阅读说明：本技术 微电流检测电路及基因测序装置 (Micro-current detection circuit and gene sequencing device ) 是由 张风体 蒋可 苏云鹏 邹耀中 于 2020-05-27 设计创作，主要内容包括：本公开实施例提供一种微电流检测电路及基因测序装置,该检测电路包括纳米孔电压施加单元,用于向纳米孔测试腔的公共电极和检测电极施加电压,驱动单个核苷酸分子通过纳米孔；积分电路单元,用于对纳米孔测试腔的检测电极输出的微电流信号进行积分放大,转换为积分电压信号；输出电路单元,用于接收该积分电路单元转换后的积分电压信号并输出；补偿电流输入单元,用于施加补偿电流到纳米孔测试腔的检测电极。该实施例实现了纳米孔基因测序的快速响应,提升测序准确度,便于大规模集成。(The embodiment of the disclosure provides a micro-current detection circuit and a gene sequencing device, wherein the detection circuit comprises a nanopore voltage applying unit, a micro-current detection unit and a gene sequencing unit, wherein the nanopore voltage applying unit is used for applying voltage to a common electrode and a detection electrode of a nanopore test cavity and driving a single nucleotide molecule to pass through a nanopore; the integrating circuit unit is used for carrying out integration amplification on a micro-current signal output by a detection electrode of the nanopore test cavity and converting the micro-current signal into an integrated voltage signal; the output circuit unit is used for receiving and outputting the integrated voltage signal converted by the integrating circuit unit; and the compensation current input unit is used for applying a compensation current to the detection electrode of the nanopore test chamber. The embodiment realizes the rapid response of nanopore gene sequencing, improves the sequencing accuracy and is convenient for large-scale integration.)

1. A microcurrent detection circuit, comprising:

the nanopore voltage applying unit is used for applying voltage to a common electrode and a detection electrode of the nanopore test cavity to drive a single nucleotide molecule to pass through the nanopore;

the integrating circuit unit is used for carrying out integration amplification on a micro-current signal output by a detection electrode of the nanopore test cavity and converting the micro-current signal into an integrated voltage signal;

the output circuit unit is used for receiving and outputting the integrated voltage signal converted by the integrating circuit unit;

and the compensation current input unit is used for applying a compensation current to the detection electrode of the nanopore test chamber.

2. The micro-current detection circuit as claimed in claim 1, wherein the nanopore voltage applying unit comprises a clamping tube, a first path end of the clamping tube is connected to the detection electrode, a second path end of the clamping tube is connected to the integrating circuit unit, and a control end of the clamping tube inputs a clamping voltage.

3. The micro-current detection circuit according to claim 1, wherein the nanopore voltage applying unit comprises a forward clamping tube and a reverse clamping tube, first path ends of the forward clamping tube and the reverse clamping tube are connected with the detection electrode, second path ends of the forward clamping tube and the reverse clamping tube are connected with the integrating circuit unit, a control end of the forward clamping tube inputs a first clamping voltage, and a control end of the reverse clamping tube inputs a second clamping voltage; the current direction of the forward clamp tube flows from the detection electrode to the integration circuit unit, and the current direction of the reverse clamp tube flows from the integration circuit unit to the detection electrode.

4. The micro-current detection circuit as claimed in claim 2, wherein the integration circuit unit comprises an integration capacitor and an integration reset switch, a first end of the integration capacitor is connected to the second pass end of the clamping tube, and a second end of the integration capacitor is grounded; and the first path end of the integral reset switch is connected with the first end of the integral capacitor, and the second path end of the integral reset switch is connected with a reset voltage and used for resetting the voltage of the integral capacitor.

5. The micro-current detection circuit as claimed in claim 3, wherein the integration circuit unit comprises an integration capacitor, an integration reset switch and a reset voltage selection circuit unit, a first end of the integration capacitor is connected to the second path ends of the forward clamping tube and the reverse clamping tube, and a second end of the integration capacitor is grounded; the first path end of the integral reset switch is connected with the first end of the integral capacitor, and the second path end of the integral reset switch is connected with a reset voltage selection circuit unit and used for resetting the voltage of the integral capacitor; the reset voltage selection circuit unit is used for switching and selecting the reset voltage input by the second path end of the integral reset switch.

6. The micro-current detection circuit of claim 2, wherein the compensation current input circuit comprises a current source having a negative terminal connected to a power source and a positive terminal connected to the detection electrode.

7. The micro-current detection circuit of claim 3, wherein the compensation current input circuit comprises a first current source, a second current source, a first switch, and a second switch; the negative end of the first current source is connected to the power supply, the positive end of the first current source is connected with the first switch in series to the detection electrode, the positive end of the second current source is grounded, the negative end of the second current source is connected with the second switch in series to the detection electrode, and the control ends of the first switch and the second switch are connected with the same control signal.

8. The micro-current detection circuit as claimed in claim 3, wherein the nanopore voltage application unit further comprises a reference voltage selection circuit unit for switching a reference voltage selected to be input to the common electrode.

9. The micro-current detection circuit as claimed in claim 3, wherein the nanopore voltage application unit further comprises a bias circuit for generating the first and second clamping voltages.

10. The micro-current detection circuit of claim 9, wherein the bias circuit comprises a first bias circuit and a second bias circuit; the first bias circuit comprises a third current source and a first MOS (metal oxide semiconductor) tube, wherein the negative end of the third current source is connected with the power supply, and the positive end of the third current source is connected with the control end and the first pass end of the first MOS tube; the second bias circuit comprises a fourth current source and a second MOS tube, the positive end of the fourth current source is grounded, the negative end of the fourth current source is connected with the control end and the first path end of the second MOS tube, and the second path ends of the first MOS tube and the second MOS tube are connected with a common-mode voltage.

11. The micro-current sensing circuit as claimed in claim 10, wherein the nanopore voltage application unit further comprises a first switch pair and a second switch pair, the first bias circuit connecting the first switch pair to selectively provide a second clamping voltage to the control terminal of the reverse clamp, the second bias circuit connecting the second switch pair to selectively provide a first clamping voltage to the control terminal of the forward clamp.

12. The micro-current detection circuit as claimed in claim 11, wherein the nanopore voltage application unit further comprises a first driving filter circuit and a second driving filter circuit, the first driving filter circuit being connected in series between the first bias circuit and the first switch pair, the second driving filter circuit being connected in series between the second bias circuit and the second switch pair.

13. The micro-current detection circuit of claim 11, wherein the first switch pair comprises a third switch and a fourth switch for selectively outputting the second clamped voltage or shorting the output to a power supply in accordance with a control signal; the second switch pair includes a fifth switch and a sixth switch for selectively outputting the first clamped voltage or shorting the output to ground according to a control signal.

14. The micro-current detection circuit according to claim 3, wherein the nanopore voltage application unit further comprises an input reset switch, a first path terminal of the input reset switch is connected to a preset voltage, a second path terminal is connected to the detection electrode, and a control terminal is connected to a reset control signal.

15. The micro-current detection circuit as claimed in claim 4 or 5, wherein the output circuit unit includes a source follower and a selection switch, an input terminal of the source follower is connected to a first terminal of the integration capacitor, an output terminal is connected to a first terminal of the selection switch, and a second terminal of the selection switch outputs the integrated voltage signal.

16. A gene sequencing device comprising a plurality of measurement units, each measurement unit comprising a nanopore test chamber and the microcurrent detection circuit of any of claims 1-15; wherein the nanopore test chamber comprises a common electrode and a detection electrode.

17. The gene sequencing apparatus of claim 16, further comprising a common signal line for receiving the voltage signal output by the microcurrent detection circuit and an analog-to-digital conversion circuit connected to the common signal line for converting the voltage signal into a digital signal.

18. The gene sequencing apparatus of claim 17, further comprising a tail current source, a negative terminal of the tail current source being connected to the common signal line, and a positive terminal of the tail current source being grounded.

Technical Field

The disclosure belongs to the technical field of electronic circuits, and particularly relates to a micro-current detection circuit and a gene sequencing device, which can be used for biological micro-current signals of gene sequencing and detection of pA-level micro-current in other application fields.

Background

The nanopore sequencing method adopts an electrophoresis technology, and realizes sequencing by driving single molecules to pass through nanopores one by means of electrophoresis. The nanopore (nanopore) is a channel with the diameter of about 1-10 nanometers and comprises a solid nanopore and a biological nanopore. Single-stranded DNA (or RNA) molecules, due to their charged nature, spontaneously pass through the nanopore in an electric field and cause a change in the nanopore resistance during the pass, resulting in a so-called blocking current. The four different bases A, T (U), C and G of DNA (RNA) have recognizable differences in the blocking effect on current flow when they pass through the nanopore due to differences in their own chemical structures, resulting in respective corresponding characteristic blocking currents. The accurate detection of the characteristic blocking current allows the type of the corresponding base to be determined and thus the nucleic acid sequence to be determined.

In the existing nanopore sequencing method, taking the sequencing technology of the Genia Technologies company as an example, modified nucleotide analogs are adopted to perform sequencing while synthesizing nucleic acid, the modified nucleotide analogs comprise nucleotides and linkers for synthesis, different linkers can generate characteristic blocking current more effective than the nucleic acid, the base recognition degree can be effectively improved by detecting the linkers, but the situation that only the linkers are detected cannot avoid reading the linker blocking current, but the nucleotides do not really participate in the synthesis reaction, and the error (inertia error) that signals are read excessively is caused. Therefore, there is a need for improvements in the existing nanopore sequencing technology that can detect not only the linker, but also the nucleotide itself to improve sequencing accuracy and rapid response capability. This requires higher sampling frequencies and more stringent noise control measures.

Disclosure of Invention

The embodiment of the disclosure provides a micro-current detection circuit and a gene sequencing device, which are used for rapidly and accurately judging the type of nucleotide molecules passing through a nanopore and completing a sequencing function.

In a first aspect, an embodiment of the present disclosure provides a micro-current detection circuit, including:

the nanopore voltage applying unit is used for applying voltage to a common electrode and a detection electrode of the nanopore test cavity to drive a single nucleotide molecule to pass through the nanopore;

the integrating circuit unit is used for carrying out integration amplification on a micro-current signal output by a detection electrode of the nanopore test cavity and converting the micro-current signal into an integrated voltage signal;

the output circuit unit is used for receiving and outputting the integrated voltage signal converted by the integrating circuit unit;

and the compensation current input unit is used for applying a compensation current to the detection electrode of the nanopore test chamber.

In an alternative embodiment, the nanopore voltage applying unit includes a clamping tube, a first path end of the clamping tube is connected to the detection electrode, a second path end of the clamping tube is connected to the integrating circuit unit, and a control end of the clamping tube inputs a clamping voltage.

In an alternative embodiment, the nanopore voltage applying unit comprises a forward clamping tube and a reverse clamping tube, first path ends of the forward clamping tube and the reverse clamping tube are connected with the detection electrode, second path ends of the forward clamping tube and the reverse clamping tube are connected with the integrating circuit unit, a control end of the forward clamping tube inputs a first clamping voltage, and a control end of the reverse clamping tube inputs a second clamping voltage; the current direction of the forward clamp tube flows from the detection electrode to the integration circuit unit, and the current direction of the reverse clamp tube flows from the integration circuit unit to the detection electrode.

In an alternative embodiment, the integration circuit unit includes an integration capacitor and an integration reset switch, a first end of the integration capacitor is connected to the second pass end of the clamping tube, and a second end of the integration capacitor is grounded; and the first path end of the integral reset switch is connected with the first end of the integral capacitor, and the second path end of the integral reset switch is connected with a reset voltage and used for resetting the voltage of the integral capacitor.

In an optional embodiment, the integration circuit unit comprises an integration capacitor, an integration reset switch and a reset voltage selection circuit unit, a first end of the integration capacitor is connected to the second path ends of the forward clamping tube and the reverse clamping tube, and a second end of the integration capacitor is grounded; the first path end of the integral reset switch is connected with the first end of the integral capacitor, and the second path end of the integral reset switch is connected with a reset voltage selection circuit unit and used for resetting the voltage of the integral capacitor; the reset voltage selection circuit unit is used for switching and selecting the reset voltage connected with the second path end of the integral reset switch.

In an alternative embodiment, the compensation current input circuit comprises a current source having a negative terminal connected to a power supply and a positive terminal connected to the detection electrode.

In an alternative embodiment, the compensation current input circuit includes a first current source, a second current source, a first switch, and a second switch; the negative end of the first current source is connected to the power supply, the positive end of the first current source is connected with the first switch in series to the detection electrode, the positive end of the second current source is grounded, the negative end of the second current source is connected with the second switch in series to the detection electrode, and the control ends of the first switch and the second switch are connected with the same control signal.

In an alternative embodiment, the nanopore voltage application unit further includes a reference voltage selection circuit unit for switching a reference voltage selected to be input to the common electrode.

In an alternative embodiment, the nanopore voltage application unit further comprises a bias circuit for generating the first clamping voltage and the second clamping voltage.

In an alternative embodiment, the bias circuit includes a first bias circuit and a second bias circuit; the first bias circuit comprises a third current source and a first MOS (metal oxide semiconductor) tube, wherein the negative end of the third current source is connected with the power supply, and the positive end of the third current source is connected with the control end and the first pass end of the first MOS tube; the second bias circuit comprises a fourth current source and a second MOS tube, the positive end of the fourth current source is grounded, the negative end of the fourth current source is connected with the control end and the first path end of the second MOS tube, and the second path ends of the first MOS tube and the second MOS tube are connected with a common-mode voltage.

In an alternative embodiment, the nanopore voltage application unit further includes a first switch pair and a second switch pair, the first bias circuit is connected to the first switch pair to selectively provide the second clamping voltage to the control terminal of the reverse clamp, and the second bias circuit is connected to the second switch pair to selectively provide the first clamping voltage to the control terminal of the forward clamp.

In an optional embodiment, the nanopore voltage applying unit further includes a first driving filter circuit and a second driving filter circuit, the first driving filter circuit is connected in series between the first bias circuit and the first switch pair, and the second driving filter circuit is connected in series between the second bias circuit and the second switch pair.

In an alternative embodiment, the first switch pair comprises a third switch and a fourth switch for selectively outputting the second clamped voltage or shorting the output to the power supply according to a control signal; the second switch pair includes a fifth switch and a sixth switch for selectively outputting the first clamped voltage or shorting the output to ground according to a control signal.

In an optional embodiment, the nanopore voltage applying unit further includes an input reset switch, a first path end of the input reset switch is connected to the preset voltage, a second path end of the input reset switch is connected to the detection electrode, and a control end of the input reset switch is connected to the reset control signal.

In an alternative embodiment, the output circuit unit includes a source follower and a selection switch, an input terminal of the source follower is connected to the integration capacitor, an output terminal of the source follower is connected to a first terminal of the selection switch, and a second terminal of the selection switch outputs the integrated voltage signal.

In a second aspect, embodiments of the present disclosure provide a gene sequencing apparatus comprising a plurality of measurement units, each measurement unit comprising a nanopore test chamber and a microcurrent detection circuit as described in any of the previous embodiments; wherein the nanopore test chamber comprises a common electrode and a detection electrode.

In an alternative embodiment, the micro-current detection circuit further comprises a common signal line and an analog-to-digital conversion circuit connected to the common signal line, wherein the common signal line is used for receiving the voltage signal output by the micro-current detection circuit, and the analog-to-digital conversion circuit is used for converting the voltage signal into a digital signal.

In an alternative embodiment, the apparatus further comprises a tail current source, a negative terminal of the tail current source is connected to the common signal line, and a positive terminal of the tail current source is grounded.

The micro-current detection circuit of the embodiment of the disclosure provides a compensation current to the detection electrode of the nanopore test chamber through the compensation current input unit, and can simultaneously identify the nucleotide and the characteristic current generated by the linker through the nanopore. The scheme has the beneficial effects of at least one of the following: 1) the precision is high; 2) the system can quickly respond and correctly identify; 3) the detection circuit unit is simple, small in area and convenient for large-scale integration.

Drawings

In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present disclosure, and it is also possible for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

FIG. 1 is a schematic diagram of the structural and electrical model of a nanopore test chamber 101 employed in embodiments of the present disclosure;

FIG. 2 is a circuit schematic of detection circuitry in a nanopore sequencing device that may be implemented;

FIG. 3 is a waveform schematic diagram illustrating the operation of the detection circuit shown in FIG. 2;

FIG. 4 is a circuit schematic of a micro-current detection circuit according to a first embodiment of the present disclosure;

fig. 5A is a schematic diagram of a first operating waveform of a micro-current detection circuit according to a first embodiment of the disclosure;

FIG. 5B is a schematic diagram of a second operating waveform of the micro-current detection circuit according to the first embodiment of the disclosure;

FIG. 6 is a circuit schematic of a microcurrent detection circuit according to a second embodiment of the present disclosure;

FIG. 7 is a schematic diagram of an operating waveform of a micro-current detection circuit according to a second embodiment of the disclosure;

FIG. 8 is a schematic structural diagram of a gene sequencing apparatus according to an embodiment of the present disclosure.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be described below in detail and completely with reference to the accompanying drawings in the embodiments of the present disclosure. It is to be understood that the described embodiments are only a few embodiments of the present disclosure, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.

In the present disclosure, it is to be understood that terms such as "including" or "having," etc., are intended to indicate the presence of the disclosed features, numbers, steps, behaviors, components, parts, or combinations thereof, and are not intended to preclude the possibility that one or more other features, numbers, steps, behaviors, components, parts, or combinations thereof may be present or added.

In a nanopore sequencing device, a nucleotide molecule and a linker for synthesis are driven to pass through a nanopore by means of a voltage applied to both ends of a test chamber, and the type of the nucleotide molecule passing through the nanopore is detected by detecting a micro-current characteristic signal output from the nanopore, thereby realizing sequencing.

FIG. 1 is a schematic diagram of the structural and electrical model of a nanopore test chamber 101 employed in embodiments of the present disclosure. As shown in fig. 1, the test chamber 101 comprises a first compartment and a second compartment separated by a phospholipid bilayer membrane 105, and an electrode 103 connected to the first compartment and an electrode 102 connected to the second compartment. The phospholipid bilayer membrane 105 has a nanopore 104 therein, and a nucleotide molecule 106 linked to a linker 107 is located in the first compartment and passes through the nanopore 104 under the application of a voltage applied to the electrodes 102 and 103. In fig. 1, the nanopore equivalent capacitance 108 and the nanopore equivalent resistance 109 may be used to simulate the electrical characteristics of the nanopore 104, and for convenience of illustration, the embodiment of the disclosure simplifies the test chamber 102 into the nanopore equivalent circuit model 113.

FIG. 2 is a schematic circuit diagram of the detection circuitry in an implementable nanopore sequencing device. As shown in fig. 2, the detection circuit includes a nanopore voltage application unit, an integration circuit unit, and an output circuit unit. Wherein the nanopore voltage applying unit is configured to apply a voltage to the common electrode of the nanopore test chamber 207 and the detection electrode 208, thereby driving a single nucleotide molecule through the nanopore by means of a voltage difference between the common electrode of the nanopore test chamber 207 and the detection electrode 208. In one embodiment, the nanopore voltage application unit includes a clamping tube 201.

The integrating circuit unit is used for integrating and amplifying the micro-current signal output by the detection electrode 208 of the nanopore test cavity 207 and converting the micro-current signal into an integrated voltage signal. In one embodiment, the integration circuit unit includes an integration capacitor 203 and an integration reset switch 202.

And the output circuit unit receives and outputs the integrated voltage signal converted by the integrating circuit unit. In one embodiment, the output circuit unit includes a source follower 204 and a selection switch 205 (implemented as a selection pipe 205 in fig. 2).

In this example of the detection circuit, a reference voltage VCMD 210 is applied to a common electrode (corresponding to the electrode 112 in fig. 1) of the nanopore test chamber 207, a source of the clamp 201 is connected to a detection electrode 208 (corresponding to the electrode 110 in fig. 1) of the nanopore test chamber 207, and a drain of the clamp 201 is connected to one end of the integrating capacitor 203. The clamping voltage VP 209 is input to the gate of the clamping tube 201, and the clamping voltage VP is a fixed voltage, so that the input voltage of the detection electrode 208 of the nanopore test chamber is ensured to be a fixed value. When a forward voltage is applied to the reference voltage VCMD, for example, about 100mV to 200mV higher than the voltage applied to the detection electrode 208, nucleotide molecules in the nanopore test chamber pass through the nanopore under the action of an electric field, a characteristic current appears in the nanopore, the characteristic current passes through the clamping tube 201 and then is input to the integrating capacitor 203, the integrating capacitor 203 integrates and amplifies the characteristic current within a certain time period to generate an integrated voltage signal 211, and the integrated voltage signal 211 is output to the common signal line 213 through the source follower 204 and the selection switch 205 and then is output to the analog-to-digital converter ADC 206 through the common signal line 213 for analog-to-digital conversion. Wherein the reset switch 202 periodically clears the charge on the integrating capacitor 203 under the control of the reset signal Rst.

Fig. 3 is a schematic diagram of an operating waveform of the detection circuit. As shown in fig. 3, 301 is a voltage waveform applied by the reference voltage VCMD; 302 is the waveform of the periodic reset signal of the integrating capacitor and 303 is the waveform of the integrated voltage on the integrating capacitor. When a forward voltage is applied to the reference voltage, the integration voltage is accumulated on the integration capacitor, and a periodic sawtooth signal is generated according to a reset signal period.

The operating principle of the detection circuit shown in fig. 2 is briefly described below.

First, when the clamp 201 operates in the sub-threshold state, the clamp voltage VP varies with the detected current.

The relation between the current and the voltage of the MOS tube in the subthreshold state is as follows:

wherein n is a constant related to the process, and can take a value of 1.5 according to experience; VT is thermal voltage, and 26mV is taken at normal temperature; ids0 is a MOS tube current parameter, and is related to the process; vth is the threshold voltage of the MOS tube; ids is the value of current flowing through the MOS tube, and Vgs is the voltage value of the MOS tube corresponding to the current value.

For different currents, such as Ids1 and Ids2, corresponding to different Vgs1 and Vgs2, respectively, there is a voltage difference Δ Vgs between them:

referring to FIG. 1, if nanopore equivalent capacitance 108 is 2pF and nanopore equivalent resistance 109 ranges from 250M Ω to 20G Ω, a current of 5pA to 800pA is generated when a voltage of 200mV is applied across the nanopore, temporarily without considering the effect of solution resistance 111.

As described above, the current range to be detected by the detection circuit is 5pA to 800pA, and when Ids1 and Ids2 are respectively the maximum and minimum, the change in Δ Vgs can be calculated as follows:

as described above, for the detection circuit shown in FIG. 2, there will be a maximum of about 200mV change in the detection electrode 208 during the detection process. Through simplified calculation, the final stable time of the structure has the following relation:

wherein n is a constant related to the process, and can take a value of 1.5 according to experience; VT is thermal voltage, and 26mV is taken at normal temperature; c is the nanopore equivalent capacitance, see nanopore equivalent capacitance 108 in fig. 1; delta Vgs is the voltage change of the detection electrode caused before and after the nanopore current mutation; i0 is the initial current value flowing through the clamp tube, namely the current value before the nanopore current suddenly changes; i1 is the final current value flowing through the clamp tube, here the current value after the nanopore current has suddenly changed.

When the detection circuit is in an unstable state, especially for the rapid change of current in the detection process, taking the example that the nucleotide molecule passes through the nanopore, the retention time of the nucleotide molecule in the nanopore is about 100us at the shortest, so that the rapid change of current cannot ensure the correct recognition by the detection circuit shown in fig. 2. In addition, different detection currents can cause different changes of the voltage value Vgs of the clamping tube, and the voltage is finally reflected at two ends of the nanopore, so that detection errors are caused.

In view of the above-mentioned drawbacks of the detection circuit, the present disclosure provides an improved micro-current detection circuit, which can rapidly and accurately determine the type of nucleotide molecules passing through the nanopore, thereby completing the sequencing function.

Fig. 4 is a circuit schematic diagram of a micro-current detection circuit according to a first embodiment of the disclosure. As shown in fig. 4, the micro-current detecting circuit of the embodiment of the present disclosure includes a nanopore voltage applying unit, an integrating circuit unit, an output circuit unit, and a compensation current input unit.

Wherein, the nanopore voltage applying unit is used for applying voltage to the common electrode of the nanopore test chamber 407 and the detection electrode 408, so that a single nucleotide molecule is driven to pass through the nanopore by means of the voltage difference between the common electrode of the nanopore test chamber 407 and the detection electrode 208, and unidirectional micro-current signal detection is realized.

The integrating circuit unit is used for integrating and amplifying the micro-current signal output by the detection electrode 408 of the nanopore test cavity 407 and converting the micro-current signal into an integrated voltage signal.

The output circuit unit is used for receiving and outputting the integrated voltage signal converted by the integrating circuit unit.

The compensation current input unit is used for applying compensation current to the detection electrode 408 of the nanopore test chamber 407 and is used for reducing voltage change caused by a micro-current signal output by the detection electrode 408, so that the dependency of the voltage of the detection electrode along with the detection current is reduced, the circuit stabilization time is shortened, and the response speed is improved.

In one embodiment, the nanopore voltage applying unit comprises a clamping tube 401, a first path end of the clamping tube 401 is connected with the detection electrode 408, a second path end of the clamping tube is connected with the integrating circuit unit, a control end of the clamping tube inputs a clamping voltage VP 409, and the clamping voltage VP acts to make the voltage of the detection electrode 408 be a fixed value. In one embodiment, the first via terminal may be a source of a MOS transistor, the second via terminal may be a drain of the MOS transistor, and the control terminal is a gate of the MOS transistor.

In one embodiment, the integrating circuit unit includes an integrating capacitor 403 and an integrating reset switch 402 (exemplarily implemented with a reset tube 402 in fig. 4). The first end of the integrating capacitor 403 is connected to the second path end of the clamping tube 401, and the second end is grounded, so as to perform integral amplification on the micro-current signal of the detection electrode. A first path terminal of the integration reset switch 402 is connected to a first terminal of the integration capacitor 403, and a second path terminal of the integration reset switch 402 is connected to a reset voltage Vpre for periodically resetting the voltage of the integration capacitor 403 under the action of a reset signal Rst.

In one embodiment, the compensation current input unit includes a current source 414. Wherein the negative terminal of the current source 414 is connected to the power supply VDD and the positive terminal is connected to the detection electrode 408 of the nanopore test chamber 407. The compensation current input unit provides compensation current for the clamp tube 401 through the current source 414, and reduces the voltage variation of the detection electrode 408 caused by the micro-current signal.

In one embodiment, the output circuit unit includes a source follower 404 and a selection switch 405 (exemplarily implemented with a selection pipe 405 in fig. 4). The input end of the source follower 404 is connected to the first end of the integrating capacitor 403, the output end is connected in series to the first end of the selection switch 405, and the second end of the selection switch 405 is used for outputting the integrated voltage signal 411. In one embodiment, the second terminal of the selection switch 405 may be connected to a common signal line 413, the integrated voltage signal 411 may be output to the common signal line 413, and the common signal line 413 may be connected to the analog-to-digital converter 406, further converting the integrated voltage signal 411 into a digital signal.

In this embodiment, as shown in the above formula (3), the settling time Tset of the detection circuit is positively correlated with Δ Vgs and I1/I0, and inversely correlated with I1, so decreasing Δ Vgs and increasing I1 will effectively decrease the settling time.

The detection electrode 408 is connected in parallel with an input compensation current 414, and the introduced compensation current 414 can reduce the voltage variation caused by the detection current (micro-current signal) output by the detection electrode, i.e. Δ Vgs described above. For example, when the offset current is 200pA, the detection current is 5pA to 800pA, the detection range is 205pA to 1000pA after the offset current is added, and the voltage difference Δ Vgs' caused by the detection current is 59.4mV, the voltage change is significantly reduced. At the same time, the compensation current also flows through the clamp 401, thereby raising the current of the clamp 401, i.e., I1 described above. Therefore, the compensation current 414 will cause the settling time of the detection circuit to be effectively reduced, enabling detection of a rapidly varying nanopore microcurrent signal.

Fig. 5A and 5B are waveforms showing the response of the conventional detection circuit shown in fig. 2 and the detection circuit of the first embodiment shown in fig. 4 to different nanopore currents, respectively.

As shown in fig. 5A, when a nucleotide molecule passes through the nanopore, the resistance change of the nanopore can be simulated by using a curve 501A, wherein 1G Ω represents that the nanopore is in an open state, and the mutation to 20G Ω represents that the nucleotide molecule enters the nanopore, the duration of the mutation can be changed within 100 us-10 ms, and then the mutation to 5G Ω represents that the nucleotide molecule linker is in the nanopore. In the graph, curve 502A represents the integration capacitor reset signal, which periodically resets the integration capacitor. Curve 503A in the figure represents the integrated voltage with the addition of the compensation current, i.e. integrated voltage 411 in the configuration of fig. 4; the graph 504A shows the integrated voltage of the conventional structure, i.e., the integrated voltage 211 in fig. 2. Comparing the curves 503A and 504A in FIG. 5A, the compensation current has a shorter settling time and allows the state of the nucleotide molecule entering the nanopore to be resolved.

FIG. 5B shows the situation where no nucleotide molecule enters the nanopore, where 1G Ω indicates that the nanopore is open, the mutation to 5G Ω indicates that only the linker enters the nanopore, and the corresponding curve 503B indicates the integrated voltage with the addition of the offset current, and curve 504B indicates the integrated voltage of the conventional structure, and neither of them detects the passage of a nucleotide molecule through the nanopore.

Curves 504A and 504B in fig. 5A and 5B represent integrated voltage signals of a conventional detection circuit, corresponding to the case where a nucleotide molecule enters a nanopore and the case where no nucleotide molecule enters a nanopore, respectively, the integrated voltage signals of curves 504A and 504B are similar, and it can be seen that the conventional detection circuit structure cannot effectively recognize the process of a nucleotide molecule passing through a nanopore, particularly at the time of rapid passing; comparing the curves 503A and 503B, when the compensation current is increased, the detection circuit can respond to the rapid detection current change and convert the current into a voltage signal, when the nucleotide molecule enters the nanopore, the voltage signal is lower, and as shown by the curve 503A, whether the nucleotide molecule passes through the nanopore can be distinguished according to the voltage signal.

Fig. 6 is a circuit schematic diagram of a micro-current detection circuit according to a second embodiment of the disclosure. As shown in fig. 6, the micro-current detecting circuit of the embodiment of the present disclosure also includes a nanopore voltage applying unit, an integrating circuit unit, an output circuit unit, and a compensation current input unit.

Wherein, the nanopore voltage applying unit is used for applying voltage to the common electrode and the detection electrode 608 of the nanopore test cavity 607, so that single nucleotide molecules are driven to pass through the nanopore by means of the voltage difference between the common electrode and the detection electrode 608 of the nanopore test cavity 607, and the bidirectional micro-current signal detection is realized.

The integrating circuit unit is used for integrating and amplifying the micro-current signal output by the detection electrode 608 of the nanopore test cavity 607 and converting the micro-current signal into an integrated voltage signal.

The output circuit unit is used for receiving and outputting the integrated voltage signal converted by the integrating circuit unit.

The compensation current input unit is used for applying compensation current to the detection electrode 608 of the nanopore test cavity 607, and is used for reducing voltage change caused by a micro-current signal output by the detection electrode 608, so that the dependency of the voltage of the detection electrode along with the detection current is reduced, the circuit stabilization time is shortened, and the response speed is improved. The embodiment has forward and reverse detection capabilities, can reduce the circuit stabilization time and improve the response speed.

In one embodiment, the nanopore voltage applying unit comprises a forward clamp 601B and a reverse clamp 601A, wherein a first path end of the forward clamp 601B and a first path end of the reverse clamp 601A are connected with the detection electrode 608, a second path end of the forward clamp 601B and a second path end of the reverse clamp 601A are connected with the integrating circuit unit, a control end of the forward clamp inputs a first clamp voltage VP, and a control end of the reverse clamp inputs a second clamp voltage VN, so that forward and reverse clamping functions are respectively completed. The current direction of the forward clamp 601B flows from the detection electrode 608 to the integration circuit unit, and the current direction of the reverse clamp 601A flows from the integration circuit unit to the detection electrode 608. In one embodiment, the first via terminal may be a source of a MOS transistor, the second via terminal may be a drain of the MOS transistor, and the control terminal is a gate of the MOS transistor.

In one embodiment, the integrating circuit unit includes an integrating capacitor 603, an integrating reset switch 602 (exemplarily implemented as a reset tube 602 in fig. 6), and a reset voltage selection circuit unit 609. A first end of the integrating capacitor 603 is connected to the second path end of the forward clamp 601B and the reverse clamp 601A, and a second end of the integrating capacitor 603 is grounded. A first path terminal of the integration reset switch 602 is connected to the first terminal of the integration capacitor, and a second path terminal of the integration reset switch 602 is connected to the reset voltage selection circuit unit 609. The reset voltage selection circuit 609 is used to select the reset voltage (Vpre 1 and Vpre2 in fig. 6) inputted to the second path terminal of the integration reset switch 602 by switching.

In one embodiment, the nanopore voltage application unit further includes a reference voltage selection circuit unit 610 for switching selection of a reference voltage (VCMD 1 and VCMD2 in fig. 6) input to the common electrode of the nanopore test chamber 607.

In one embodiment, the compensation current input unit includes current sources 614B and 614A, selection switches 615B and 615A. The negative terminal of the current source 614B is connected to the power supply VDD, and the positive terminal is connected in series to the selection switch 615B to the detection electrode 608 of the nanopore test chamber 607. The positive terminal of the current source 614A is connected to ground, the negative terminal is connected to the select switch 615A in series to the detection electrode 608 of the nanopore test chamber 607, and the control terminals of the select switches 615B and 615A are connected to the same control signal CMD. The compensation current input unit provides compensation current for the forward clamp and the reverse clamp through current sources 614B and 614A respectively.

In one embodiment, the nanopore voltage application unit further comprises a bias circuit for generating the first clamping voltage VP and the second clamping voltage VN. The bias circuits may include, among other things, a first bias circuit 619A and a second bias circuit 619B. The first bias circuit 619A comprises a first current source and a first MOS transistor, and the second bias circuit 619B comprises a second current source and a second MOS transistor. The first MOS tube and the second MOS tube are in diode configuration, respective control ends are connected with the second channel end, and the first channel ends of the first MOS tube and the second MOS tube are connected with a common-mode voltage VCM; the negative end of the first current source is connected with a power supply VDD, the positive end of the first current source is connected with the control end of the first MOS tube and the second path end, the positive end of the second current source is grounded, and the negative end of the second current source is connected with the control end of the second MOS tube and the second path end. In one embodiment, the first via terminal may be a source of a MOS transistor, the second via terminal may be a drain of the MOS transistor, and the control terminal is a gate of the MOS transistor. In one embodiment, the first MOS transistor and the second MOS transistor may be NMOS transistors or PMOS transistors.

In one embodiment, the nanopore voltage applying unit further includes a first selection switch pair 617A and a second selection switch pair 617B, the first bias circuit 619A is connected to the first selection switch pair 617A, the second bias circuit 619B is connected to the second selection switch pair 617B, and the first selection switch pair 617A and the second selection switch pair 617B are matched to switch to output the first clamping voltage VP and the second clamping voltage VN to the control ends of the forward clamping tube 601B and the reverse clamping tube 601A, respectively, so as to switch forward and reverse detection. Control terminals of the first selection switch pair 617A and the second selection switch pair 617B are connected to the same control signal CMD. The first selection switch pair 617A and the second selection switch pair 617B each include a pair of selection switches, one of the selection switches of the first selection switch pair 617A is used for selectively outputting the second clamping voltage VN according to the control signal CMD, and the other selection switch is used for short-circuiting the output to the power supply; one of the selection switches of the second selection switch pair 617B is used to select the output of the first clamping voltage VP according to the control signal CMD, and the other selection switch is used to short the output to ground.

In an embodiment, as shown in fig. 6, in consideration of the weak driving capability of the voltages generated by the first bias circuit 619A and the second bias circuit 619B, a first driving filter circuit 618A may be added between the first bias circuit 619A and the first selection switch pair 617A, and a second driving filter circuit 618B may be added between the second bias circuit 619B and the second selection switch pair 617B, so as to implement the driving enhancement and the noise reduction functions.

In one embodiment, the nanopore voltage application unit further comprises an input reset switch 616, a first path terminal of the input reset switch 616 is connected to the preset voltage VCM, a second path terminal is connected to the detection electrode 608 of the nanopore test chamber, and a control terminal of the input reset switch 616 is connected to the reset control signal Rst _ cmd. The input reset switch 616 is used for fixing the detection electrode 608 at the preset voltage VCM during the positive and negative detection switching, so that the response speed of the detection circuit during the positive and negative detection switching can be increased, and the fast establishment of the circuit working point is facilitated.

In one embodiment, the output circuit unit includes a source follower 604 and a selection switch 605 (exemplarily implemented with a selection pipe 605 in fig. 6). The input terminal of the source follower 604 is connected to the first terminal of the integrating capacitor 603, the output terminal is connected to the first terminal of the selection switch 605 in series, and the second terminal of the selection switch 605 outputs the integrated voltage signal 611. In one embodiment, the second terminal of the selection switch 605 may be connected to the common signal line 613, and the common signal line 613 may be connected to the analog-to-digital converter 606, further converting the integrated voltage signal 611 into a digital signal.

Fig. 7 is a schematic diagram of an operating waveform of the micro-current detection circuit according to the second embodiment of the disclosure, and as shown in fig. 7, 701 is a waveform of the integrated reset signal Rst, 702 is a waveform of the control signal CMD for switching the detection direction, 703 is a waveform of the reset control signal Rst _ CMD inputted to the reset switch, 704 is a waveform of the reset voltage Vpre, and 705 is a waveform of the reference voltage VCMD. The reference voltage VCMD is synchronously switched with the control signal CMD, and when the detection direction is switched, the input reset switch 616 in fig. 6 is controlled by Rst _ CMD to fix the detection electrode of the nanopore to the preset voltage VCM.

FIG. 8 is a schematic structural diagram of a gene sequencing apparatus according to an embodiment of the present disclosure. As shown in fig. 8, the gene sequencing apparatus of the present embodiment includes a plurality of measurement units, each of which includes a nanopore test chamber 802 and a detection circuit unit 805 connected correspondingly; therein, the nanopore test chamber 802 comprises an electrode 808 in a first compartment connected to the common electrode 801 and a detection electrode 809 in a second compartment. The plurality of detection circuit units 805 are implemented by the micro-current detection circuit described in the foregoing first embodiment or second embodiment.

In one embodiment, the output voltage of the detection circuit unit 805 is output to the shared common signal line 806 through a selection switch in the detection circuit unit, and is converted into a digital signal by an analog-to-digital converter 807 and then output.

In one embodiment, the gene sequencing apparatus may further comprise a tail current source 810, the negative terminal of the tail current source 810 being connected to the common signal line 806 and the positive terminal being connected to ground.

It should be noted that the above embodiments can be freely combined as required, devices involved in the circuit are illustrated as CMOS devices, and other devices, such as BJTs, JFETs, etc., can also implement the technical solution of the present disclosure. The foregoing is merely a preferred embodiment of the present disclosure, and it should be noted that changes and modifications can be made by those skilled in the art without departing from the principle of the present disclosure, and such changes and modifications should also be considered as falling within the scope of the present disclosure.