CN112708544A - Measuring device and measuring method for gene sequencing - Google Patents
Measuring device and measuring method for gene sequencing Download PDFInfo
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- CN112708544A CN112708544A CN201911024083.7A CN201911024083A CN112708544A CN 112708544 A CN112708544 A CN 112708544A CN 201911024083 A CN201911024083 A CN 201911024083A CN 112708544 A CN112708544 A CN 112708544A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The embodiment of the disclosure provides a measuring device and a measuring method for gene sequencing, wherein the measuring device comprises at least one detection unit, and the detection unit comprises a test cavity and a measuring circuit unit connected to the test cavity; the measurement circuit unit comprises a first amplification circuit and a second amplification circuit, and is used for modulating and amplifying a current signal caused by bidirectional movement of the nucleotide molecule between the first compartment and the second compartment through the nanopore on the membrane, and outputting a first characteristic voltage signal and a second characteristic voltage signal for judging the type of the nucleotide molecule and whether the nucleotide molecule participates in a synthesis reaction and further completing sequencing.
Description
Technical Field
The disclosure belongs to the technical field of biological detection, and particularly relates to a measuring device and a measuring method for gene sequencing, which can be used for detecting the base type of nucleotide so as to realize sequencing.
Background
The concept of nanopore-based nucleic acid sequencing was proposed in 1995. Researchers have found that certain transmembrane proteins, such as the bacterial toxin α -hemlysin, form stable channels on phospholipid membranes, called nanopores, with diameters of about 1-2 nm. 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. DNA (RNA) four different bases A, T (U), C and G have recognizable difference on the blocking effect of current when passing through the nanopore due to the difference of self chemical structures, and generate respective corresponding characteristic blocking currents. The accurate detection of the characteristic blocking current can determine the type of the corresponding base, thereby determining the nucleic acid sequence.
The existing methods for sequencing through a Nanopore are mainly two, one method is represented by the system of Oxford Nanopore Technologies, and a DNA single-stranded molecule is directly passed through the Nanopore and the current is blocked by reading the characteristic corresponding to the base in turn. However, because the difference of the characteristic current given by different bases is small, a plurality of bases can stay in the nanopore at the same time, so that the blocking current characterization is very complicated, and the extremely high requirement is provided for the current data analysis in the later period of sequencing. More importantly, this system has difficult to overcome difficulties in determining a stretch of contiguous DNA sequences of the same base (homopolymer). Another approach is represented by the system adopted by Genia Technologies (currently in Roche Sequencing Solutions), using modified nucleotide analogs for Sequencing while synthesizing nucleic acids. Although tagging nucleotides for replication can improve the recognition of characteristic blocking currents corresponding to different bases, and the time interval of a single nucleotide tag entering a nanopore is also helpful for determining a nucleic acid sequence (homo polymer) of continuous same bases, the system has difficulty in ensuring that each tag of nucleotides for synthesis enters the nanopore to give a blocking current, thereby causing missed reading (deletion error) in the sequencing process; it is also difficult to avoid the situation where the nucleotide tag blocks the current being read, but the nucleotide itself does not actually participate in the synthesis reaction, causing an error in which the signal is read redundantly (interruption error).
Disclosure of Invention
The embodiment of the disclosure provides a measuring device and a measuring method for gene sequencing, which are used for improving the accuracy of gene sequencing.
In a first aspect, an embodiment of the present disclosure provides a measurement apparatus for gene sequencing, including:
at least one detection unit including a test chamber and a measurement circuit unit connected to the test chamber;
the test chamber comprises a first compartment and a second compartment which are separated by a membrane, and a first electrode connected to the first compartment and a second electrode connected to the second compartment, wherein the first electrode is connected with a common electrode terminal, and the second electrode is connected with the measurement circuit unit;
the measurement circuit unit includes a first amplification circuit and a second amplification circuit for modulating and amplifying a current signal caused by bidirectional movement of a nucleotide molecule between the first compartment and the second compartment through a nanopore on the membrane and outputting a first or second characteristic voltage signal.
According to a preferred embodiment of the present disclosure, the bidirectional movement of the nucleotide molecule between the first and second compartments results in a change in the resistive-capacitive properties characterized by the membrane and nanopore.
According to a preferred embodiment of the present disclosure, the measurement apparatus further includes a multiplexer connected to the measurement circuit unit of the at least one detection unit, and an analog-to-digital converter connected to the multiplexer for analog-to-digital converting the first or second characteristic voltage signal output by the measurement circuit unit of the at least one detection unit.
According to a preferred embodiment of the present disclosure, the measuring apparatus further includes at least one analog-to-digital converter, which is correspondingly connected to the measuring circuit unit of the at least one detecting unit, and is configured to perform analog-to-digital conversion on the first or second characteristic voltage signal output by the measuring circuit unit of the at least one detecting unit.
According to a preferred embodiment of the present disclosure, the first amplifying circuit includes a first operational amplifier, a first capacitor, and a first reset switch; the first electrode inputs a reference voltage, the positive phase input end of the first operational amplifier inputs a working voltage, the negative phase input end of the first operational amplifier is connected with the second electrode, and the first capacitor and the first reset switch are connected in parallel with the negative phase input end and the output end of the first operational amplifier; the first operational amplifier performs integral amplification on the current signal under the action of the first capacitor and outputs a third voltage signal, and the first reset switch is used for resetting the first capacitor.
According to a preferred embodiment of the present disclosure, the second amplifying circuit includes a second operational amplifier, a second capacitor, and a second reset switch; the positive phase input end of the second operational amplifier is connected to the output end of the first operational amplifier through a third capacitor, and the second capacitor and the second reset switch are connected in parallel to the negative phase input end and the output end of the second operational amplifier; the second operational amplifier amplifies the third voltage signal under the action of the second capacitor and outputs the first or second characteristic voltage signal, and the second reset switch is used for resetting the second capacitor.
According to a preferred embodiment of the present disclosure, the first operational amplifier includes a first chopper-stabilized circuit, and the second operational amplifier includes a second chopper-stabilized circuit; the first chopper stabilization circuit is used for modulating a signal to be measured in the current signal input to the first operational amplifier into a high-frequency signal, and the second chopper stabilization circuit is used for modulating the amplified signal to be measured back into a low-frequency signal and modulating the amplified noise signal into a high-frequency signal.
According to a preferred embodiment of the present disclosure, the measurement circuit unit further comprises a low-pass filter for filtering a high-frequency signal of the first or second characteristic voltage signal.
According to a preferred embodiment of the present disclosure, the device further comprises a variable voltage source for generating an assembly voltage to drive insertion of the nanopore on the membrane.
According to a preferred embodiment of the present disclosure, the variable voltage source is further adapted to generate a working voltage to drive the bi-directional movement of the nucleotide molecule between the first compartment and the second compartment through the nanopore on the membrane.
According to a preferred embodiment of the present disclosure, the membrane comprises a phospholipid bilayer membrane.
In a second aspect, the embodiments of the present disclosure provide a measurement method for gene sequencing, which is based on the measurement device of any one of the foregoing embodiments, and the method includes:
applying a positive working voltage between the first electrode and the second electrode to drive the nucleotide molecule from the first compartment into the second compartment through the nanopore;
modulating and amplifying a first current signal generated by the nucleotide molecule passing from the first compartment into the second compartment through the nanopore, and outputting a first characteristic voltage signal;
applying a reverse working voltage between the first electrode and the second electrode to drive the nucleotide molecule from the second compartment back to the first compartment through the nanopore;
modulating and amplifying a second current signal generated by returning the nucleotide molecule from the second compartment to the first compartment through the nanopore, and outputting a second characteristic voltage signal;
and sequencing the nucleotide sequence to be detected according to the first characteristic voltage signal and the second characteristic voltage signal.
According to a preferred embodiment of the present disclosure, the method further comprises: an assembly voltage is applied between the first electrode and the second electrode to drive insertion of the nanopore in the membrane.
According to a preferred embodiment of the present disclosure, the method further comprises: detecting a change in the resistive-capacitive characteristics of the film during insertion of the nanopore in the film, thereby controlling the process and amount of insertion of the nanopore.
According to a preferred embodiment of the present disclosure, the method further comprises: the nucleotide molecule in the first or second compartment is modified with a biopolymer.
The measuring device and the measuring method for gene sequencing of the embodiment modulate and amplify characteristic current signals generated when nucleotide molecules enter a second compartment from a first compartment or enter the first compartment from the second compartment through a nanopore respectively, so that characteristic voltage signals are output, and the base type of the nucleotide molecules is judged according to the characteristic voltage signals; meanwhile, sequencing is carried out according to the bidirectional characteristic voltage formed by whether the nucleotide molecules participate in synthesis, so that the missing reading and multiple reading errors in the sequencing process can be avoided, and the sequencing accuracy is improved.
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 structural diagram of a measurement apparatus 100 for gene sequencing according to an embodiment of the present disclosure;
FIG. 2 is a schematic diagram of an equivalent circuit model of a test chamber in a measurement apparatus for gene sequencing according to an embodiment of the present disclosure;
FIG. 3 is a schematic diagram of a first process of sequencing implemented by the measuring apparatus for gene sequencing according to the embodiment of the disclosure;
FIG. 4 is a schematic diagram of a second process of implementing sequencing by the measuring device for gene sequencing according to the embodiment of the disclosure;
FIG. 5 is a schematic diagram of a third process of sequencing implemented by the gene sequencing measuring device according to the embodiment of the disclosure;
FIG. 6 is a circuit schematic of a measurement circuit cell 400 according to an embodiment of the present disclosure;
FIG. 7 is a signal waveform diagram of the measurement circuit unit shown in FIG. 6;
FIG. 8 is a schematic diagram of the signal modulation principle of the measuring circuit unit shown in FIG. 6;
fig. 9 is a schematic flow diagram of a measurement method of gene sequencing according to an embodiment of the disclosure.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the 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 invention.
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.
The present disclosure proposes a novel nucleic acid sequencing method that utilizes a nanopore to transport nucleotides for synthesis in both directions, thereby ensuring that each nucleotide involved in synthesis must pass through the nanopore and record its characteristic blocking current, thereby avoiding missed and multiple read errors.
The method separates four kinds of modified deoxynucleotide triphosphates for nucleic acid synthesis and other components on two sides of a membrane with nanopores, transports the modified deoxynucleotide triphosphates on two sides of the membrane through the nanopores embedded on the membrane under the action of voltage and performs synthesis reaction, and determines the base type of the nucleotide according to the change of the electrical properties of the nanopores caused in the process of transporting the nucleotide by the nanopores so as to realize sequencing.
The present disclosure also provides a measuring device for gene sequencing, which measures characteristic current signals in a nanopore by using an alternating current sampling method, thereby determining the type of nucleotide passing through the nanopore to achieve nucleic acid sequencing.
Fig. 1 is a schematic structural diagram of a measurement apparatus 100 for gene sequencing according to an embodiment of the present disclosure. As shown in fig. 1, a measurement apparatus 100 for gene sequencing according to an embodiment of the present disclosure includes:
at least one detection unit comprising a test chamber 102 and a measurement circuit unit 105 connected to the test chamber 102.
Wherein the test chamber 102 comprises a first compartment 111 and a second compartment 112 separated by a membrane 103, and a first electrode 108 connected to the first compartment 111 and a second electrode 109 connected to the second compartment 112, the first electrode 108 being connected to the common electrode terminal 101, the second electrode 109 being connected to the measurement circuit unit 105.
The measurement circuit unit 105 is used for detecting a current signal caused by bidirectional movement of a nucleotide molecule between the first compartment 111 and the second compartment 112 through the nanopore 104 on the membrane 103, and outputting a characteristic voltage signal.
Wherein the nucleotide molecule is initially retained in the first compartment 111 and is driven from the first compartment 111 into the second compartment 112 by an operating voltage applied to the first electrode 108 and the second electrode 109. It may also be initially left in the second compartment 112 and driven by a reverse operating voltage applied to the first electrode 108 and the second electrode 109 to pass from the second compartment 112 into the first compartment 111.
In an alternative embodiment, the measurement device 100 further comprises a variable voltage source (not shown in the figures) that can generate an assembly voltage to be applied to the membrane 103 in order to insert the nanopore 104 on the membrane 103. In addition, the variable voltage source can also generate working voltage with variable direction to drive the nucleotide molecules to move in the nanopore 104 on the membrane 103 in two directions. Application of the operating voltage to drive bidirectional movement of a nucleotide molecule in a nanopore 104 on the membrane 103 will result in a change in the resistive-capacitive properties characterized by the membrane 103 and the nanopore 104.
In an alternative embodiment, the measurement device 100 can also detect changes in the resistive-capacitive properties of the film 103 during insertion of the nanopore 104 on the film 103, thereby controlling the process and amount of nanopore 104 insertion.
In an alternative embodiment the assembly voltage comprises a level or pulse voltage, the level ranging from 500mv to 1v, the peak level of which may be higher than the peak level of the operating voltage.
In an alternative embodiment the operating voltage may comprise different direction levels or pulse voltages, the level range from 0mv to 250mv, preferably from 100mv to 150 mv.
The gene sequencing measuring device disclosed by the embodiment of the disclosure modulates and amplifies a characteristic current signal generated when a nucleotide molecule enters a second compartment from a first compartment through a nanopore or enters the first compartment from the second compartment, so as to output a characteristic voltage signal, and accordingly, the base type of the nucleotide molecule is judged and sequencing is performed by whether the nucleotide molecule participates in a synthesis reaction, so that the missing and multiple reading errors in the sequencing process can be avoided, and the sequencing accuracy is improved.
In an alternative embodiment, as shown in fig. 1, the measurement apparatus 100 for gene sequencing may further include a multiplexer 110 and an analog-to-digital converter 107. The multiplexer 110 is connected to the measurement circuit unit 105 of the at least one detection unit, and the analog-to-digital converter 107 is connected to the multiplexer 110 for performing analog-to-digital conversion on the voltage signal output by the measurement circuit unit 105 of the at least one detection unit. When there are a plurality of detection units, the plurality of measurement circuit units 105 can share the analog-to-digital converter 107 in a time-division multiplexing manner through the multiplexer 110 to realize analog-to-digital conversion.
In an alternative embodiment, the measuring apparatus 100 for gene sequencing may further include at least one analog-to-digital converter 107 disposed corresponding to the measurement circuit unit in the at least one detection unit, where the at least one analog-to-digital converter 107 is correspondingly connected to the measurement circuit unit in the at least one detection unit, and is configured to perform analog-to-digital conversion on the voltage signal output by the measurement circuit unit in the at least one detection unit.
In an alternative embodiment, the membrane 103 separating the first compartment 111 and the second compartment 112 in the test chamber 102 described above may comprise a phospholipid bilayer membrane. The side wall of the test chamber 102 may be made of a hydrophobic material, and the test chamber 102 is divided into a first compartment and a second compartment by a phospholipid bilayer membrane, and a nanopore 104 is inserted into the phospholipid bilayer membrane 103 to serve as a passage for the test nucleotide to pass through.
Fig. 2 is a schematic diagram of an equivalent circuit model of a test chamber in a measurement apparatus for gene sequencing according to an embodiment of the present disclosure. As shown in fig. 2, one implementation of electrodes 202 and 203 electrically connecting the first and second compartments of the test chamber 201, respectively, is a non-faraday electrode, and electrodes 202 and 203, respectively, may be equivalent to a capacitance (on the order of 300pF or more). Nanopore 205 may be equivalent to a parallel connection of nanopore equivalent capacitance 208 (on the order of pF) and nanopore equivalent resistance 209 (on the order of 1-20G Ω). In the following description of the embodiment of the measurement circuit unit, the entire test chamber is replaced with a nanopore equivalent circuit model 210 for simplicity.
As shown in FIG. 3, in an alternative embodiment, the nucleotide molecule 306 and the linker 305 are linked and initially remain in the first compartment 307 or the second compartment 308 of the test chamber 301.
As shown in fig. 4, a voltage difference, preferably in the order of 100-150 mv, is formed by the control circuit between the electrodes 303, 304 connected to the first compartment 307 and the second compartment 308, respectively. The nucleotide molecule 306 connected by linker 305 passes from the first compartment 307 through the nanopore 304 into the second compartment 308. Or conversely, a negative pressure difference is created by the control circuit, preferably in the order of-100 mv to-150 mv, and the nucleotide molecule 306 connected by the linker 305 passes from the second compartment 308 into the first compartment 307 through the nanopore 304.
As shown in FIG. 5, if the linker 305 connects the nucleotide molecules 306 to participate in the synthesis reaction, the linker 305 and the nucleotide molecules 306 may be separated. The voltage difference formed by the control circuit is preferably in the order of 100mv to 150 mv. Linker 305 and nucleotide molecule 306 may pass from first compartment 307 into second compartment 308 through nanopore 304, respectively. Or conversely, a negative pressure difference, preferably in the order of-100 mv to-150 mv, is created by the control circuit, and the linker 305 and the nucleotide molecule 306, respectively, may pass from the second compartment 308 into the first compartment 307 through the nanopore 304.
The nucleotide molecule 306 has four base types (ATCGs), and different base types cause different degrees of change in the electrical signal through the nanopore 304. The embodiments of the present disclosure can determine the base type of the nucleotide molecule 306 and whether the nucleotide molecule 306 participates in the synthesis reaction by detecting the change of the electric signal caused by the movement of the linker 305 and the nucleotide molecule 306 from the first compartment 307 to the second compartment 308 through the nanopore 304, or from the second compartment 308 to the first compartment 307, thereby realizing gene sequencing.
Fig. 6 is a circuit schematic of a measurement circuit cell 400 according to an embodiment of the disclosure. As shown in fig. 6, the measurement circuit unit 400 includes a first amplification circuit and a second amplification circuit.
The first amplification circuit includes an operational amplifier 404, an integrating feedback capacitor 405, and a reset switch 406. A reference voltage 402 is input to a first electrode (e.g., the electrode 303 in fig. 5) at one end of the nanopore equivalent model 401, a second electrode (e.g., the electrode 304 in fig. 5) at the other end of the nanopore equivalent model 401 is connected to an inverting input terminal of an operational amplifier 404, and a working voltage 403 is input to a non-inverting input terminal of the operational amplifier 404. An integrating feedback capacitor 405 and a reset switch 406 are connected in parallel to the inverting input and output of the operational amplifier 404.
The operational amplifier 404 integrates and amplifies the current signal generated in the nanopore equivalent circuit 401 under the action of the integral feedback capacitor 405, and outputs a characteristic voltage signal. The reset switch 406 is used to periodically reset the integrating feedback capacitor 405.
In a preferred embodiment, the operational amplifier 404 may be a transconductance operational amplifier.
The first amplifying circuit is connected to a second amplifying circuit through a capacitor 408, and the second amplifying circuit includes an operational amplifier 409, a feedback capacitor 410, and a reset switch 411. The operating voltage 403 is input to a non-inverting input terminal of the operational amplifier 409, an inverting input terminal of the operational amplifier 404 is connected to an output terminal of the operational amplifier 404 through the capacitor 408, the feedback capacitor 410 and the reset switch 411 are connected in parallel to the inverting input terminal and the output terminal of the operational amplifier 409, and the capacitor 408 plays a role in isolating a direct current signal. The operational amplifier 409 further amplifies the characteristic voltage signal output by the operational amplifier 404 under the action of the feedback capacitor 410, so as to further amplify the micro-current signal to be detected output by the nanopore equivalent model 401.
Fig. 7 is a signal waveform diagram of the measurement circuit unit shown in fig. 6. One form of the operating voltage input to the non-inverting input of the operational amplifier 404 is shown as the operating voltage 501, and the reset switch 406 periodically resets the integrating feedback capacitor 405, where the reset signal has a waveform shown as the reset signal 502. Different nucleotide molecules produce different characteristic current signals when passing through the nanopore. The output of the operational amplifier 404 can detect different characteristic voltage signals, as shown by characteristic voltage 503.
It should be noted that the nucleotide molecule moves between the first compartment and the second compartment of the test chamber under the action of the working voltage 403, and the characteristic voltage signal 503 has two kinds of positive and negative under the action of the working voltage 501 according to whether the synthesis reaction occurs. For simplicity and ease of presentation, the characteristic voltage 503 shown in fig. 8 is the absolute value of the voltage signal.
In the embodiment of the present disclosure, the operational amplifier 404 further includes a chopper stabilization circuit and is controlled to be turned on by 407, and the operational amplifier 409 includes a chopper stabilization circuit and is controlled to be turned on by 412. The measuring circuit unit further comprises a low pass filter 413, to which low pass filter 413 the output of the operational amplifier 409 is connected. Because the micro-current signal to be detected output by the nanopore equivalent model 401 belongs to a pA-level micro-current signal and has low signal-to-noise ratio, the detection circuit is not expected to introduce excessive noise to cover an effective signal. In the low-noise low-bandwidth detection circuit, generally speaking, low-frequency noise such as 1/f noise and offset of the operational amplifier itself has the largest influence on signal detection, and in the embodiment of the present disclosure, the operational amplifiers 404 and 409 with chopper stabilization circuits are used for modulating and amplifying signals to be detected, and low-pass filtering is performed through a low-pass filter, so that low-frequency noise signals introduced by the detection circuit are effectively filtered, and thus, the amplified effective signals to be detected are detected.
In an alternative embodiment, the output voltage of the operational amplifier 409 may be further stored and processed after passing through the low pass filter 413 and analog-to-digital conversion by the analog-to-digital converter 414. Although not shown in fig. 1 and 6, the measurement device for gene sequencing of the present disclosure may include a circuit unit or system that stores, transmits, processes, and analyzes data processed by an analog-to-digital converter.
Fig. 8 is a schematic diagram of the signal modulation principle of the measuring circuit unit shown in fig. 6. As shown IN fig. 8, an input signal 601 (including a signal to be measured and a noise floor, refer to the input signal and noise distribution 612 IN fig. 8) is first modulated to a high frequency (refer to AMP _ IN and noise distribution 613 IN fig. 8), input operational amplifiers 404 and 409 introduce low frequency noise such as 1/f noise and offset after amplification, and an output signal refers to AMP _ OUT and noise distribution 614 IN fig. 8. It can be seen that the signal under test, the 1/f noise and the low frequency noise are all amplified, and only the signal under test is modulated. Subsequently, the output signal is further modulated, the amplified 1/f noise and low frequency noise are modulated to high frequency, and the low frequency part of the signal to be measured is restored to an effective signal (refer to LPF _ IN and noise distribution 615 IN fig. 9). Finally, after the modulated signal passes through the low pass filter 610 (corresponding to 413 in fig. 6), the modulated 1/f noise and low frequency noise signal are filtered OUT, and only the low frequency effective signal to be measured is output (refer to LPF _ OUT and noise distribution 616 in fig. 9). Therefore, the measuring circuit unit improves the signal-to-noise ratio of the output detection signal by modulating and filtering the input signal.
Fig. 9 is a schematic flow diagram of a measurement method of gene sequencing according to an embodiment of the disclosure. As shown in fig. 9, the method for measuring gene sequencing according to the embodiment of the present disclosure is implemented based on the apparatus 100 for measuring gene sequencing according to any of the foregoing embodiments, and mainly includes the following steps:
step S110 of applying a positive working voltage between the first electrode and the second electrode to drive the nucleotide molecule from the first compartment to the second compartment through the nanopore;
step S120, modulating and amplifying a first current signal generated by the nucleotide molecule entering a second compartment from a first compartment through the nanopore, and outputting a first characteristic voltage signal;
step S130 of applying a reverse working voltage between the first electrode and the second electrode to drive the nucleotide molecule from the second compartment back to the first compartment through the nanopore;
step S140, modulating and amplifying a second current signal generated by returning the nucleotide molecule from the second compartment to the first compartment through the nanopore, and outputting a second characteristic voltage signal;
and S150, sequencing the nucleotide sequence to be detected according to the first characteristic voltage signal and the second characteristic voltage signal.
In some embodiments, the measurement method further comprises: an assembly voltage is applied between the first electrode and the second electrode to drive insertion of the nanopore in the membrane.
In some embodiments, the measurement method further comprises: the nucleotide molecule in the first or second compartment is modified with a biopolymer.
The gene sequencing measurement method of the embodiment modulates and amplifies a characteristic current signal generated when a nucleotide molecule enters a second compartment from a first compartment through a nanopore or enters the first compartment from the second compartment, so as to output a characteristic voltage signal, and accordingly, the base type of the nucleotide molecule is judged; meanwhile, whether the nucleotide molecule participates in synthesis can be judged by comparing the difference of the characteristic current signals of two times of operation, so that the reading missing and the reading multiple errors in the sequencing process can be avoided, and the sequencing accuracy is improved.
It should be noted that the above embodiments can be freely combined as necessary. The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, changes and modifications can be made without departing from the principle of the present invention, and such changes and modifications should also be considered as falling within the scope of the present invention.
Claims (15)
1. A measuring device for gene sequencing, comprising:
at least one detection unit comprising a test chamber and a measurement circuit unit connected to the test chamber;
the test chamber comprises a first compartment and a second compartment which are separated by a membrane, and a first electrode connected to the first compartment and a second electrode connected to the second compartment, wherein the first electrode is connected with a common electrode terminal, and the second electrode is connected with the measurement circuit unit;
the measurement circuit unit includes a first amplification circuit and a second amplification circuit for modulating and amplifying a current signal caused by bidirectional movement of a nucleotide molecule between the first compartment and the second compartment through a nanopore on the membrane and outputting a first or second characteristic voltage signal.
2. The measurement device of claim 1, wherein the bi-directional movement of the nucleotide molecule between the first and second compartments results in a change in the resistive-capacitive properties characterized by the membrane and nanopore.
3. The measurement device according to claim 1, further comprising a multiplexer connected to the measurement circuit unit of the at least one detection unit, and an analog-to-digital converter connected to the multiplexer for analog-to-digital converting the first or second characteristic voltage signal output from the measurement circuit unit of the at least one detection unit.
4. The measuring apparatus according to claim 1, further comprising at least one analog-to-digital converter, wherein the at least one analog-to-digital converter is correspondingly connected to the measuring circuit unit of the at least one detecting unit, and is configured to perform analog-to-digital conversion on the first or second characteristic voltage signal output by the measuring circuit unit of the at least one detecting unit.
5. The measurement device according to claim 3 or 4, wherein the first amplification circuit comprises a first operational amplifier, a first capacitor and a first reset switch; the first electrode inputs a reference voltage, the non-inverting input end of the first operational amplifier inputs a working voltage, the inverting input end of the first operational amplifier is connected with the second electrode, and the first capacitor and the first reset switch are connected in parallel to the inverting input end and the output end of the first operational amplifier; the first operational amplifier performs integral amplification on the current signal under the action of the first capacitor and outputs a third voltage signal, and the first reset switch is used for resetting the first capacitor.
6. The measurement device of claim 5, wherein the second amplification circuit comprises a second operational amplifier, a second capacitor, and a second reset switch; the non-inverting input end of the second operational amplifier is connected to the output end of the first operational amplifier through a third capacitor, and the second capacitor and the second reset switch are connected in parallel to the inverting input end and the output end of the second operational amplifier; the second operational amplifier amplifies the third voltage signal under the action of the second capacitor and outputs the first or second characteristic voltage signal, and the second reset switch is used for resetting the second capacitor.
7. The measurement device of claim 6, wherein the first operational amplifier comprises a first chopper-stabilized circuit and the second operational amplifier comprises a second chopper-stabilized circuit; the first chopper stabilization circuit is used for modulating a signal to be detected in the current signal input to the first operational amplifier into a high-frequency signal, and the second chopper stabilization circuit is used for modulating the amplified signal to be detected back into a low-frequency signal and modulating the amplified noise signal into a high-frequency signal.
8. The measurement device according to claim 7, wherein the measurement circuit unit further comprises a low-pass filter for filtering a high-frequency signal of the first or second characteristic voltage signal.
9. The measurement device of claim 1, further comprising a variable voltage source for generating an assembly voltage to drive insertion of the nanopore on the membrane.
10. The measurement device of claim 9, wherein the variable voltage source is further configured to generate an operating voltage to drive bidirectional movement of the nucleotide molecule between the first compartment and the second compartment through a nanopore in the membrane.
11. A measurement device as claimed in claim 1, wherein the membrane comprises a phospholipid bilayer membrane.
12. A method for measuring gene sequencing, which is based on the measuring apparatus according to any one of claims 1 to 11, and which comprises:
applying a positive working voltage between the first electrode and the second electrode to drive the nucleotide molecule from the first compartment into the second compartment through the nanopore;
modulating and amplifying a first current signal generated by the nucleotide molecule passing from the first compartment into the second compartment through the nanopore, and outputting a first characteristic voltage signal;
applying a reverse working voltage between the first electrode and the second electrode, driving the nucleotide molecule from the second compartment back to the first compartment through the nanopore;
modulating and amplifying a second current signal generated by returning the nucleotide molecule from the second compartment to the first compartment through the nanopore, and outputting a second characteristic voltage signal;
and sequencing the nucleotide sequence to be detected according to the first characteristic voltage signal and the second characteristic voltage signal.
13. The measurement method according to claim 12, further comprising: an assembly voltage is applied between the first electrode and the second electrode to drive insertion of the nanopore on the membrane.
14. The measurement method according to claim 13, further comprising: detecting a change in the resistive-capacitive characteristics of the membrane during insertion of the nanopore in the membrane, thereby controlling the process and amount of nanopore insertion.
15. The measurement method according to claim 12, further comprising: the nucleotide molecules in the first or second compartment are modified with a biopolymer.
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