A method for current compensation and frequency tracking in a nanopore single-molecule sequencing platform

Abstract

This invention discloses a method for current compensation and frequency tracking in a nanopore single-molecule sequencing platform. The method involves placing a nanopore in two chambers with different concentrations and applying a command voltage. This command voltage causes biological single molecules to move and pass through the nanopore. The movement of the biological single molecules changes the number of ions in the chambers, generating an ion current. The stray capacitance and parasitic capacitance inherent in the nanopore itself also generate perturbation currents. Furthermore, the movement speed of the biological single molecules driven by the command voltage can cause traditional circuits to fail to keep up, resulting in signal loss. This compensation method and design compensates for the perturbation current and frequency by changing the resistance value connected in the regulating circuit. This invention can effectively regulate the perturbation current during nanopore single-molecule sequencing signal detection, promptly follow signal changes, prevent signal loss, and achieve precise control of the movement of biological single molecules, which is beneficial for the repetitive measurement of single bases in DNA sequencing.

Description

Technical Field

[0001] This invention belongs to the field of electronic information technology, specifically relating to a current compensation method and a frequency tracking method for a nanopore single-molecule sequencing platform, as well as the compensation circuit for implementing the method and design. Background Technology

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

[0003] Typical nanopores are natural nanoporous materials composed of protein molecules embedded in phospholipid membranes, which have unique conical cavity structures with diameters varying between 1.4 nm and 4.6 nm. These materials can effectively recognize and transfer single-stranded DNA molecules and resolve individual nucleotides when double-stranded DNA is temporarily fixed in the narrow pores. Alternatively, nanoporous films with uniform and controllable pore sizes can be prepared on two-dimensional materials such as tungsten disulfide (WS2), molybdenum disulfide (MoS2), and silicon nitride (SiNx) using focused ion beams, focused electron beams, and liquid-phase chemical vapor deposition (LPCVD). Typically, a small atomic layer of film material, such as graphene, boron nitride, or molybdenum sulfide, is transferred onto a silicon substrate with pyramidal windows to create a suspended film. Subsequently, a focused electron beam is used to create a pore of a few nanometers in the suspended film. Chips with such nanopores are generally cut into small chips approximately 3 mm square. The nanopore portion is typically placed within a storage cell with two chambers, usually containing a conductive ionic solution such as potassium chloride (KCl) solution. It is connected to a signal acquisition device via a silver / silver chloride (Ag / AgCl) electrode to collect data and record the ionic current of the nanopore. Applying an external voltage to the silver / silver chloride (Ag / AgCl) electrode causes negatively charged DNA molecules in the ionic solution to migrate and pass through the nanopore. When a positive voltage is applied, a baseline current is generated before the DNA molecules migrate. As the negatively charged DNA molecules migrate through the nanopore under the influence of a positive voltage, the blocking effect of the molecules on the pores affects the number of ions passing through, causing current fluctuations. This results in spikes in the ionic current. Expanding these signals reveals various signal morphologies, representing different molecular structures within the pore. Currently, the diffusion coefficient, mobility, conformation, and other characteristics of molecules can be obtained by statistically observing the magnitude of the ion current and the degree of change in the current during pore perforation, as well as by observing a large number of pore perforation events, in order to identify and judge the corresponding base information.

[0004] A silver / silver chloride (Ag / AgCl) electrode, when subjected to an applied voltage, causes negatively charged DNA molecules in an ionic solution to migrate and pass through nanopores. Without any additional treatment, the DNA molecules can migrate at speeds up to 10 molecules per second. 6 Each base can be used to specifically bind to the DNA molecules on the nanopore by adding proteins to the ends of the DNA molecules, or by controlling the movement of the DNA with optical or magnetic tweezers, scaling up to five orders of magnitude. Modulation of the pores can also scale up to two orders of magnitude.

[0005] The main reasons for the inaccurate signal are: first, the average time of the single-base via is too short, which makes it impossible for the acquisition circuit system bandwidth to meet the requirements and 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] To achieve sequencing while addressing the insufficient resolution of nanopores, a proposed approach is to apply a reverse driving voltage immediately after the DNA molecule passes through the nanopore. The aim is to reverse the DNA molecule's movement back into the nanopore, allowing for multiple measurements of the same DNA molecule. If the reaction time delay for the DNA molecule can be reduced or eliminated, stopping the driving voltage before the DNA molecule has completely passed through the nanopore, then the DNA molecule can remain within the nanopore.

[0007] The advantages of this approach are twofold: First, it effectively confines molecules to the vicinity of nanopores, allowing for multiple measurements of the same DNA molecule. This improves the resolution of the DNA sequence, especially when nanopore resolution is insufficient, as this method can provide more data to compensate for the lack of resolution. Second, it allows for the capture of 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: nanopores inherently possess parasitic and stray capacitances. Their presence inevitably leads to a transient capacitance charging and discharging process when the driving voltage reverses, generating a disturbance current. This disturbance current typically lasts for tens to hundreds of microseconds. Before the disturbance current completely dissipates, it affects the DNA molecule in a way that differs from the initial expectation. The electric field force experienced by the DNA molecule in the ionic solution becomes even greater than theoretically expected, shortening the time it takes for the DNA molecule to return to the nanopore. Simultaneously, the amplitude of this disturbance current spike is very large, often causing the operational amplifier (op-amp) of the data acquisition system to saturate. When the data acquisition system saturates, firstly, it causes output distortion, making it unable to adapt to changes in the input signal. Secondly, the op-amp gain becomes practically zero, unable to amplify the input signal. Thirdly, it introduces additional noise, affecting signal clarity. If the signal generated when the DNA molecule returns to the nanopore happens to fall within the data acquisition system's packet and region, the signal cannot be recorded, thus failing to trigger the next change in driving voltage. Although there have been reports of thousands of consecutive DNA ping-pong motion experiments, no one has yet been able to reduce the latency to below milliseconds and achieve continuous ping-pong motion control. Therefore, to achieve rapid and precise motion control of the same DNA molecule for sequencing purposes, it is essential to solve the problem of perturbation current within the nanopores. Simultaneously, the information loss caused by the excessively high speed of DNA molecules passing through the pores must also be addressed. Summary of the Invention

[0009] The technical problem solved by this invention is to overcome the shortcomings of existing technologies and provide a current compensation method, a frequency following method, and a circuit structure for a nanopore single-molecule sequencing platform. This method can completely compensate for perturbation currents in the nanopore while simultaneously expanding the circuit bandwidth to ensure the signal acquisition system meets the required frequency. Ultimately, this achieves precise control of the movement of biological single molecules, which is helpful for the accurate measurement of individual bases in DNA sequencing.

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

[0011] A command voltage is applied to two chambers with different concentrations within a nanopore. This command voltage causes biological molecules to move and pass through the nanopore. The movement of the biological molecules changes the number of ions in the chambers, thus generating an ionic current. The stray capacitance and parasitic capacitance inherent in the nanopore also generate a perturbation current. Simultaneously, the rapid movement of the biological molecules driven by the command voltage also places demands on the frequency of the detection circuit. The perturbation current and frequency are compensated by changing the resistance value connected to the detection circuit until the frequency completely matches and the perturbation current is reduced.

[0012] The technical solution of this invention is as follows: This nanopore single-molecule sequencing platform uses a current compensation method and a frequency tracking design. A voltage difference is generated by connecting a driving voltage and a ground terminal to the two ends of the nanopore electrode. Under the influence of this voltage difference, DNA molecules move through the nanopore, generating changes in ionic current. The parasitic and stray capacitances inherent in the nanopore itself also generate perturbation currents. Simultaneously, under the driving voltage, DNA molecules rapidly pass through the nanopore, which places high bandwidth requirements on the information acquisition system. This compensation method uses a compensation circuit to compensate for the perturbation current and adjust the bandwidth until both are achieved.

[0013] This compensation method compensates for the parasitic and stray capacitances generated by the nanopores by designing corresponding current compensation circuits one and two. The time constant is adjusted by regulating the values ​​of the variable resistor and capacitor to compensate for the disturbance current. The bandwidth of the signal acquisition system is extended to meet the required specifications through a frequency compensation circuit. The driving voltage ensures that the current change generated by the movement of DNA molecules remains consistent with the expected process, without altering the ionic current in the solution itself due to the addition of the compensation circuit. The signal-to-noise ratio is improved under the action of the compensation circuit, reducing information loss caused by disturbance currents and insufficient bandwidth. This contributes to the accurate measurement of individual bases in DNA sequencing. Both current compensation circuits one and two perform current compensation, specifically compensating for the current generated by fast and slow capacitors, respectively. The parasitic capacitances existing in the nanopore equivalent circuit can be categorized into fast and slow capacitors based on the magnitude of their time constants. The phospholipid membrane capacitance Cm is a slow capacitor, while the separator capacitance Cg is a fast capacitor; therefore, the current generated by each type of capacitor must be compensated separately.

[0014] This invention provides a circuit structure for implementing a current compensation method and frequency tracking for a nanopore single-molecule sequencing platform, including an equivalent circuit, current compensation circuit one and two, and a frequency compensation circuit. The current compensation circuit one and two are connected before the signal amplification section, and the frequency compensation circuit is connected after the signal amplification section. The current compensation circuit 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.

[0015] Specifically, current compensation circuit one and current compensation circuit two are connected to the solution resistance and series resistance of the equivalent circuit, respectively. The equivalent circuit is connected to the signal detection and amplification circuit to detect and amplify the signal. The signal detection and amplification circuit is then connected to the frequency compensation circuit to achieve frequency compensation.

[0016] More specifically, the equivalent circuit of the nanopore detection device consists of: a solution resistor Rr, a nanopore membrane resistor Rm, a phospholipid membrane capacitor Cm, a series resistor Rs, a separator capacitor Cg, a separator capacitor Cg connected in series with the solution resistor Rr, a nanopore membrane resistor Rm connected in parallel with the phospholipid membrane capacitor Cm and then connected in series with the series resistor Rs, and finally a portion connected in parallel with the separator capacitor Cg connected in series with the solution resistor Rr. The current compensation circuit includes variable resistors Rp1, Ri, Rp2, Rx, capacitor Cp, and operational amplifiers A1 and A2. One end of the variable resistor Rp1 and one end of the capacitor Cp are connected to the inverting input of operational amplifier A1. Rp2 is connected to the non-inverting input of A1 and to the inverting input of operational amplifier A2. Ri is connected in parallel between Rp1 and Rp2, and Rx is connected in parallel between the inverting input and output of operational amplifier A2. The second current compensation circuit includes Rm1 connected to the inverting input of operational amplifier A3; Rm2, Rm3, and Cm1 connected across the inverting inputs and outputs of operational amplifiers A3, A4, and A5, respectively; Rp3 and Rm4 connected to the inverting inputs and outputs of A3, A4, and A5, respectively; Rp4 connected to the inverting inputs of A3 and A6, respectively; and the command voltage connected to the non-inverting input of A6. This compensation circuit can compensate for the disturbance current generated by the nanopores. The frequency compensation circuit includes a variable resistor Rp6 connected in parallel with capacitor C, with its two ends connected to the inverting input of operational amplifier A7 and one end of resistor R, respectively. The other end of resistor R is connected in series with C4 and then connected to the non-inverting input of A7. The variable resistor Rp5 is connected to the inverting input and output of A7. Connecting one end of Rp6 in the compensation circuit to the detection device circuit performs frequency compensation.

[0017] This invention can effectively regulate the perturbation current during nanopore single-molecule sequencing signal detection, promptly follow signal changes, and prevent signal loss. It enables precise control of the movement of biological single molecules and facilitates the repeated measurement of single bases in DNA sequencing. Attached Figure Description

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

[0019] Figure 2 Equivalent electrical diagram of a nanopore signal generation device;

[0020] Figure 3 Design of a nanopore disturbance current compensation circuit (Part 1);

[0021] Figure 4 Design of a nanopore disturbance current compensation circuit (Part 2);

[0022] Figure 5 Design of a nanopore frequency compensation circuit;

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

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

[0025] Figure 8 The overall connection diagram is shown. Detailed Implementation

[0026] This invention provides a current compensation method and frequency tracking design for a nanopore single-molecule sequencing platform to accurately measure the current signals generated by the motion of individual biomolecules. This invention is suitable for measuring signals generated by the motion of single chain molecules such as DNA.

[0027] Example 1: Current compensation method and frequency tracking design for a nanopore single-molecule sequencing platform

[0028] Figure 1 A schematic diagram of the nanopore sequencing platform device is shown. Figure 1 As can be seen, a typical nanopore sequencing platform places the nanopore device in two chambers filled with electrolyte (such as KCl or Ag / AgCl systems). Under the influence of an applied voltage, when a single-stranded nucleotide sequence passes through the nanopore and through the membrane, the base molecules impede the flow of ions, causing changes in the current signal characteristics as the sequence passes through the nanopore. Since different bases impede ion flow to varying degrees, analyzing the current fluctuation signal can identify the bases passing through the nanopore, thus completing the sequence reading.

[0029] Figure 2 The equivalent electrical diagram of the nanopore signal generation device is shown. When an external driving voltage is applied, due to the presence of the separator capacitance Cg and the phospholipid membrane capacitance Cm, it forms multiple RC circuits with its own solution resistance Rr, nanopore membrane resistance Rm, or other dielectric loss series resistance Rs, thus exhibiting a significant disturbance current phenomenon. This disturbance current typically lasts for hundreds of microseconds or even tens of milliseconds. The presence of this disturbance current can cause the data acquisition device to saturate, resulting in blank recording areas. This means the device cannot record data normally, causing information loss.

[0030] Simultaneously, this perturbation current also hinders the recapture of returning molecules, preventing the continuous ping-pong motion of DNA from lasting for an extended period. This affects the continuity and efficiency of sequencing. During the period of perturbation current, a strong electric field is generated, effectively pulling back 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, thus impacting sequencing accuracy. Due to the influence of the perturbation current, the system signal-to-noise ratio may decrease, and the accuracy of current measurement may decline, which is crucial for the accurate identification of DNA sequences. In some cases, the perturbation current may also limit the maximum sampling frequency of the sequencing circuit.

[0031] This invention provides a method for compensating for disturbance current and frequency tracking in nanopores. A driving voltage interface and a grounding point are set at both ends of the nanopore to generate a driving voltage. This driving voltage drives the movement of biological single molecules passing through the nanopore, generating changes in ion current. The nanopore itself has capacitance, generating a disturbance current. This compensation method compensates for the disturbance current by connecting a compensation circuit to the signal generation section of the nanopore and successively changing the time constant of the compensation circuit until all disturbance current is compensated. Simultaneously, a frequency compensation circuit is connected after the signal detection and amplification circuit. The circuit frequency is compensated by adjusting the time constant of the compensation circuit until the circuit can follow the signal changes.

[0032] Figure 3 , 4 Section 5 shows detailed schematic diagrams of the current compensation circuit and the frequency compensation circuit. These will be explained in detail below.

[0033] Figure 3 The circuit structure of a current compensation circuit is shown. One end of the variable resistor Rp1 and capacitor Cp is connected to the inverting input of operational amplifier A1. Rp2 is connected to the non-inverting input of A1 and to the inverting input of operational amplifier A2. Ri is connected in parallel between Rp1 and Rp2. Rx is connected in parallel between the inverting input and output of A2.

[0034] Figure 4 The specific circuit structure of the current compensation circuit 2 is shown. Rm1 is connected to the inverting input terminal of operational amplifier A3. Rm2, Rm3, and Cm1 are connected across the inverting input and output terminals of operational amplifiers A3, A4, and A5, respectively. Rp3 and Rm4 are connected to the inverting input and output terminals of A3, A4, and A5, respectively. Rp4 is connected to the inverting input terminals of A3 and A6, respectively. 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] This design realizes the precise control of biomolecular movement by connecting a current compensation circuit and a frequency regulation compensation circuit, and by adjusting the corresponding parameters of the compensation circuit, the disturbance current can be compensated, the frequency can change with the signal, and the parameters of the compensation circuit are continuously adjusted until all the disturbance currents are compensated and the circuit frequency completely follows the signal change.

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

[0038] (1) Monitor / record the ionic current of the nanopore in real time, observe the generated disturbance current while detecting the frequency of the circuit, and compare the frequency required to ensure that the signal of the nanopore detection circuit is not lost.

[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, there is: After simplification, the compensation gain is obtained as: Calculate the time constant τ = R r C g . According to Figure 3 As shown in the current compensation circuit 1, the relationship between the output voltage and the input voltage is: 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, there is: [[ID=�9]] Since Rs << Rm, it simplifies 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, there is: 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, the current compensation circuit 2 shown in Figure 4 can be established. In Figure 4If Rm1 = Rm2 is set in the condition, then the relationship between voltage V0 and command voltage is as follows: Based on gain matching: By combining and sorting, the parameter relationships in the second current compensation circuit can be obtained as follows:

[0040]

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

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

[0043] (5) An active filter was constructed using operational amplifiers and RC components to improve frequency response, particularly for compensation in high-frequency or low-frequency conditions. A design was developed... Figure 5 In the frequency compensation circuit, the feedback network of operational amplifier A7 consists of R, C, and C4. The combination of Rp6 and C4 primarily sets the time constant of the filter, adjusting the frequency response of the signal. Figure 5 The circuit shown yields the following transfer function H(s) for the feedback network: s = jw is the Laplace transform variable, and Rp6C sets a low-pass cutoff frequency: The RC4 has a set high-pass cutoff frequency: Calculate the corresponding resistance and capacitance parameters based on the formula and time constant;

[0044] (6) Based on the parameter relationships obtained in steps (3)(4)(5), set the corresponding values ​​and connect the circuit for testing;

[0045] (7) Determine whether the disturbance current in step (6) has been fully compensated. If yes, proceed to step (9); otherwise, proceed to steps (3) and (4).

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

[0047] (9) End.

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

[0049] In step (4), the disturbance current is adjusted by regulating the resistance values ​​of variable resistors Rp3 and Rp4. Adjusting variable resistor Rp3 can compensate for the influence of the series resistor on the charging and discharging current of the film capacitor. The parameters of Rp4 and Rp3 are completely independent and can both be obtained through theoretical calculations.

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

[0051] The paper also provides a current compensation method and frequency tracking design for a nanopore single-molecule sequencing platform. The equivalent circuit in the nanopore detection device consists of: a solution resistor Rr, a nanopore membrane resistor Rm, a phospholipid membrane capacitor Cm, a series resistor Rs, and a separator capacitor Cg. The separator capacitor Cg is connected in series with the solution resistor Rr. The nanopore membrane resistor Rm is connected in parallel with the phospholipid membrane capacitor Cm, and then connected in parallel with a portion of the separator capacitor Cg connected in series with the solution resistor Rr, before being connected in series with the resistor Rs. The compensation circuit includes variable resistors Rp1, Ri, Rp2, Rx, capacitor Cp, and operational amplifiers A1 and A2. One end of the variable resistor Rp1 and capacitor Cp is connected to the inverting input of operational amplifier A1. Rp2 is connected to the non-inverting input of A1 and the inverting input of operational amplifier A2. Ri is connected in parallel between Rp1 and Rp2, and Rx is connected in parallel between the inverting input and output of A2. Rm1 is connected to the inverting input of operational amplifier A3. Rm2, Rm3, and Cm1 are connected across the inverting inputs and outputs of operational amplifiers A3, A4, and A5, respectively. Rp3 and Rm4 are connected to the inverting inputs and outputs of A3, A4, and A5, respectively. Rp4 is connected to the inverting inputs of A3 and A6, respectively. The command voltage is connected to the non-inverting input of A6. The compensation circuit compensates for the disturbance current generated by the nanopores. A variable resistor Rp6 is connected in parallel with capacitor C, and its two ends are connected to the inverting input of operational amplifier A7 and one end of resistor R, respectively. The other end of resistor R is connected in series with C4 and then connected to the non-inverting input of A7. A sliding rheostat Rp5 is connected to the inverting input and output of A7. One end of Rp6 in the compensation circuit is connected to the detection device circuit for frequency compensation.

[0052] Example of application effect in Example 2

[0053] Figure 8The diagram shows the overall structure. Specifically, current compensation circuit one and current compensation circuit two are connected to the solution resistance Rr and series resistance Rs of the nanopore equivalent circuit, respectively. In the actual compensation circuit design, in order to improve the signal processing capability of the detection device, the input voltage applied to current compensation circuit one and current compensation circuit two is 10 times the command voltage. The nanopore equivalent circuit is connected to the signal detection amplification circuit for signal detection and amplification. The signal detection amplification circuit is then connected to the frequency compensation circuit to achieve frequency compensation.

[0054] The application examples illustrate the basic design idea of ​​compensation: to compensate for disturbance current and frequency by using a compensation circuit. The compensation circuit is designed to generate a current that is equal in magnitude and opposite in direction to the parasitic capacitance. The frequency compensation circuit is designed to achieve high-frequency compensation by using operational amplifiers and RC circuits in the circuit.

[0055] As an example, a comparison diagram of the disturbance current before and after compensation is obtained according to the present invention is shown below. Figure 6 As shown. Under the conditions of a command voltage amplitude of 100mV, an input voltage amplitude of 1V for the current compensation circuit, a duty cycle of 50%, no bias, correct circuit connection, no external interference, and the devices in ideal condition, simulations were conducted with and without compensation circuits one and two connected. A significant difference is clearly visible between the conditions before and after compensation. Figure 6 The left figure shows the current situation before compensation. Figure 6 The right figure shows the current situation after compensation. For general nanopores, the method according to the present invention can effectively reduce the interference of disturbance current.

[0056] A comparison diagram of the circuit frequencies before and after compensation, obtained according to the present invention, is shown below. Figure 7 As shown. Under AC input voltage conditions, with a command voltage amplitude of 100mV, a current compensation circuit input voltage amplitude of 1V, a duty cycle of 50%, no bias, correct circuit connection, no external interference, and the device in ideal condition, simulations were performed with and without the frequency compensation circuit. A significant difference is clearly visible between before and after compensation. Before compensation, the frequency is below 1kHz, failing to meet the frequency requirements for signal detection. After compensation, the frequency is above 50kHz, perfectly tracking signal changes. For typical nanopores, the method according to this invention can essentially achieve circuit detection that follows signal changes without information loss.

[0057] In summary, these are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for current compensation and frequency following in a nanopore single-molecule sequencing platform, characterized in that, Includes the following steps: When a nanopore is placed in two chambers with different concentrations, a command voltage is applied. The command voltage causes biological molecules to move and pass through the nanopore. The movement of biological molecules causes changes in the number of ions in the left and right chambers, thereby generating ion current. The stray capacitance and parasitic capacitance of the nanopore itself also generate perturbation current. At the same time, the rapid movement of biological molecules driven by the command voltage also puts forward requirements on the frequency of the detection circuit. The disturbance current and frequency are compensated by changing the resistance value of the detection circuit until the frequency is fully followed and the disturbance current is reduced. The method specifically includes the following steps: (1) Record the ion current of the nanopore in real time, and observe the magnitude of the generated disturbance current and frequency by changing the resistance value connected in the detection circuit; (2) According to the formula The time constants τ and τ' are used to derive the parameter relationships and adjust the resistors and capacitors in the circuit. Where A and B are the gain, Vc is the command voltage, Vm is the node voltage, Cc and Cc2 are the reference capacitors of the design compensation part, Ci is the stray capacitance, Rr is the ion solution resistance, and Rs is the phospholipid bilayer film resistance. (3) According to the formula Calculate the parameters corresponding to the resistance and capacitance values ​​using the time constant; (4) Based on the time constant obtained in step (2), adjust the resistor connected to the compensation circuit and observe the change in the disturbance current. (5) Calculate the resistance change of the access circuit based on the compensation parameters obtained in step (3), and observe the cutoff frequency. (6) Determine whether the disturbance current in step (4) has been fully compensated. If yes, end the process; otherwise, proceed to step (2). (7) Determine whether the cutoff frequency in step (5) meets the requirements. If yes, end the process; otherwise, proceed to step (3).

2. The method according to claim 1, characterized in that: In step (4), the time constant is adjusted by adjusting the resistance and capacitance values ​​of Rp1 and Cp, the compensation gain is adjusted by adjusting the resistance values ​​of Rx and Rp2, and the delay circuit is compensated by adjusting the resistance values ​​of Ri and Rx. After forming the compensation circuit, it is output to the front-end detection device.

3. The method according to claim 1, characterized in that: In step (4), the disturbance current is adjusted by adjusting the resistance values ​​of Rp3 and Rp4; Among them, the adjustable rheostat Rp3 is used to compensate for the influence of the series resistance on the charging and discharging current of the film capacitor. The parameters of the sliding rheostat Rp4 and Rp3 are completely independent of each other, and their specific values ​​are obtained through theoretical calculations.

4. The method according to claim 2, characterized in that: In step (5), the resistance values ​​of Rp5 and Rp6 in the circuit are adjusted. C, R, and C4 are resistors and capacitors of fixed size, and their specific values ​​are determined according to the feedback resistance value of the preamplifier.

5. A circuit structure for implementing the method according to any one of claims 1 to 4, characterized in that, It includes an equivalent circuit, current compensation circuits one and two, and a frequency compensation circuit. The current compensation circuits one and two are connected before the signal amplification section, and the frequency compensation circuit is connected after the signal amplification section. 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.

6. The circuit structure as described in claim 5, characterized in that, Current compensation circuit one and current compensation circuit two are respectively connected to the solution resistance and series resistance of the equivalent circuit. The equivalent circuit is connected to the signal detection and amplification circuit to detect and amplify the signal. The signal detection and amplification circuit is then connected to the frequency compensation circuit to achieve frequency compensation.

7. The circuit structure as described in claim 5, characterized in that, The equivalent circuit is as follows: solution resistance Rr, nanoporous membrane resistance Rm, phospholipid membrane capacitance Cm, series resistance Rs, separator capacitance Cg, separator capacitance Cg is connected in series with solution resistance Rr, nanoporous membrane resistance Rm is connected in parallel with phospholipid membrane capacitance Cm, and then connected in parallel with the separator capacitance Cg in series with solution resistance Rr, and then connected in series with resistance Rs. The current compensation circuit is used to compensate for the disturbance current generated by the nanopore fast capacitor. It includes variable resistors Rp1, Ri, Rp2, Rx, capacitor Cp, and operational amplifiers A1 and A2. One end of the variable resistor Rp1 and one end of the 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 to the inverting input terminal of operational amplifier A2. Ri is connected in parallel between Rp1 and Rp2. 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 for the disturbance current generated by the slow capacitor in the nanopore. It includes Rm1 connected to the inverting input terminal of operational amplifier A3, Rm2, Rm3, and Cm1 connected across the inverting input and output terminals of operational amplifiers A3, A4, and A5 respectively, Rp3 and Rm4 connected to the inverting input and output terminals of A3, A4, and A5 respectively, Rp4 connected to the inverting input terminals of A3 and A6 respectively, and the non-inverting input terminal of A6 connected to the command voltage. The frequency compensation circuit is used to change the frequency in the circuit to prevent signal loss. It includes a sliding rheostat Rp6 connected in parallel with a capacitor C, and its two ends connected to the inverting input terminal 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 then connected to the non-inverting input terminal of A7. The two ends of the sliding rheostat Rp5 are connected to the inverting input terminal and the output terminal of A7. One end of the compensation circuit Rp6 is connected to the detection device circuit to perform frequency compensation. Observe the waveform changes of the disturbance current before and after connecting the current compensation circuit and the waveform changes of the cutoff frequency before and after connecting the frequency compensation circuit to determine the effectiveness of the compensation circuit.

8. A nanopore single-molecule sequencing platform detection device having the circuit structure as described in claim 5, 6, or 7.

9. The application of the detection device as described in claim 8 in gene sequencing.

Images