation circuit until the circuit can follow the change of the signal.

[0032] Figure 3 , 4 5 shows the schematic diagram of the current compensation circuit and the frequency compensation circuit in detail. The following is a detailed introduction.

[0033] Figure 3 The specific circuit structure of the current compensation circuit is shown, where one end of the variable resistor Rp1 and the capacitor Cp are connected to the inverting input terminal of the operational amplifier A1, Rp2 is connected to the non-inverting input terminal of A1 and connected to the inverting input terminal of the operational amplifier A2, Ri is connected in parallel between Rp1 and Rp2, and Rx is connected in parallel between the inverting input terminal and the output terminal of A2;

[0034] Figure 4 The specific circuit structure of the second current compensation circuit is shown, Rm1 is connected to the inverting input terminal of the operational amplifier A3, Rm2, Rm3, and Cm1 are respectively connected across the inverting input terminal and output terminal of the operational amplifier A3, A4, and A5, Rp3 and Rm4 are respectively connected to the inverting input terminal and output terminal of A3, A4 and A4, A5, Rp4 is respectively connected to the inverting input terminal of A3 and A6, and the non-inverting input terminal of A6 is connected to the command voltage.

[0035] Figure 5The specific circuit structure of the frequency compensation circuit is shown. After the sliding rheostat Rp6 is connected in parallel with the capacitor C, both ends are respectively connected to the inverting input terminal of the operational amplifier A7 and one end of the resistor R. The other end of the resistor R is connected to the non-inverting input terminal of A7 after being connected in series with C4. Both ends of the sliding rheostat Rp5 are connected to the inverting input terminal and the output terminal of A7.

[0036] Through the access of the current compensation circuit and the frequency regulation compensation circuit, by adjusting the corresponding parameters of the compensation circuit, the disturbance current can be compensated, so that the frequency can change with the signal. Continuously adjust the parameters of the compensation circuit until all the disturbance currents are compensated and the circuit frequency completely follows the signal change, realizing the precise control of the biological single-molecule movement.

[0037] Specifically, the method for current compensation and frequency tracking includes the following steps:

[0038] (1) Monitor / record the ionic current of the nanopore in real time, observe the generated disturbance current and simultaneously detect the frequency of the circuit, and compare the frequency required for the nanopore detection circuit to achieve signal non-loss;

[0039] (2) Assume that the total gain of the fast capacitor compensation circuit is A, and the voltage of the compensation path is taken from the command voltage. Then at the compensation node: After simplification, the compensation gain is obtained as: Calculate the time constant τ = R r C g . According to Figure 3 The current compensation circuit 1 shown, the relationship between the output voltage and the input voltage can be obtained as: According to the gain matching, it can be obtained that: After combining and simplifying, the parameter relationship in the current compensation circuit 1 can be obtained as: R p1 C p = τ = R r C g , Let Vm be the membrane potential of the resistance-capacitance network composed of the membrane capacitance and the membrane resistance. Then at the membrane potential node: Since Rs << Rm, it is simplified to: Assume that the total gain of the slow capacitor compensation circuit is B, and the voltage of the compensation path is taken from the command voltage. Then at the compensation node: Calculate the time constant τ' = (R m / / R s )C m ≈ R s C m , and after simplification, it is obtained that: After the above analysis, Figure 4 The current compensation circuit 2 shown can be established. At Figure 4If Rm1=Rm2 is set in , the relationship between voltage V0 and command voltage is: According to gain matching: The parameter relationship in the current compensation circuit 2 can be obtained by combining the following:

[0040]

[0041] (3) setting corresponding resistance and capacitance values ​​according to the parameter relationship of the current compensation circuit 1 obtained in step (2);

[0042] (4) setting corresponding resistance and capacitance values ​​according to the parameter relationship of the current compensation circuit 2 obtained in step (2);

[0043] (5) An active filter is formed using an operational amplifier and RC elements to improve frequency response, especially compensation in high or low frequency situations. Figure 5 Frequency compensation circuit, the feedback network of operational amplifier A7 is composed of R, C, C4, where the combination of Rp6 and C4 mainly sets the time constant of the filter and adjusts the frequency response of the signal. Figure 5 The transfer function H(s) of the feedback network can be obtained from the circuit shown as: s=jw is the Laplace transform variable, and Rp6C sets a low-pass cutoff frequency: RC4 sets a high-pass cutoff frequency: Calculate the corresponding resistance and capacitance parameters according to the formula and time constant;

[0044] (6) setting corresponding values ​​according to the parameter relationship obtained in steps (3), (4), and (5) and connecting the circuit for testing;

[0045] (7) Determine whether the disturbance current in step (6) is fully compensated, if yes, execute step (9), otherwise execute steps (3) and (4);

[0046] (8) Determine whether the circuit frequency in step (6) can follow the change of the signal. If yes, execute step (9); otherwise, execute step (5);

[0047] (9)End.

[0048] In the step (3), the time constant is adjusted by adjusting the resistance value and capacitance value of the variable resistor Rp1 and the variable capacitor Cp of the compensation circuit, the compensation gain is adjusted by adjusting the resistance value of the fixed resistor Rx and the variable resistor Rp2, and the delay link is compensated by adjusting the resistance value of the variable resistor Ri and the fixed resistor Rx. After the compensation circuit is formed, it is output to the front-end detection device.

[0049] In step (4), the perturbation current is adjusted by adjusting the resistance values ​​of the variable resistor Rp3 and the variable resistor Rp4. The adjustment of the variable resistor Rp3 can be used to compensate for the influence of the series resistor on the charge and discharge current of the membrane capacitor. The parameters of Rp4 and Rp3 do not interfere with each other at all, and specific values ​​can be obtained through theoretical calculation.

[0050] In step (5), the feedback network of the operational amplifier A7 is composed of R, C, and C4, wherein the combination of Rp6 and C4 mainly sets the time constant of the filter and adjusts the frequency response of the signal.

[0051] Also provided is a design for realizing current compensation method and frequency following of nanopore single molecule sequencing platform, wherein the equivalent circuit in the nanopore detection device is: solution resistance Rr, nanopore membrane resistance Rm, phospholipid membrane capacitance Cm, series resistance Rs, partition capacitance Cg, partition capacitance Cg series resistance Rr, nanopore membrane resistance Rm connected in parallel with phospholipid membrane capacitance Cm and then connected in parallel with the part of partition capacitance Cg connected in series with solution resistance Rr and then connected in series with resistance Rs. The compensation circuit comprises variable resistor Rp1, Ri, Rp2, Rx capacitor Cp and operational amplifiers A1 and A2, wherein one end of variable resistor Rp1 and capacitor Cp are connected to the inverting input terminal of operational amplifier A1, Rp2 is connected to the non-inverting input terminal of A1 and connected to the inverting input terminal of operational amplifier A2, Ri is connected in parallel between Rp1 and Rp2, and Rx is connected in parallel between the inverting input terminal and output terminal of A2. Rm1 is connected to the inverting input terminal of operational amplifier A3, Rm2, Rm3, and Cm1 are respectively connected across the inverting input terminal and output terminal of operational amplifiers A3, A4, and A5, Rp3 and Rm4 are respectively connected to the inverting input terminal and output terminal of A3, A4 and A4, A5, Rp4 is respectively connected to the inverting input terminal of A3 and A6, and the non-inverting input terminal of A6 is connected to the command voltage. The perturbation current generated by the nanopore can be compensated by the compensation circuit. After the variable resistor Rp6 is connected in parallel with the capacitor C, the two ends are respectively connected to the inverting input terminal of the operational amplifier A7 and one end of the resistor R, the other end of the resistor R is connected in series with C4 and connected to the non-inverting input terminal of A7, and the two ends of the sliding variable resistor Rp5 are connected to the inverting input terminal and output terminal of A7. One end of Rp6 of the compensation circuit is connected to the detection device circuit to compensate the frequency.

[0052] Example 2 Application Effect Example

[0053] Figure 8The overall structural schematic diagram is shown. The specific connection diagram is that the current compensation circuit 1 and the current compensation circuit 2 are respectively connected to the solution resistance Rr and the series resistance Rs of the nanopore equivalent circuit. In the actual compensation circuit design, in order to improve the ability of the detection device to process signals, the input voltage applied to the current compensation circuit 1 and the current compensation circuit 2 is 10 times the command voltage. The nanopore equivalent circuit is connected to the signal detection amplifier circuit to perform signal detection and amplification. The signal detection amplifier circuit is then connected to the frequency compensation circuit to realize frequency compensation.

[0054] The application example explains in detail that the basic idea of ​​compensation design is to compensate the disturbance current and frequency of the circuit through the compensation circuit. The compensation of the disturbance current is to design the compensation circuit to generate a current equal to the magnitude and opposite to the direction of the parasitic capacitance. The design of the frequency compensation circuit is to achieve the purpose of high-frequency compensation through the operational amplifier and RC circuit in the circuit.

[0055] As an example, the comparison diagram of the disturbance current before and after compensation obtained according to the present invention is as follows: Figure 6 As shown. When the command voltage amplitude is 100mV, the current compensation circuit input voltage amplitude is 1V, the duty cycle is 50%, there is no bias, the line connection is correct, there is no external interference, and the device is in an ideal state, simulation verification is carried out under the conditions of not connecting to compensation circuits one and two and connecting to current compensation circuits one and two. It can be clearly seen that there is a significant gap between before and after compensation. Figure 6 The left picture shows the current situation before compensation. Figure 6 The right figure shows the current after compensation. For general nanopores, the method according to the present invention can basically effectively reduce the interference of the perturbation current.

[0056] According to the present invention, the comparison diagram of the circuit frequency before and after compensation is as follows: Figure 7 As shown. When the input voltage is AC, the command voltage amplitude is 100mV, the current compensation circuit input voltage amplitude is 1V, the duty cycle is 50%, there is no bias, the line connection is correct, there is no external interference, and the device is in an ideal state, the simulation verification is carried out under the conditions of not connecting to the frequency compensation circuit and connecting to the frequency compensation circuit. It can be clearly seen that there is a significant gap between before and after compensation. Before the compensation band is not performed, the frequency is within 1khz, which fails to meet the frequency requirements for signal detection. After the compensation circuit, it can be seen that the frequency is above 50khz and can completely follow the changes in the signal. For general nanopores, the method according to the present invention can basically achieve circuit detection following signal changes without losing information.

[0057] To sum up, the above are only preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for current compensation and frequency following of a nanopore single-molecule sequencing platform, characterized in that: The steps include: Applying a command voltage to the left and right chambers of the nanopore with different concentrations will cause the biological single molecule to move and pass through the nanopore. The movement of the biological single molecule will cause the number of ions in the left and right chambers to change, thereby generating an ion current. The stray capacitance and parasitic capacitance of the nanopore itself will also generate a disturbance current. At the same time, the rapid movement of the biological single molecule driven by the command voltage also puts forward requirements for the detection circuit frequency. The disturbance current and frequency are compensated by changing the resistance value of the connected detection circuit until the frequency completely follows and the disturbance current is reduced.

2. The method according to claim 1, characterized in that: Specifically include the following steps: (1) Record the ionic current of the nanopore in real time and observe the disturbance current and frequency generated by changing the resistance value connected in the detection circuit; (2) According to the formula The time constants τ and τ' are used to obtain the parameter relationship and adjust the resistance and capacitance in the circuit. Where A and B are gains, Vc is the command voltage, Vm is the node voltage, Cc and Cc2 are the reference capacitors for the designed compensation part, Ci is the stray capacitance, Rr is the ionic solution resistance, and Rs is the phospholipid bilayer membrane resistance; (3) According to the formula Calculate the corresponding resistance and capacitance parameters according to the formula and time constant; (4) According to the time constant obtained in step (2), adjust the resistance connected to the compensation circuit and observe the change of the disturbance current; (5) Calculating the resistance change of the access circuit according to the compensation parameters obtained in step (3) and observing the cut-off frequency; (6) Determine whether the disturbance current in step (4) is fully compensated, if yes, then end; otherwise, execute step (2); (7) Determine whether the cutoff frequency of step (5) meets the requirement. If yes, the process ends. Otherwise, the process proceeds to step (3).

3. The method according to claim 2, characterized in that: In step (4), the time constant is adjusted by adjusting the resistance value and capacitance value of Rp1 and Cp, the compensation gain is adjusted by adjusting the resistance value of Rx and Rp2, and the delay link is compensated by adjusting the resistance value of Ri and Rx. After forming a compensation circuit, it is output to the front-end detection device.

4. The method according to claim 2, characterized in that: In the step (4), the disturbance current is adjusted by adjusting the resistance values ​​of Rp3 and Rp4; Among them, the adjustable variable resistor Rp3 is used to compensate for the influence of the series resistance on the charging and discharging current of the membrane capacitor. The parameters of the sliding variable resistor Rp4 and Rp3 do not interfere with each other at all, and the specific values ​​are obtained through theoretical calculation.

5. The method according to claim 3, characterized in that: In the step (5), the resistance values ​​of Rp5 and Rp6 connected to the circuit are adjusted, and C, R, and C4 are fixed resistors and capacitors, and the specific sizes are determined according to the feedback resistance value of the preamplifier.

6. A circuit structure for implementing the method according to any one of claims 1 to 5, characterized in that: It includes an equivalent circuit, current compensation circuits one and two, and a frequency compensation circuit, wherein the current compensation circuits one and two are connected in front of the signal amplification part, and the frequency compensation circuit is connected after the signal amplification part; wherein the current compensation circuits one and two compensate for the current generated by the fast capacitor and the current generated by the slow capacitor respectively; the frequency compensation circuit is used to change the frequency in the circuit to prevent signal loss.

7. The circuit structure according to claim 6, characterized in that: The current compensation circuit 1 and the current compensation circuit 2 are respectively connected to the solution resistance and the series resistance of the equivalent circuit. The equivalent circuit is connected to the signal detection amplifier circuit to detect and amplify the signal. The signal detection amplifier circuit is then connected to the frequency compensation circuit to realize frequency compensation.

8. The circuit structure according to claim 6, characterized in that: The equivalent circuit is: solution resistance Rr, nanopore membrane resistance Rm, phospholipid membrane capacitance Cm, series resistance Rs, and partition capacitance Cg. The partition capacitance Cg is connected in series with the solution resistance Rr. The nanopore membrane resistance Rm is connected in parallel with the phospholipid membrane capacitance Cm and then connected in parallel with the part of the partition capacitance Cg connected in series with the solution resistance Rr and then connected in series with the resistance Rs. The current compensation circuit 1 is used to compensate the disturbance current generated by the nanopore fast capacitor, including variable resistor Rp1, Ri, Rp2, Rx capacitor Cp and operational amplifiers A1 and A2, wherein one end of the variable resistor Rp1 and the capacitor Cp are connected to the inverting input terminal of the operational amplifier A1, Rp2 is connected to the non-inverting input terminal of A1 and the inverting input terminal of the operational amplifier A2, Ri is connected in parallel between Rp1 and Rp2, and Rx is connected in parallel between the inverting input terminal and the output terminal of A2; The second current compensation circuit is used to compensate the disturbance current generated by the nanopore slow capacitor, including Rm1 connected to the reverse input terminal of the operational amplifier A3, Rm2, Rm3, and Cm1 respectively connected across the inverting input terminal and output terminal of the operational amplifier A3, A4, and A5, Rp3 and Rm4 are respectively connected to the inverting input terminal and output terminal of A3, A4 and A4, A5, Rp4 is respectively connected to the inverting input terminal of A3 and A6, and the non-inverting input terminal of A6 is connected to the command voltage. The frequency compensation circuit is used to change the frequency in the circuit to prevent signal loss, including a sliding rheostat Rp6 connected in parallel with a capacitor C, with both ends connected to the inverting input of an operational amplifier A7 and one end of a resistor R respectively, the other end of the resistor R is connected in series with C4 and connected to the non-inverting input of A7, both ends of the sliding rheostat Rp5 are connected to the inverting input and output of A7, and one end of Rp6 of the compensation circuit is connected to the detection device circuit to perform frequency compensation. Observe the change in the disturbance current waveform before and after the current compensation circuit is connected and the change in the cutoff frequency waveform before and after the frequency compensation circuit is connected to determine the effect of the compensation circuit.

9. A nanopore single-molecule sequencing platform detection device having the circuit structure as described in claim 6, 7 or 8.

10. Use of the detection device as claimed in claim 9 in gene sequencing.

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