CN113176317A - Single-layer membrane double-nanopore DNA detection device and detection method - Google Patents
Single-layer membrane double-nanopore DNA detection device and detection method Download PDFInfo
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Abstract
The invention discloses single-layer membrane double-nanopore DNA detection equipment, which comprises a DNA modification cavity, a DNA detection cavity, an electric field electrode and a signal processing terminal, wherein the DNA modification cavity is formed by a plurality of DNA modification cavities; the DNA modification cavity is used for connecting the tail end of the DNA to be detected with the magnetic small ball; and the electric field electrode is used for generating an external electric field in the DNA detection cavity, so that the head end of the DNA to be detected additionally provided with the probe and the magnetic ball passes through the front surface of the single-layer film double-nano chip firstly, the nano hole A reaches the back surface of the single-layer film double-nano chip and then passes through the nano hole B from the back surface, and after the single-time DNA via hole detection is completed, the electric field electrode is closed to enable the DNA to be detected to retract. According to the invention, the two DNA detection results are obtained simultaneously, the accuracy of the detection result is improved, the detection time is saved, and the detection efficiency is improved. The invention also provides a single-layer membrane double-nanopore DNA detection method with the advantages.
Description
Technical Field
The invention relates to the field of DNA detection, in particular to single-layer membrane double-nanopore DNA detection equipment and a detection method.
Background
The gene sequencing technology is one of the important means for human beings to explore the life secret, and plays a great role in promoting the technical development in the fields of biology, life science, medicine and the like. The solid-state nanopore sequencing technology is used as a novel fourth-generation gene sequencing technology and has the advantages of low cost, high read length, easiness in integration and the like. The fourth generation solid state nanopore gene sequencing technology is based on the principle that DNA molecule electrophoresis is conducted through a thin and small nanopore, two ends of DNA are fixed, a nanometer displacement platform is moved, due to the difference of physicochemical properties of different bases, the current blocking effect of the nanometer displacement platform on the holes is different, and the sequence information of bases in the DNA molecule can be obtained through the discrimination of the blocking current.
However, the current solid-state nanopore sequencing technology also faces many problems, firstly, the DNA to be detected is only subjected to single-pass hole passing, if the detection result needs to be corrected, the nano displacement platform needs to be reset and then re-measured, so that the test efficiency and the measurement accuracy cannot be obtained at the same time, secondly, in the prior art, when the single-pass hole detection is completed and the probe of the DNA to be detected needs to be replaced, the DNA needs to be taken down from the fixed end, the probe is replaced and the probe is re-fixed, the steps are complex, and the detection efficiency is further reduced.
In summary, how to improve the result accuracy between DNA detections on the premise of ensuring the testing efficiency is an urgent problem to be solved by those skilled in the art.
Disclosure of Invention
The invention aims to provide single-layer membrane double-nanopore DNA detection equipment and a detection method, and aims to solve the problem that in the prior art, the test efficiency and the test accuracy cannot be obtained at the same time.
In order to solve the technical problem, the invention provides a single-layer membrane double-nanopore DNA detection device, which comprises a DNA modification cavity, a DNA detection cavity, an electric field electrode and a signal processing terminal;
the DNA detection cavity comprises a single-layer film double-nanopore chip;
the DNA modification cavity is used for additionally installing a probe on DNA to be detected and connecting a magnetic small ball to the tail end of the DNA to be detected, and the diameter of the magnetic small ball is larger than the aperture of a nanopore A on the single-layer membrane double-nanopore chip;
the electric field electrode is used for generating an external electric field in the DNA detection cavity, so that the head end of the DNA to be detected, which is additionally provided with the probe and the magnetic ball, firstly passes through the nanopore A from the front side of the single-layer film double-nano chip to reach the back side of the single-layer film double-nano chip and then passes through the nanopore B from the back side, one-time detection of the through hole of the DNA to be detected is completed, and after the single-time detection of the through hole of the DNA is completed, the electric field electrode is closed, so that the DNA to be detected retracts;
and the signal processing terminal is used for collecting electric signals of the nano-pores when the DNA to be detected passes through the nano-pores A and B, and determining a DNA detection result through the electric signals of the nano-pores.
Optionally, in the single-layer membrane double-nanopore DNA detection device, the DNA detection chamber comprises a two-channel microfluidic component;
the double-channel micro-fluidic component comprises a flow guide channel, and the flow guide channel and the single-layer film double-nanopore chip are enclosed to form a flow guide pipe;
and the guide pipe is used for respectively connecting the nanopore A and the nanopore B with the DNA modification cavity.
Optionally, in the single-layer membrane double-nanopore DNA detection device, the single-layer membrane double-nanopore chip sequentially comprises a silicon substrate, a silicon nitride layer and a protective layer from bottom to top;
the silicon substrate comprises a substrate through hole, and the protective layer comprises two protective through holes;
the substrate through hole and the silicon nitride layer form a lower groove, and the protection through hole and the silicon nitride layer form two upper grooves;
the nano-hole A and the nano-hole B are respectively positioned at the bottoms of the two upper grooves and at the bottom of the lower groove.
Optionally, in the single-layer membrane double-nanopore DNA detection device, the method for manufacturing the single-layer membrane double-nanopore chip includes:
cleaning the silicon substrate;
sequentially arranging the silicon nitride layer and the protective layer on the front surface of the cleaned silicon substrate;
respectively carrying out photoetching and etching on the back surface of the silicon substrate and the protective layer to obtain a patterned substrate through hole and a patterned protective through hole;
and etching the silicon nitride layer through a transmission electron microscope to obtain the nanopore A and the nanopore B.
Optionally, in the single-layer membrane double-nanopore DNA detection device, the electric field electrode includes an inlet electrode, a bottom electrode, and an outlet electrode;
the inlet electrode is disposed at the nanopore A;
the bottom electrode is arranged below the single-layer film double-nanopore chip and corresponds to the positions of the nanopore A and the nanopore B;
the outlet electrode is arranged at the nanopore B;
the potential of the entrance electrode, the potential of the bottom surface electrode, and the potential of the exit electrode are sequentially increased.
Optionally, in the single-layer membrane double-nanopore DNA detection device, the DNA detection chamber includes an electrode slot assembly;
the electrode groove assembly comprises an electrode channel, and an electrode cavity is defined by the electrode channel and the single-layer film double-nanopore chip;
the bottom electrode is arranged in the electrode cavity.
Optionally, in the single-layer membrane double-nanopore DNA detection apparatus, the pore diameters of the nanopore a and the nanopore B range from 1 nm to 10 nm, inclusive.
Optionally, in the single-layer membrane double-nanopore DNA detection device, the diameter of the magnetic bead ranges from 70 nm to 200 nm, inclusive.
Optionally, in the single-layer membrane double-nanopore DNA detection device, the distance between the nanopore a and the nanopore B ranges from 0.5 micrometers to 10 micrometers, inclusive.
A single-layer membrane double-nanopore DNA detection method comprises the following steps:
acquiring a list of probes to be loaded, and connecting a magnetic small ball to the tail end of the DNA to be tested;
according to the probe list to be installed, installing a first probe for the DNA to be detected;
enabling the head end of the DNA to be detected to firstly penetrate through the nanopore A from the front side of the single-layer film double-nanopore chip to reach the back side of the single-layer film through an external electric field, then penetrating through the nanopore B from the back side, and collecting nanopore electric signals when the DNA to be detected penetrates through the nanopore A and the nanopore B; wherein the diameter of the magnetic bead is larger than the pore diameter of the nanopore A;
closing the external electric field to enable the DNA to be detected to retract, and washing off the first probe;
according to the probe list, circularly adding probes to wash-off the probes to obtain a nanopore signal set corresponding to the probe list;
and determining a DNA detection result according to the nanopore signal set.
The single-layer film double-nanopore DNA detection equipment provided by the invention comprises a DNA modification cavity, a DNA detection cavity, an electric field electrode and a signal processing terminal; the DNA detection cavity comprises a single-layer film double-nanopore chip; the DNA modification cavity is used for additionally installing a probe on DNA to be detected and connecting a magnetic small ball to the tail end of the DNA to be detected, and the diameter of the magnetic small ball is larger than the aperture of a nanopore A on the single-layer membrane double-nanopore chip; the electric field electrode is used for generating an external electric field in the DNA detection cavity, so that the head end of the DNA to be detected, which is additionally provided with the probe and the magnetic ball, firstly passes through the nanopore A from the front side of the single-layer film double-nano chip to reach the back side of the single-layer film double-nano chip and then passes through the nanopore B from the back side, one-time detection of the through hole of the DNA to be detected is completed, and after the single-time detection of the through hole of the DNA is completed, the electric field electrode is closed, so that the DNA to be detected retracts; and the signal processing terminal is used for collecting electric signals of the nano-pores when the DNA to be detected passes through the nano-pores A and B, and determining a DNA detection result through the electric signals of the nano-pores.
The invention sets an external electric field in a DNA detection cavity through an electric field electrode, so that DNA to be detected sequentially passes through two nanopores (namely a nanopore A and a nanopore B) at adjacent positions on the same film layer, and electric signals of the two nanopores when the DNA to be detected passes through the pores are respectively obtained, thus simultaneously obtaining two times of DNA detection results, further eliminating measurement errors caused by mismatched probes and the like, realizing the check of single measurement, greatly improving the accuracy of the detection results while ensuring the detection efficiency, in addition, the invention also connects a magnetic small ball with the diameter larger than the pore diameter of the nanopore A at the tail end of the DNA to be detected, so that the DNA to be detected can not pass through the pores completely, but the tail end is clamped at the nanopore A, when one time of DNA via detection is completed (namely, the part of the DNA to be detected passes through the nanopore A and the nanopore B), the electric field electrode is closed, the external electric field is disappeared, and the DNA retracts under the action of entropy, the probe can be directly replaced in the DNA modification cavity by rebounding the DNA modification cavity, so that the detection time is further saved, and the detection efficiency is improved. The invention also provides a single-layer membrane double-nanopore DNA detection method with the beneficial effect.
Drawings
In order to more clearly illustrate the embodiments or technical solutions of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIGS. 1 to 7 are schematic structural diagrams of single-layer membrane double-nanopore DNA detection devices provided by the present invention;
FIG. 8 is a schematic diagram of the position relationship between the DNA to be detected and the single-layer double-nanopore DNA detection device after one-time via detection is completed according to an embodiment of the present invention;
FIG. 9 is a schematic flow chart of a single-layer membrane double-nanopore DNA detection method according to an embodiment of the present invention.
Detailed Description
In order that those skilled in the art will better understand the disclosure, the invention will be described in further detail with reference to the accompanying drawings and specific embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The core of the invention is to provide a single-layer membrane double-nanopore DNA detection device, the structural schematic diagram of one specific embodiment of which is shown in FIG. 1, and is called as the first specific embodiment, and the device comprises a DNA modification cavity 100, a DNA detection cavity 200, an electric field electrode 300 and a signal processing terminal 400;
the DNA detection chamber 200 includes a single-layer membrane double-nanopore chip 210;
the DNA modification cavity 100 is used for additionally installing a probe on DNA to be detected and connecting a magnetic small ball to the tail end of the DNA to be detected, and the diameter of the magnetic small ball is larger than the aperture of a nanopore A on the single-layer membrane double-nanopore chip 210;
the electric field electrode 300 is configured to generate an external electric field in the DNA detection chamber 200, so that the head end of the DNA to be detected, to which the probe and the magnetic bead are attached, first passes through the nanopore a from the front side of the single-layer membrane double-nanochip to reach the back side of the single-layer membrane double-nanochip, and then passes through the nanopore B from the back side, thereby completing one-time detection of the DNA via hole to be detected, and after completing one-time detection of the DNA via hole, the electric field electrode 300 is turned off, so that the DNA to be detected retracts;
the signal processing terminal 400 is configured to collect nanopore electrical signals when the DNA to be detected passes through the nanopore a and the nanopore B, and determine a DNA detection result according to the nanopore electrical signals.
It should be noted that, while the description of the electrode electric field in the foregoing only describes the flow of a single DNA via detection, it is easy to find that only a single type of probe may be detected by a single DNA via detection, in the actual DNA detection, a plurality of probes are usually required to be tested, which requires that after the completion of the single DNA via detection, the probe is washed away, another type of probe is replaced, and the above detection process is repeated until all types of probes are tested, and the whole DNA sequence is tested.
As a preferred embodiment, the single-layer double-nanopore chip 210 sequentially comprises, from bottom to top, a silicon substrate 211, a silicon nitride layer 212, and a protective layer 215, and a cross-sectional view of an embodiment thereof is shown in fig. 2;
the silicon substrate 211 comprises a substrate through hole, and the protection layer 215 comprises two protection through holes;
the substrate through hole and the silicon nitride layer 212 form a lower groove, and the protection through hole and the silicon nitride layer 212 form two upper grooves;
the nano-hole A and the nano-hole B are respectively positioned at the bottoms of the two upper grooves and at the bottom of the lower groove.
In the present embodiment, the silicon nitride layer 212 is sandwiched by the silicon substrate 211 and the protective layer 215, and the silicon nitride layer 212 is exposed by providing the through holes on the silicon substrate 211 and the protective layer 215, which is equivalent to changing the silicon nitride layer 212 into a suspended layer, and thus the nano-holes are easy to be formed. Of course, the single-layer film double-nano chip with other structures can be adopted according to the actual situation.
Furthermore, the protection layer 215 may comprise, from bottom to top, a sacrificial layer 213 of silicon dioxide and a mask 214 of silicon nitride, which are illustrated in fig. 3, wherein A, B in fig. 3 is the nanopore a and nanopore B.
Accordingly, as a specific embodiment, the method for manufacturing the single-layer film double-nanopore chip 210 includes:
s1: the silicon substrate 211 is cleaned.
S2: the silicon nitride layer 212 and the protective layer 215 are sequentially disposed on the front surface of the cleaned silicon substrate 211.
S3: and respectively photoetching and etching the back surface of the silicon substrate 211 and the protective layer 215 to obtain a patterned substrate through hole and a patterned protective through hole.
S4: and etching the silicon nitride layer 212 through a transmission electron microscope to obtain the nanopore A and the nanopore B.
The high-precision holes with the sizes at the nanometer level can be quickly obtained through the transmission electron microscope, and certainly, other schemes can be selected according to actual conditions to arrange the nano holes.
The following describes an example of a manufacturing process of the single-layer double-nanopore chip 210, including:
1) and cleaning the silicon wafer.
And respectively cleaning the silicon wafer by using deionized water, acetone, alcohol and deionized water.
2) And depositing silicon nitride, silicon dioxide and silicon nitride on two sides of the silicon wafer in sequence.
Depositing three layers of films on two surfaces of a silicon wafer simultaneously by LPCVD (low pressure chemical vapor deposition), wherein the first layer of film is a suspended film for preparing nano holes subsequently, the second layer of silicon dioxide is a sacrificial layer, and the third layer of silicon nitride film is a mask.
3) And (4) photoresist homogenizing, photoetching and developing.
Uniformly and rotationally coating photoresist by using a spin coater, performing pre-film-frying, photoetching, developing, performing microscopic examination, judging whether the developing is sufficient, and then performing film-frying. A pre-etch site pattern is obtained.
4) And (5) etching.
And etching the third silicon nitride film on the first surface and the three films on the second surface by using a plasma etching machine.
5) Removing the photoresist, and corroding the second surface by KOH.
And heating by adopting acetone to remove the photoresist. And then respectively corroding the silicon layer on the second surface by adopting a single-surface corrosion clamp. When exposed to a KOH solution, an inverted pyramid-structured V-shaped groove is formed due to the anisotropy of the etch rate along different crystal axes.
6) The BOE etches the silicon dioxide of the first side.
7) And (6) slicing.
8) Two nanopores are etched in the exposed silicon nitride film by transmission electron microscopy.
In the specific flow, two adjacent nanopores are prepared by adopting an MEMS technology and a TEM (transmission electron microscope) punching technology, and in actual production, other manufacturing processes can be selected according to actual conditions.
The pore diameters of the nanopore a and the nanopore B range from 1 nm to 10 nm, inclusive, such as any of 1.0 nm, 5.3 nm, or 10.0 nm; accordingly, the magnetic beads have a diameter in a range of 70 nanometers to 200 nanometers, inclusive, such as any of 70.0 nanometers, 152.3 nanometers, or 200.0 nanometers; additionally, the nanopore a is spaced from the nanopore B by a distance in a range from 0.5 microns to 10 microns, inclusive, such as any of 0.50 microns, 5.36 microns, or 10.00 microns. Of course, all the parameters can be adjusted correspondingly according to actual conditions.
As a preferred embodiment, the DNA detection chamber 200 includes a dual-channel microfluidic assembly 220;
the dual-channel microfluidic component 220 comprises a flow guide channel 221, and a flow guide pipe is formed by the flow guide channel 221 and the single-layer film double-nanopore chip 210 in a surrounding mode;
the draft tube connects nanopore a and nanopore B with the DNA modification chamber 100, respectively.
The microfluidic component 220 may also be a silicon wafer with the flow guide channel 221, and a schematic structural diagram of a specific embodiment of the microfluidic component is shown in fig. 4, and the microfluidic component can be well attached to the film of the double-nanopore chip, is low in connection difficulty and small in occupied space, and provides a channel for the DNA to be detected to flow, so that the spatial layout in the device is more flexible.
The single-layer film double-nanopore DNA detection equipment provided by the invention comprises a DNA modification cavity 100, a DNA detection cavity 200, an electric field electrode 300 and a signal processing terminal 400; the DNA detection chamber 200 includes a single-layer membrane double-nanopore chip 210; the DNA modification cavity 100 is used for additionally installing a probe on DNA to be detected and connecting a magnetic small ball to the tail end of the DNA to be detected, and the diameter of the magnetic small ball is larger than the aperture of a nanopore A on the single-layer membrane double-nanopore chip 210; the electric field electrode 300 is configured to generate an external electric field in the DNA detection chamber 200, so that the head end of the DNA to be detected, to which the probe and the magnetic bead are attached, first passes through the nanopore a from the front side of the single-layer membrane double-nanochip to reach the back side of the single-layer membrane double-nanochip, and then passes through the nanopore B from the back side, thereby completing one-time detection of the DNA via hole to be detected, and after completing one-time detection of the DNA via hole, the electric field electrode 300 is turned off, so that the DNA to be detected retracts; the signal processing terminal 400 is configured to collect nanopore electrical signals when the DNA to be detected passes through the nanopore a and the nanopore B, and determine a DNA detection result according to the nanopore electrical signals. In the invention, an external electric field is arranged in a DNA detection cavity 200 through an electric field electrode 300, so that DNA to be detected sequentially passes through two nanopores (namely a nanopore A and a nanopore B) at adjacent positions on the same membrane layer, and electric signals of the two nanopores are respectively obtained when the DNA to be detected passes through the pores, thus two times of DNA detection results can be simultaneously obtained, further measurement errors caused by mismatched probes and the like are eliminated, the verification of single measurement is realized, the detection efficiency is ensured, and the accuracy of the detection results is greatly improved at the same time, in addition, the tail end of the DNA to be detected is connected with a magnetic small ball with the diameter larger than the pore diameter of the nanopore A, so that the DNA to be detected can not pass through the pores completely, but the tail end of the DNA to be detected is clamped at the nanopore A, when one time of DNA passing through pore detection is finished (namely, the part of the DNA to be detected passes through the nanopore A and the nanopore B), the electric field electrode 300 is closed, the external electric field disappears, and the DNA retracts under the action of entropy, the probe can be directly replaced in the DNA modification cavity 100 by rebounding the DNA modification cavity 100, so that the detection time is further saved, and the detection efficiency is improved.
On the basis of the first embodiment, the DNA detection chamber 200 is further improved to obtain a second embodiment, and a schematic diagram of a partial structure thereof is shown in fig. 5, and includes a DNA modification chamber 100, a DNA detection chamber 200, an electric field electrode 300 and a signal processing terminal 400;
the DNA detection chamber 200 includes a single-layer membrane double-nanopore chip 210;
the DNA modification cavity 100 is used for additionally installing a probe on DNA to be detected and connecting a magnetic small ball to the tail end of the DNA to be detected, and the diameter of the magnetic small ball is larger than the aperture of a nanopore A on the single-layer membrane double-nanopore chip 210;
the electric field electrode 300 is configured to generate an external electric field in the DNA detection chamber 200, so that the head end of the DNA to be detected, to which the probe and the magnetic bead are attached, first passes through the nanopore a from the front side of the single-layer membrane double-nanochip to reach the back side of the single-layer membrane double-nanochip, and then passes through the nanopore B from the back side, thereby completing one-time detection of the DNA via hole to be detected, and after completing one-time detection of the DNA via hole, the electric field electrode 300 is turned off, so that the DNA to be detected retracts;
the signal processing terminal 400 is configured to collect nanopore electrical signals when the DNA to be detected passes through the nanopore a and the nanopore B, and determine a DNA detection result according to the nanopore electrical signals;
the electric field electrode 300 includes an entrance electrode 310, a bottom electrode 320, and an exit electrode 330;
the inlet electrode 310 is disposed at the nanopore a;
the bottom electrode 320 is disposed below the single-layer film double-nanopore chip 210, and corresponds to the positions of the nanopore a and the nanopore B;
the outlet electrode 330 is disposed at the nanopore B;
the potential of the entrance electrode 310, the potential of the bottom electrode 320, and the potential of the exit electrode 330 increase in this order.
This embodiment has further limited the arrangement of electric field electrode 300 on the basis of the above-mentioned embodiment, because the DNA molecule is negatively charged, therefore, in order to make the DNA to be measured pass through nanopore a, reach the back of the single-layer film, and pass through nanopore B from the back of the single-layer film, the configuration of using inlet electrode 310, bottom electrode 320, and outlet electrode 330 in this embodiment is the simplest, the easiest scheme to set up, can greatly simplify the design of the applied electric field, reduce the installation difficulty of electric field electrode 300.
It should be noted that the lower side of the single-layer double-nanopore chip 210 refers to the side where the DNA molecule to be detected reaches after passing through the nanopore a, and preferably, the bottom electrode 320 is disposed right below the midpoint of the line connecting the nanopore a and the nanopore B; the inlet electrode 310 and the outlet electrode 330 may be installed at any position of the corresponding nanopore, or even plated around the corresponding nanopore, or may be selected by the user according to the actual situation, as long as the potential relationship among the nanopore a, the lower side of the single-layer film double-nanopore chip 210, and the nanopore B is ensured.
On the basis of the above embodiments, as a preferable scheme, the DNA detection chamber 200 includes an electrode slot assembly 230;
the electrode tank assembly 230 comprises an electrode channel 231, and the electrode channel 231 and the single-layer film double-nanopore chip 210 enclose an electrode cavity;
the bottom electrode 320 is disposed within the electrode cavity.
Preferably, the electrode slot assembly 230 is a silicon wafer assembly, and a schematic structural diagram of a specific embodiment of the electrode slot assembly is shown in fig. 6, the electrode slot assembly 230 is disposed below the single-layer film double-nanopore chip 210, the electrode channel 231 and the single-layer film double-nanopore chip 210 enclose the electrode cavity to protect the bottom electrode 320, and meanwhile, the silicon wafer assembly is more easily attached to the single-layer film double-nanopore chip 210, which saves more space compared with other protection schemes.
The schematic structural diagram of the electrode slot assembly 230, the microfluidic assembly 220 and the single-layer film double-nano chip after combination is shown in fig. 7, wherein the channels 1 and 2 in the figure are the flow guide tubes, and the channel 3 is the electrode cavity.
The invention also provides a single-layer membrane double-nanopore DNA detection method, wherein the flow diagram of one specific embodiment is shown in FIG. 9, which is called as a third specific embodiment and comprises the following steps:
s101: and acquiring a list of probes to be loaded, and connecting the tail end of the DNA to be tested with a magnetic small ball.
S102: and adding a first probe for the DNA to be detected according to the probe list to be detected.
When the first probe is installed, the first probe can be installed firstly, and then the magnetic ball is installed.
S103: enabling the head end of the DNA to be detected to firstly pass through the nanopore A from the front side of the single-layer film double-nanopore chip 210 to reach the back side of the single-layer film through an external electric field, then pass through the nanopore B from the back side, and collecting nanopore electric signals when the DNA to be detected passes through the nanopore A and the nanopore B; wherein the diameter of the magnetic bead is larger than the pore diameter of the nanopore A.
S104: and closing the external electric field to retract the DNA to be detected, and washing the first probe.
After the external electric field is closed, the DNA to be detected which loses the action of the electric field rebounds, returns to the DNA modification cavity 100 from the nanopore A again, and can be directly grabbed and the probe is replaced.
In addition, when the external electric field is not turned off after the detection of the DNA via hole to be detected is completed once, a schematic diagram of the position of the DNA to be detected in the DNA detection chamber 200 is shown in fig. 8, and the magnetic small ball is clamped at the nanopore a.
S105: and according to the probe list, circularly adding the probe to the step of washing off the probe to obtain a nanopore signal set corresponding to the probe list.
The loop in this step refers to S102, S103, and S104; the nanopore signal set refers to a set of all nanopore electrical signals obtained by the nanopore A and the nanopore B after all probes in the probe list are detected.
S106: and determining a DNA detection result according to the nanopore signal set.
The single-layer membrane double-nanopore DNA detection method provided by the invention corresponds to the single-layer membrane double-nanopore DNA detection device, and all the steps are executed by the single-layer membrane double-nanopore DNA detection device.
The single-layer membrane double-nanopore DNA detection method provided by the invention obtains a list of probes to be installed, and connects a magnetic ball at the tail end of the DNA to be detected; according to the probe list to be installed, installing a first probe for the DNA to be detected; enabling the head end of the DNA to be detected to firstly pass through the nanopore A from the front side of the single-layer film double-nanopore chip 210 to reach the back side of the single-layer film through an external electric field, then pass through the nanopore B from the back side, and collecting nanopore electric signals when the DNA to be detected passes through the nanopore A and the nanopore B; wherein the diameter of the magnetic bead is larger than the pore diameter of the nanopore A; closing the external electric field to enable the DNA to be detected to retract, and washing off the first probe; according to the probe list, circularly adding probes to wash-off the probes to obtain a nanopore signal set corresponding to the probe list; and determining a DNA detection result according to the nanopore signal set. In the invention, an external electric field is arranged in a DNA detection cavity 200 through an electric field electrode 300, so that DNA to be detected sequentially passes through two nanopores (namely a nanopore A and a nanopore B) at adjacent positions on the same membrane layer, and electric signals of the two nanopores are respectively obtained when the DNA to be detected passes through the pores, thus two times of DNA detection results can be simultaneously obtained, further measurement errors caused by mismatched probes and the like are eliminated, the verification of single measurement is realized, the detection efficiency is ensured, and the accuracy of the detection results is greatly improved at the same time, in addition, the tail end of the DNA to be detected is connected with a magnetic small ball with the diameter larger than the pore diameter of the nanopore A, so that the DNA to be detected can not pass through the pores completely, but the tail end of the DNA to be detected is clamped at the nanopore A, when one time of DNA passing through pore detection is finished (namely, the part of the DNA to be detected passes through the nanopore A and the nanopore B), the electric field electrode 300 is closed, the external electric field disappears, and the DNA retracts under the action of entropy, the probe can be directly replaced in the DNA modification cavity 100 by rebounding the DNA modification cavity 100, so that the detection time is further saved, and the detection efficiency is improved. The invention also provides a single-layer membrane double-nanopore DNA detection method with the beneficial effect.
The embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same or similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
It is to be noted that, in the present specification, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The single-layer membrane double-nanopore DNA detection device and the detection method provided by the invention are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (10)
1. A single-layer film double-nanopore DNA detection device is characterized by comprising a DNA modification cavity, a DNA detection cavity, an electric field electrode and a signal processing terminal;
the DNA detection cavity comprises a single-layer film double-nanopore chip;
the DNA modification cavity is used for additionally installing a probe on DNA to be detected and connecting a magnetic small ball to the tail end of the DNA to be detected, and the diameter of the magnetic small ball is larger than the aperture of a nanopore A on the single-layer membrane double-nanopore chip;
the electric field electrode is used for generating an external electric field in the DNA detection cavity, so that the head end of the DNA to be detected, which is additionally provided with the probe and the magnetic ball, firstly passes through the nanopore A from the front side of the single-layer film double-nano chip to reach the back side of the single-layer film double-nano chip and then passes through the nanopore B from the back side, one-time detection of the through hole of the DNA to be detected is completed, and after the single-time detection of the through hole of the DNA is completed, the electric field electrode is closed, so that the DNA to be detected retracts;
and the signal processing terminal is used for collecting electric signals of the nano-pores when the DNA to be detected passes through the nano-pores A and B, and determining a DNA detection result through the electric signals of the nano-pores.
2. The single-layer membrane double-nanopore DNA detection device of claim 1, wherein the DNA detection chamber comprises a dual-channel microfluidic component;
the double-channel micro-fluidic component comprises a flow guide channel, and the flow guide channel and the single-layer film double-nanopore chip are enclosed to form a flow guide pipe;
and the guide pipe is used for respectively connecting the nanopore A and the nanopore B with the DNA modification cavity.
3. The single-layer double-nanopore DNA detection device of claim 1, wherein the single-layer double-nanopore chip comprises, from bottom to top, a silicon substrate, a silicon nitride layer, and a protective layer in that order;
the silicon substrate comprises a substrate through hole, and the protective layer comprises two protective through holes;
the substrate through hole and the silicon nitride layer form a lower groove, and the protection through hole and the silicon nitride layer form two upper grooves;
the nano-hole A and the nano-hole B are respectively positioned at the bottoms of the two upper grooves and at the bottom of the lower groove.
4. The single-layer membrane double-nanopore DNA detection device of claim 3, wherein the single-layer membrane double-nanopore chip manufacturing method comprises:
cleaning the silicon substrate;
sequentially arranging the silicon nitride layer and the protective layer on the front surface of the cleaned silicon substrate;
respectively carrying out photoetching and etching on the back surface of the silicon substrate and the protective layer to obtain a patterned substrate through hole and a patterned protective through hole;
and etching the silicon nitride layer through a transmission electron microscope to obtain the nanopore A and the nanopore B.
5. The single-layer membrane double-nanopore DNA detection device of claim 1, wherein said electric field electrode comprises an inlet electrode, a bottom electrode, and an outlet electrode;
the inlet electrode is disposed at the nanopore A;
the bottom electrode is arranged below the single-layer film double-nanopore chip and corresponds to the positions of the nanopore A and the nanopore B;
the outlet electrode is arranged at the nanopore B;
the potential of the entrance electrode, the potential of the bottom surface electrode, and the potential of the exit electrode are sequentially increased.
6. The single-layer membrane double-nanopore DNA detection device of claim 5, wherein the DNA detection chamber comprises an electrode well assembly;
the electrode groove assembly comprises an electrode channel, and an electrode cavity is defined by the electrode channel and the single-layer film double-nanopore chip;
the bottom electrode is arranged in the electrode cavity.
7. The single-layer membrane double-nanopore DNA detection device according to claim 1, wherein the pore diameters of the nanopore a and the nanopore B range from 1 nm to 10 nm, inclusive.
8. The single-layer membrane double-nanopore DNA detection device according to claim 7, wherein the magnetic bead has a diameter in a range of 70 nm to 200 nm, inclusive.
9. The single-layer membrane double-nanopore DNA detection device of claim 1, wherein the nanopore a is spaced from the nanopore B by a distance in a range of 0.5 microns to 10 microns, inclusive.
10. A single-layer membrane double-nanopore DNA detection method is characterized by comprising the following steps:
acquiring a list of probes to be loaded, and connecting a magnetic small ball to the tail end of the DNA to be tested;
according to the probe list to be installed, installing a first probe for the DNA to be detected;
enabling the head end of the DNA to be detected to firstly penetrate through the nanopore A from the front side of the single-layer film double-nanopore chip to reach the back side of the single-layer film through an external electric field, then penetrating through the nanopore B from the back side, and collecting nanopore electric signals when the DNA to be detected penetrates through the nanopore A and the nanopore B; wherein the diameter of the magnetic bead is larger than the pore diameter of the nanopore A;
closing the external electric field to enable the DNA to be detected to retract, and washing off the first probe;
according to the probe list, circularly adding probes to wash-off the probes to obtain a nanopore signal set corresponding to the probe list;
and determining a DNA detection result according to the nanopore signal set.
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