Molecular detection unit, chip, preparation method and application

Abstract

The invention discloses a molecular detection unit, a chip, a preparation method and application. The molecular detection unit comprises a substrate, a first structural layer, a second structural layer, a sensing electrode, a third structural layer, a first buffer solution flow channel, a sample flow channel, a single-hole liquid storage cavity, a liquid resistance flow channel and a second buffer solution flow channel, the liquid resistance flow channel and the single-hole liquid storage cavity are arranged in the first structure layer, and the bottom of the single-hole liquid storage cavity is communicated with one end of the liquid resistance flow channel; the sample runner is arranged in the second structural layer and is communicated with the single-hole liquid storage cavity; the first buffer solution runner is arranged in the third structural layer; the bottom of the second buffer solution runner is communicated with the other end of the liquid resistance runner, and the top of the second buffer solution runner is communicated with the first buffer solution runner; the sensing electrode is arranged at the communication position of the single-hole liquid storage cavity and the liquid resistance flow channel. According to the invention, the arrangement density of the nanopore device in unit area can be improved, the chip manufacturing efficiency is improved, and the flux of the nanopore sequencing device is improved.

Description

Technical Field

[0001] This application relates to the field of biological detection technologies, and in particular, to a molecular detection unit, a chip, a preparation method, and uses thereof. Background Art

[0002] A nanopore sequencing device is a device that determines the base pair sequence of a DNA molecule by detecting changes in electrical signals generated when a biological molecule (e.g., a DNA molecule) passes through a nanopore-sized hole. This type of device generally includes a thin film that can isolate two liquid reservoirs and a nanopore embedded in the thin film. The nanopore can use a protein molecule with a pore structure that exists in nature or is artificially compiled (e.g., a nanopore protein), or can be formed by physical processing means such as an ion beam to create a hole in the thin film. When using a nanopore sequencing device to sequence a biological molecule (e.g., a DNA molecule), the liquid reservoirs on both sides of the thin film are at different potentials to drive the DNA molecule through the nanopore. When the target DNA to be sequenced passes through the nanopore, the electrical signals between the two liquid reservoirs are simultaneously recorded. By analyzing these signals, the sequencing of the DNA structure can be completed.

[0003] In the prior art, the main method used in nanopore devices is to monitor the change in ion current through a nanopore protein between two liquid reservoirs to calculate the base pair sequence of the target DNA to be sequenced. The length of a DNA molecule in a stretched state is relatively long, and it takes a certain amount of time for it to pass through the nanopore. During a specific time period, only a small portion of the DNA molecule's base sequence is within the nanopore. Since each base has a different impact on the magnitude of the change in ion current, the magnitude of the ion current will change accordingly due to the change in a small segment of the base sequence within the nanopore. Therefore, by analyzing the change in ion current throughout the entire process of DNA passing through the nanopore and using corresponding calculation methods, the base sequence of the target DNA to be sequenced can be calculated.

[0004] When using a sequencing method based on ion current, this type of nanopore device has relatively high requirements for the detection accuracy of ion current. The nanopore protein used in existing biological nanopore sequencing devices has a resistance of approximately 1 gigaohm in the sequencing environment, which makes the magnitude of the ion current during sequencing approximately 100 picoamperes. Therefore, changes in the device structure and the test environment will significantly affect the accuracy of ion current measurement, thereby posing relatively high requirements for the amplification performance and stability of the device circuit. At the same time, the measurement of ion current is affected by the size of the electrode area, so the total number of nanopore devices per unit area is limited, thus limiting the throughput of commercial nanopore devices.

[0005] In addition, in the voltage sequencing method using the voltage divider principle in the prior art, a voltage divider is formed by adding a buffer solution flow channel connected by a liquid resistance flow channel to a liquid storage cavity on one side of the thin film embedded with nanopores, so that the device can obtain the change in resistance inside the nanopore by detecting the change in potential of the liquid storage cavity on one side of the thin film, and then calculate the base sequence of the DNA to be sequenced through corresponding methods.

[0006] In the prior art, the liquid resistance flow channel and the buffer solution flow channel are usually realized by making through holes in the substrate. However, in the field of micro-nano processing technology, the commonly used through hole manufacturing methods have certain requirements for the thickness of the material and the geometric shape of the through hole. Most of the nanopore sequencing devices in the prior art are based on silicon wafers, with a thickness of several hundred micrometers, while the required hole diameter in this structure is often in the range of several micrometers. In the existing relatively mature deep aspect ratio micro-nano processing technology, due to factors such as the loading effect and the area of the mask layer, the maximum aspect ratio of deep hole etching that can be effectively completed in actual production is only 15:1. Therefore, how to effectively manufacture such a substrate through hole structure has become an important technical problem in the current production of voltage sequencing principle chips. Summary of the Invention

[0007] Embodiments of the present disclosure provide a molecular detection unit, a chip, a preparation method, and uses.

[0008] In a first aspect, embodiments of the present disclosure provide a molecular detection unit, which includes: a substrate, a first structural layer, a second structural layer, a sensing electrode, a third structural layer, a first buffer solution flow channel, a sample flow channel, a single-hole liquid storage cavity, a liquid resistance flow channel, and a second buffer solution flow channel;

[0009] The first structural layer is disposed on the top of the substrate;

[0010] The second structural layer is disposed above the first structural layer and the insulating layer;

[0011] The third structural layer is disposed above the second structural layer;

[0012] The liquid resistance flow channel and the single-hole liquid storage cavity are disposed in the first structural layer;

[0013] The bottom of the single-hole liquid storage cavity is communicated with one end of the liquid resistance flow channel;

[0014] The sample flow channel is disposed in the second structural layer, above the single-hole liquid storage cavity and communicated with the single-hole liquid storage cavity;

[0015] The first buffer solution flow channel is disposed in the third structural layer;

[0016] The bottom of the second buffer channel is communicated with the other end of the liquid resistance channel; the top of the second buffer channel is communicated with the first buffer channel;

[0017] The sensing electrode is arranged at the connection of the single-hole liquid storage cavity and the liquid resistance channel.

[0018] In a second aspect, an embodiment of the present disclosure provides a molecular detection chip, which includes: a molecular detection array;

[0019] The molecular detection array includes at least one molecular detection unit group; each molecular detection unit group includes a plurality of the molecular detection units described in any one of the above;

[0020] Wherein, the sample channels of each molecular detection unit are communicated with each other to form a common sample channel; and / or,

[0021] The first buffer channels of each molecular detection unit are communicated with each other to form a first common buffer channel; and / or,

[0022] The second buffer channels of each molecular detection unit are communicated with each other to form a second common buffer channel.

[0023] In a third aspect, an embodiment of the present disclosure provides a method for manufacturing a molecular detection chip, which includes:

[0024] A substrate is prepared, and sensing electrodes of each molecular detection unit in the molecular detection chip are respectively formed on the top of the substrate;

[0025] A first structural layer is formed on the substrate, and a corresponding single-hole liquid storage cavity and a bonding hole for the second buffer channel are formed for each molecular detection unit in the molecular detection chip in the first structural layer;

[0026] On the substrate material for the second structural layer, a cavity of the corresponding sample channel and the second buffer channel are formed for each molecular detection unit in the molecular detection chip to form the second structural layer;

[0027] The second structural layer and the first structural layer are bonded to form a bonding structure;

[0028] A corresponding liquid resistance channel is formed for each molecular detection unit in the molecular detection chip in the first structural layer;

[0029] On the substrate material for the third structural layer, a corresponding first buffer channel is formed for each molecular detection unit in the molecular detection chip to form the third structural layer;

[0030] The third structural layer and the bonding structure are bonded.

[0031] Fourthly, embodiments of the present disclosure provide the use of the molecular detection unit, the molecular detection chip, the preparation method of the molecular detection chip, or the chip prepared by the preparation method of the molecular detection chip in any of the above embodiments in preparing a nanopore sensor or in nanopore characterization and analysis of analytes.

[0032] It should be understood that the content described in this part is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it used to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings are used to better understand the solution and do not constitute a limitation to the present disclosure. Among them:

[0034] Figure 1 It is a schematic structural diagram of a molecular detection unit in a specific embodiment of the present disclosure.

[0035] Figure 2 is Figure 1 the schematic AA' cross-sectional view of.

[0036] Figure 3 It is a schematic structural diagram of a molecular detection unit in another specific embodiment of the present disclosure.

[0037] Figure 4 It is a schematic structural diagram of a molecular detection unit in another specific embodiment of the present disclosure.

[0038] Figure 5 It is a schematic structural diagram of a molecular detection unit provided with a film layer in a specific embodiment of the present disclosure.

[0039] Figure 6 It is a schematic diagram of the working principle of a molecular detection unit in a specific embodiment of the present disclosure.

[0040] Figure 7 It is a schematic structural diagram of a molecular detection unit in another specific embodiment of the present disclosure.

[0041] Figure 8 is Figure 7 the schematic BB' cross-sectional view of.

[0042] Figure 9 It is a schematic structural diagram of a molecular detection unit provided with a film layer in another specific embodiment of the present disclosure.

[0043] Figure 10 It is a schematic structural diagram of a molecular detection array in a specific embodiment of the present disclosure.

[0044] Figure 11 is Figure 10 the schematic CC' cross-sectional view of.

[0045] Figure 12 Schematic structural diagram of the molecular detection array in another specific embodiment of the present disclosure.

[0046] Figure 13 is Figure 12 Schematic cross-sectional view of DD'.

[0047] Figure 14 Schematic structural diagram of the molecular detection array provided with a film layer in another specific embodiment of the present disclosure.

[0048] Figure 15 Schematic structural diagram of the molecular detection array in another specific embodiment of the present disclosure.

[0049] Figure 16 is Figure 15 Schematic cross-sectional view of EE'.

[0050] Figure 17 Schematic structural diagram of the molecular detection array provided with a film layer in another specific embodiment of the present disclosure.

[0051] Figure 18 Array schematic diagram of the molecular detection array in another specific embodiment of the present disclosure.

[0052] Figure 19 Schematic flow chart of the preparation method of the molecular detection chip in a specific embodiment of the present disclosure.

[0053] Figure 20 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 1 .

[0054] Figure 21 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 2 .

[0055] Figure 22 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 3 .

[0056] Figure 23 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 4 .

[0057] Figure 24 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 5 .

[0058] Figure 25 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 6 .

[0059] Figure 26 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 7 。

[0060] Figure 27 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 8 。

[0061] Figure 28 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 9 。

[0062] Figure 29 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 10 。

[0063] Figure 30 Schematic of the preparation process of the molecular detection chip in a specific embodiment of the present disclosure Figure 10 One. Detailed implementation manners

[0064] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present disclosure. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present disclosure belongs.

[0065] Note that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present disclosure. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0066] In the case of no conflict, the embodiments in the present disclosure and the features in the embodiments can be combined with each other.

[0067] To make the technical solutions and advantages of the present disclosure clearer and more understandable, the present disclosure will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0068] A molecular detection unit is proposed in the present disclosure.

[0069] Such as Figure 1 and Figure 2As shown, in a specific embodiment of the present disclosure, the molecular detection unit may include: a substrate 1, a first structural layer 2, a second structural layer 3, a sensing electrode 4, a third structural layer 5, a first buffer liquid channel 101, a sample channel 102, a single-hole liquid storage cavity 103, a liquid resistance channel 104, and a second buffer liquid channel 105;

[0070] The first structural layer 2 is disposed on the top of the substrate 1;

[0071] The second structural layer 3 is disposed above the first structural layer 2;

[0072] The third structural layer 5 is disposed above the second structural layer 3;

[0073] The liquid resistance channel 104 and the single-hole liquid storage cavity 103 are disposed in the first structural layer 2;

[0074] The bottom of the single-hole liquid storage cavity 103 is communicated with one end of the liquid resistance channel 104;

[0075] The sample channel 102 is disposed in the second structural layer 3, above the single-hole liquid storage cavity 103 and communicated with the single-hole liquid storage cavity 103;

[0076] The first buffer liquid channel 101 is disposed in the third structural layer 5;

[0077] The bottom of the second buffer liquid channel 105 is communicated with the other end of the liquid resistance channel 104; the top of the second buffer liquid channel 105 is communicated with the first buffer liquid channel 101;

[0078] The sensing electrode 4 is disposed at the communication position between the single-hole liquid storage cavity 103 and the liquid resistance channel 104.

[0079] In addition, in the technical solution of the present disclosure, corresponding membrane layers and nanopores may be further disposed in the above-mentioned molecular detection unit according to the needs of the actual application scenario;

[0080] Wherein, the membrane layer can separate the single-hole liquid storage cavity from the sample channel, and the nanopore can liquid-connect the sample channel and the single-hole liquid storage cavity.

[0081] For example, by way of example, as Figure 5 shown, in a specific embodiment of the present disclosure, the above-mentioned molecular detection unit may further include: a membrane layer 301;

[0082] The membrane layer 301 is disposed at the connection position between the sample channel 102 and the single-hole liquid storage cavity 103; nanopores 302 are disposed on the membrane layer 301.

[0083] In addition, in the technical solution of the present disclosure, different film layers may also be formed in the above-mentioned molecular detection unit according to the needs of the actual application scenario.

[0084] For example, as an example, in a specific embodiment of the present disclosure, the film layer 301 may be a bilayer or a monolayer.

[0085] For example, when the material of the film layer is phospholipid, the film layer 301 is a bilayer; while when the material of the film layer is other polymer materials, the film layer 301 is a monolayer.

[0086] In addition, as an example, as Figure 3 and Figure 4 shown, in a specific embodiment of the present disclosure, the first structural layer 2 may include: a first insulating layer 21 and a second insulating layer 22;

[0087] The first insulating layer 21 is disposed on the top of the substrate 1;

[0088] The second insulating layer 22 is disposed on the top of the first insulating layer 21;

[0089] The liquid resistance flow channel 104 is disposed in the first insulating layer 21;

[0090] The single-hole liquid storage cavity 103 is disposed in the second insulating layer 22.

[0091] In the technical solution of the present disclosure, the first insulating layer 21 and the second insulating layer 22 may be further disposed in the above-mentioned first structural layer 2, and then the liquid resistance flow channel 104 and the single-hole liquid storage cavity 103 are respectively disposed in the first insulating layer 21 and the second insulating layer 22.

[0092] In addition, in the technical solution of the present disclosure, the specific positional relationship between the second structural layer 3 and the second buffer liquid flow channel 105 may also be set according to the needs of the actual application scenario.

[0093] For example, as an example, as Figure 3 shown, in a specific embodiment of the present disclosure, the second structural layer 3 may be disposed on the top of the second insulating layer 22;

[0094] The second buffer liquid flow channel 105 penetrates through the second structural layer 3, the second insulating layer 22, and the first insulating layer 21.

[0095] In this embodiment, the second structural layer 3 is disposed on the top of the first structural layer, and the bottom of the second structural layer 3 abuts against the top of the second insulating layer 22 of the first structural layer.

[0096] For another example, as an example, as Figure 4As shown, in a specific embodiment of the present disclosure, the second structural layer 3 may be disposed above the first insulating layer 21 and the second insulating layer 22, and the bottom of the second structural layer 3 passes through the second insulating layer 22 and abuts against the top of the first insulating layer 21;

[0097] The second buffer fluid channel 105 penetrates through the second structural layer 3 and the first insulating layer 21.

[0098] In this embodiment, the bottom of the second structural layer 3 is embedded in the first structural layer, passes through the second insulating layer 22 and abuts against the top of the first insulating layer 21.

[0099] In addition, as an example, as Figure 5 shown, in a specific embodiment of the present disclosure, the above-mentioned molecule detection unit may further include: a driving electrode 6;

[0100] The driving electrode 6 is disposed in the third structural layer 5 and is connected to the first buffer fluid channel 101.

[0101] Before using the above-mentioned molecule detection unit, considering requirements such as storage and transportation, buffer solutions (a polar solution containing required electrolytes) can be injected into and filled in the sample channel 102, the first buffer fluid channel 101, and the second buffer fluid channel 105 respectively. Since the single-hole liquid storage cavity 103 is communicated with the second buffer fluid channel 105 through the liquid resistance channel 104, the single-hole liquid storage cavity 103 and the liquid resistance channel 104 will also be filled with buffer solutions.

[0102] When it is necessary to use the above-mentioned molecule detection unit, a sample solution can be injected into the sample channel 102 to replace the original buffer solution in the sample channel 102.

[0103] During this process, the liquid can be driven to flow by the positive pressure or negative pressure provided by an external pump. For example, when using positive pressure to drive the sample solution into the sample channel 102, the sample solution in the sample solution storage device (such as a storage bottle, etc.) will enter the sample channel 102 from one end of the sample channel 102 under the pressure provided by the external pump, flow through the sample channel 102 and leave the sample channel 102 from the other end of the sample channel 102, and be collected by a waste liquid collection device (such as a waste liquid bottle, etc.) to prevent pollution.

[0104] Under the pressure drive, the Reynolds number of the sample solution when moving in the sample channel 102 is relatively low, showing a laminar flow state. At this time, the sample solution will not pass through the membrane layer 301 and enter the single-hole liquid storage cavity 103. When there is no pressure drive, the membrane layer 301, as a thin film between the single-hole liquid storage cavity 103 and the sample channel 102, can also prevent the mutual diffusion between the sample solution in the sample channel 102 and the buffer solution in the single-hole liquid storage cavity 103.

[0105] When the above-mentioned molecular detection unit is in the working state, a voltage can be applied to one side of the sample flow channel 102 (for example, applying a voltage to the sample solution in the sample flow channel 102, which can be denoted as V3, as shown in Figure 5 ), and a voltage can be applied to one side of the first buffer flow channel 101 (for example, the buffer in the first buffer flow channel 101 can be applied with a voltage through the driving electrode 6, which can be denoted as V2, as shown in Figure 5 ). Since the first buffer flow channel 101 can communicate with the single-hole liquid storage cavity 103 through the second buffer flow channel 105 and the liquid resistance flow channel 104, there will be a certain difference between the voltage V1 finally applied to one side of the single-hole liquid storage cavity 103 and the voltage V2. Since different voltages can be applied to different liquids, the basic principle of voltage sequencing can be ensured to be realized. Therefore, only when the preset voltages V2 and V3 are applied to the buffer and the sample solution, the electrolyte and the target sample molecules will pass through the nanopore 302 and the liquid resistance flow channel 104 under the action of the electric field force.

[0106] When the DNA molecule 303 in the sample solution passes through the nanopore (for example, nanopore protein) 302 embedded in the membrane layer 301, the resistance R1 of the nanopore 302 can be regarded as a variable resistance when the DNA molecule 303 passes through, and the resistance of the liquid resistance flow channel 104 can be regarded as a fixed resistance. As the base sequence of the DNA molecule 303 passes through the nanopore 302 in sequence, the resistance R1 will change correspondingly, so that the voltage V1 between the two resistances will change. Therefore, according to the change law of the voltage V1 detected by the sensing electrode 4, the base arrangement of the DNA molecule 303 can be calculated to realize the sequencing of the DNA molecule 303.

[0107] For example, as an example, in another specific embodiment of the present disclosure, the resistance between the above-mentioned voltages V2 and V3 is mainly composed of the resistance R1 of the membrane layer 301 embedded with the nanopore and the resistance R2 of the liquid resistance flow channel 2. The voltage V1 will be collected and amplified by an amplifier circuit 43 and transmitted and recorded into the electrical signal database of the sequencing system 44 (such as a computer, etc.), as shown in Figure 6 . Since during the sequencing process, the DNA molecule will pass through the nanopore, and the DNA base combinations passing through the nanopore at different times are different, resulting in the overall resistance R1 of the membrane layer 301 embedded with the nanopore changing with time, that is, the resistance R1 can be regarded as a variable resistance. Therefore, through the voltage V1 at a given moment, the total resistance of the membrane layer 301 embedded with the nanopore and the base combination currently passing through the nanopore can be determined, and then by using a deep learning algorithm, the base sequence of the DNA molecule passing through the pore protein can be calculated.

[0108] In the above-mentioned molecular detection unit of the present disclosure, the first buffer fluid channel 101 is disposed in the third structural layer 5, the sample fluid channel 102 is disposed in the second structural layer 3, and the single-hole liquid storage cavity 103 is disposed in the first structural layer 2, forming a stacked multi-layer structure. Since the first buffer fluid channel 101, the sample fluid channel 102, and the single-hole liquid storage cavity 103 are respectively disposed in different layers, and the first buffer fluid channel 101 is located in the third structural layer 5 above the sample fluid channel 102 and the single-hole liquid storage cavity 103, the arrangement density of nanopore devices per unit area can be effectively increased (for example, the channel density of a nanopore chip can be increased to the level of one hundred thousand devices per square centimeter), greatly improving the throughput of nanopore sequencing devices, so that ultra-high-throughput nanopore gene sequencing can be achieved. In addition, since the above-mentioned molecular detection unit is a stacked multi-layer structure, mature semiconductor processing techniques can be used to separately fabricate each layer structure, and then the various layer structures are formed into the above-mentioned multi-layer structure by bonding, thereby effectively improving the production and manufacturing efficiency.

[0109] In addition, the single-hole liquid storage cavity 103, the liquid resistance fluid channel 104, the first buffer fluid channel 101, the second buffer fluid channel 105, and the sample fluid channel 102 in the above-mentioned molecular detection unit are all disposed on the same side of the substrate 1. Therefore, a liquid resistance fluid channel and a buffer fluid channel can be constructed on the same surface of the substrate, thereby further effectively improving the chip manufacturing efficiency based on the voltage sequencing method and further enhancing the throughput of nanopore sequencing devices.

[0110] Furthermore, in the above-mentioned molecular detection unit of the present disclosure, the sample fluid channel 102 and the first buffer fluid channel 101 are isolated from each other. The sample solution can flow into one end of the sample fluid channel 102 and then flow out from the other end of the sample fluid channel 102, and the sample solution will not enter the first buffer fluid channel 101; similarly, the buffer fluid can flow into one end of the first buffer fluid channel 101 and then flow out from the other end of the first buffer fluid channel 101, and the buffer fluids in the first buffer fluid channel 101 and the second buffer fluid channel 105 will not enter the sample fluid channel 102.

[0111] Since the above two fluid channels are separated, different voltages can be applied to the different liquids in the two fluid channels, thus ensuring the realization of the basic principle of voltage sequencing. In addition, the above two separated fluid channels can also greatly reduce the cross-contamination and leakage between samples and improve the signal-to-noise ratio of the signal.

[0112] In addition, as an example, such as Figure 7 and Figure 8 shown, in a specific embodiment of the present disclosure, the above-mentioned molecular detection unit may further include: two sample solution inlets and outlets 1021 and two buffer fluid inlets and outlets 1011;

[0113] The two sample solution inlets and outlets 1021 are respectively communicated with the sample flow channel 102;

[0114] The two buffer solution inlets and outlets 1011 are respectively communicated with the first buffer solution flow channel 101.

[0115] For example, as Figure 7 and Figure 8 shown, the above-mentioned sample solution inlets and outlets 1021 can penetrate through the third structural layer 5 and be communicated with the sample flow channel 102 in the second structural layer 3; while the buffer solution inlets and outlets 1011 can be arranged in the third structural layer 5 and be communicated with the first buffer solution flow channel 101 in the third structural layer 5.

[0116] In the above-mentioned molecular detection unit, the sample flow channel 102 and the two sample solution inlets and outlets 1021 can form a microfluidic system for the sample, while the first buffer solution flow channel 101, the second buffer solution flow channel 105 and the two buffer solution inlets and outlets 1011 can form a microfluidic system for the buffer solution. The liquids in the two microfluidic systems both enter from one inlet and outlet of the system and then flow out from the other inlet and outlet, and are isolated from each other. For example, the sample solution only enters the sample flow channel 102 from one sample solution inlet and outlet 1021 and then flows out of the sample flow channel 102 from the other sample solution inlet and outlet 1021; during this process, the sample solution does not enter the microfluidic system of the buffer solution. Similarly, the buffer solution can enter the first buffer solution flow channel 101 and the second buffer solution flow channel 105 from one buffer solution inlet and outlet 1011 and then flow out of the first buffer solution flow channel 101 from the other buffer solution inlet and outlet 1011; during this process, the buffer solution in the microfluidic system of the buffer solution also does not enter the microfluidic system of the sample.

[0117] Since the above two microfluidic systems are separated, different voltages can be applied to the different liquids in the two microfluidic systems, so as to ensure the realization of the basic principle of voltage sequencing. In addition, the above two separated microfluidic systems can also greatly reduce the cross-contamination and leakage between samples and improve the signal-to-noise ratio.

[0118] In addition, as an example, as Figure 9 shown, in a specific embodiment of the present disclosure, the above-mentioned molecular detection unit may further include: a sample flow channel interface 12 and a buffer solution flow channel interface 11;

[0119] The sample flow channel interface 12 is arranged in the sample solution inlet and outlet 1021, and the top of the sample flow channel interface 12 extends out from the top of the sample solution inlet and outlet 1021; the bottom of the sample flow channel interface 12 is communicated with the sample flow channel 102;

[0120] The buffer flow channel interface 11 is disposed through the buffer inlet / outlet 1011, and the top of the buffer flow channel interface 11 extends out from the top of the buffer inlet / outlet 1011; the bottom of the buffer flow channel interface 11 communicates with the first buffer flow channel 101.

[0121] In the technical solution of the present disclosure, a sample flow channel interface 12 can be provided in each sample solution inlet / outlet 1021, and a buffer flow channel interface 11 can be provided in each buffer inlet / outlet 1011, so that the sample flow channel interface 12 can communicate with the sample solution inlet / outlet 1021, and the buffer flow channel interface 11 can communicate with the buffer inlet / outlet 1011.

[0122] In addition, as an example, in a specific embodiment of the present disclosure, a first driving electrode may be further provided in the sample flow channel interface, and a second driving electrode may be further provided in the buffer flow channel interface.

[0123] At this time, the above sample flow channel interface and buffer flow channel interface can be a structure that allows liquid to enter and exit and driving electrodes to be connected, so that the corresponding liquid can be injected into the sample solution inlet / outlet 1021 or the corresponding liquid can flow out from the sample solution inlet / outlet 1021 through the sample flow channel interface, and a corresponding voltage V3 can be applied to the liquid in the sample flow channel through the first driving electrode in the sample flow channel interface. Similarly, the corresponding liquid can be injected into the buffer inlet / outlet 1011 or the corresponding liquid can flow out from the buffer inlet / outlet 1011 through the above buffer flow channel interface, and a corresponding voltage V2 can be applied to the liquid in the first buffer flow channel through the second driving electrode in the buffer flow channel interface.

[0124] In addition, in the technical solution of the present disclosure, the shape of the liquid resistance flow channel 104 can be set according to the needs of the actual application scenario.

[0125] For example, in a specific embodiment of the present disclosure, the shape of the liquid resistance flow channel 104 can be linear (as shown in Figure 1 ), curved, arc-shaped or loop-shaped.

[0126] For another example, in a specific embodiment of the present disclosure, a part of the liquid resistance flow channel 104 may be in a curved or arc-shaped form, while the other part is in a linear form.

[0127] In addition, in the technical solution of the present disclosure, a molecular detection chip is further proposed.

[0128] For example, as an example, in another specific embodiment of the present disclosure, the molecular detection chip includes: a molecular detection array;

[0129] The molecular detection array includes at least one group of molecular detection units; each group of molecular detection units includes a plurality of molecular detection units; the molecular detection units can be the molecular detection units in any of the above embodiments.

[0130] Wherein, the sample channels of each molecular detection unit are interconnected to form a common sample channel; and / or,

[0131] the first buffer channels of each molecular detection unit are interconnected to form a first common buffer channel and / or,

[0132] the second buffer channels of each molecular detection unit are interconnected to form a second common buffer channel.

[0133] In this molecular detection chip, it can include a molecular detection array, and one or more groups of molecular detection units are arranged in the molecular detection array, and each group of molecular detection units can contain a plurality of molecular detection units.

[0134] Since the sample channels in each molecular detection unit are all arranged in the same structural layer (i.e., the second structural layer), therefore, in each group of molecular detection units, the sample channels of each molecular detection unit can be interconnected according to their respective arrangement orders to form a common sample channel in the second structural layer; or, the sample channels of all the molecular detection units in the molecular detection array can also be interconnected to form a common sample channel in the second structural layer. Similarly, since the first buffer channels in each molecular detection unit are all arranged in the same structural layer (i.e., the third structural layer), therefore, the first buffer channels of each molecular detection unit in each group of molecular detection units can also be interconnected according to their respective arrangement orders to form a first common buffer channel in the third structural layer; or, the first buffer channels of all the molecular detection units in the molecular detection array can also be interconnected to form a first common buffer channel in the third structural layer; similarly, the second buffer channels of each molecular detection unit in the molecular detection array can also be interconnected according to their respective arrangement orders to form a second common buffer channel in the second structural layer; or, the second buffer channels of all the molecular detection units in the molecular detection array can also be interconnected to form a second common buffer channel in the second structural layer. Therefore, each molecular detection unit in each group of molecular detection units or in the entire molecular detection array can share the above-mentioned common sample channel, and / or share the above-mentioned first common buffer channel, and / or share the above-mentioned second common buffer channel. Therefore, the required liquid (such as buffer and / or sample solution) can be injected into each molecular detection unit respectively through the above-mentioned common sample channel, and the required liquid (such as buffer) can also be injected into each molecular detection unit respectively through the above-mentioned first common buffer channel.

[0135] In addition, in the technical solution of the present disclosure, the number of molecular detection units in a molecular detection unit group can be set according to the needs of the actual application scenario.

[0136] For example, as an example, in another specific embodiment of the present disclosure, a molecular detection unit group may include 2, 4, 6, 8 or more molecular detection units. In the technical solution of the present disclosure, the number of molecular detection units in the molecular detection unit group can be set according to the length of the second buffer channel and / or the second common buffer channel and the size of the molecular detection unit group, so they will not be listed one by one here.

[0137] For example, as Figure 10 shown, in another specific embodiment of the present disclosure, a molecular detection unit group may include 4 molecular detection units.

[0138] Of course, the number of molecular detection units in a molecular detection unit group can also be other suitable values, which will not be listed one by one here.

[0139] In addition, as an example, in a specific embodiment of the present disclosure, the molecular detection chip may further include: two common sample solution inlets and outlets 51 and / or two common buffer inlets and outlets 52; the two common sample solution inlets and outlets 51 are respectively communicated with the common sample channel; the two common buffer inlets and outlets 52 are respectively communicated with the first common buffer channel.

[0140] That is to say, when each molecular detection unit in the molecular detection chip shares the common sample channel and shares the first common buffer channel, only two common sample solution inlets and outlets and / or two common buffer inlets and outlets can be set in the entire molecular detection chip, and the two common sample solution inlets and outlets are respectively communicated with the shared common sample channel, and the two common buffer inlets and outlets are respectively communicated with the shared first common buffer channel, as Figure 12 and Figure 13 shown. Therefore, it is equivalent that all the molecular detection units in the molecular detection array share two common sample solution inlets and outlets and / or two common buffer inlets and outlets. When the above-mentioned molecular detection array needs to be used, the buffer solution or sample solution can be respectively injected into the sample channels of each molecular detection unit from one common sample solution inlet and outlet, and flow out of the sample channel from the other common sample solution inlet and outlet; the buffer solution can be respectively injected into the first buffer channel and the second buffer channel of each molecular detection unit from one common buffer inlet and outlet, and flow out of the first buffer channel from the other common buffer inlet and outlet.

[0141] In addition, as an example, as Figure 14As shown, in a specific embodiment of the present disclosure, the molecular detection chip may further include: a common sample flow channel interface 501 and / or a common buffer solution flow channel interface 502;

[0142] The common sample flow channel interface 501 is disposed through the common sample solution inlet / outlet 51, and the top of the common sample flow channel interface 501 extends out from the top of the common sample solution inlet / outlet 51; the bottom of the common sample flow channel interface 501 communicates with the common sample flow channel;

[0143] The common buffer solution flow channel interface 502 is disposed through the common buffer solution inlet / outlet 52, and the top of the common buffer solution flow channel interface 502 extends out from the top of the common buffer solution inlet / outlet 52; the bottom of the common buffer solution flow channel interface 502 communicates with the first common buffer solution flow channel.

[0144] In the technical solution of the present disclosure, a common sample flow channel interface 501 can be provided in each common sample solution inlet / outlet 51, and a common buffer solution flow channel interface 502 can be provided in each common buffer solution inlet / outlet 52, so that the common sample flow channel interface 501 can communicate with the common sample solution inlet / outlet 51, and the common buffer solution flow channel interface 502 can communicate with the common buffer solution inlet / outlet 52.

[0145] In addition, as an example, in a specific embodiment of the present disclosure, a first common driving electrode may be further provided in the common sample flow channel interface 501, and a second common driving electrode may be further provided in the common buffer solution flow channel interface 502.

[0146] At this time, the above-mentioned common sample flow channel interface 501 and common buffer solution flow channel interface 502 can be a structure that allows liquid to enter and exit and driving electrodes to be connected. Thus, the corresponding liquid can be injected into the common sample solution inlet / outlet 51 or the corresponding liquid can be made to flow out of the common sample solution inlet / outlet 51 through the common sample flow channel interface 501, and a corresponding voltage V3 can be applied to the liquid in the common sample flow channel and the sample flow channel through the first common driving electrode in the common sample flow channel interface 501. Similarly, the corresponding liquid can be injected into the common buffer solution inlet / outlet 52 or the corresponding liquid can be made to flow out of the common buffer solution inlet / outlet 52 through the above-mentioned common buffer solution flow channel interface 502, and a corresponding voltage V2 can be applied to the liquid in the first common buffer solution flow channel and the first buffer solution flow channel through the second common driving electrode in the common buffer solution flow channel interface 502.

[0147] In addition, in the technical solution of the present disclosure, the second buffer solution flow channels in the respective molecular detection units in the molecular detection array may be independent of each other or directly communicate with each other.

[0148] For example, as an example, in another specific embodiment of the present disclosure, the first buffer channels in each of the molecular detection units in the molecular detection array are interconnected to form a first common buffer channel, and the second buffer channels in each of the molecular detection units can be respectively connected to the first common buffer channel.

[0149] For example, in another specific embodiment of the present disclosure, the first buffer channels in each of the molecular detection units in the molecular detection array can be interconnected in the third structural layer to form a first common buffer channel; while the second buffer channels in each of the molecular detection units are independently and respectively connected to the first common buffer channel. At this time, the second buffer channels in each of the molecular detection units are equivalent to hollow needles or pipes extending downward from the first common buffer channel, passing through the second structural layer and inserted into the liquid resistance channels of each of the molecular detection units, so as to connect the liquid resistance channels of each of the molecular detection units to the first common buffer channel.

[0150] In addition, in the technical solution of the present disclosure, multiple molecular detection units in a molecular detection unit group can have various different arrangements.

[0151] For example, as an example, in another specific embodiment of the present disclosure, multiple molecular detection units in the same molecular detection unit group are evenly arranged around a common center.

[0152] For example, in another specific embodiment of the present disclosure, multiple molecular detection units in the same molecular detection unit group can be arranged around a common center in a circular or polygonal shape (for example, quadrilateral, pentagon, hexagon, etc.).

[0153] In addition, as an example, in another specific embodiment of the present disclosure, when multiple molecular detection units in the same molecular detection unit group are evenly arranged around a common center (for example, 6 molecular detection units are evenly arranged in a hexagon, as shown in Figure 15 and Figure 18 ), multiple molecular detection units in the same molecular detection unit group can share a first buffer channel and share a second buffer channel.

[0154] This arrangement can also be regarded as: the first buffer channels of each of the molecular detection units in the same molecular detection unit group are interconnected to form a first common buffer channel, and the second buffer channels of each of the molecular detection units in the same molecular detection unit group are interconnected to form a second common buffer channel.

[0155] Figure 15 and Figure 18The shown arrangement can form a group of molecular detection units through a hexagonal close-packed pattern, and a vertical second buffer fluid channel or a second common buffer fluid channel can be arranged at the central position of the group of molecular detection units, so as to save the total volume occupied by the second buffer fluid channel or the second common buffer fluid channel, and further improve the arrangement density of the nanopore devices per unit area.

[0156] In addition, in the technical solution of the present disclosure, since a liquid resistance flow channel 104 is arranged in each molecular detection unit, therefore, in the array design of the molecular detection chip, a dummy hole 61 can be introduced to achieve the consistency of the array. Of course, the above dummy hole may not be introduced.

[0157] In addition, in the technical solution of the present disclosure, the sample flow channels in each molecular detection unit in the molecular detection array can be independent of each other and are connected to a common sample flow channel through corresponding channels; or they can be directly connected to each other, and the sample flow channels in each molecular detection unit are directly used as a part of the common sample flow channel.

[0158] For example, as an example, as Figure 15 and Figure 18 shown, in another specific embodiment of the present disclosure, the shape of the sample flow channels in each molecular detection unit can be cylindrical. Therefore, the sample flow channels in each molecular detection unit are independent of each other, and each sample flow channel is connected to a common sample flow channel through a corresponding channel (not shown in the figure) arranged in the second structural layer. The shape of the above sample flow channel is a non-parallel flow channel design, and each columnar sample flow channel actually forms a wide flow channel of a columnar array, rather than a series of parallel narrow flow channels, so as to simplify the introduction of the sample solution in the sample flow channel.

[0159] In addition, in the technical solution of the present disclosure, the first buffer fluid channels in each molecular detection unit in the molecular detection array can be independent of each other and are connected to a first common buffer fluid channel through corresponding channels; or they can be directly connected to each other, and the first buffer fluid channels in each molecular detection unit are directly used as a part of the first common buffer fluid channel.

[0160] For example, as an example, as Figure 15 and Figure 16As shown, in another specific embodiment of the present disclosure, the shapes of the first buffer flow channel and the second buffer flow channel in each molecule detection unit can also be cylindrical. Multiple molecule detection units in the same molecule detection unit group share a first buffer flow channel and a second buffer flow channel; alternatively, the first buffer flow channels of the respective molecule detection units in the same molecule detection unit group are interconnected to form a first common buffer flow channel, and the second buffer flow channels of the respective molecule detection units in the same molecule detection unit group are interconnected to form a second common buffer flow channel.

[0161] For another example, as an illustration, in another specific embodiment of the present disclosure, the shape of the first buffer flow channel in each molecule detection unit can also be cylindrical. The first buffer flow channels in each molecule detection unit are independent of each other, and the respective first buffer flow channels are interconnected through corresponding channels (not shown in the figure) provided in the third structural layer to form a first common buffer flow channel. The shape of this first buffer flow channel is also a design of a non-parallel flow channel, rather than a series of parallel narrow flow channels, which can simplify the introduction of the buffer solution in the first buffer flow channel.

[0162] In addition, as an illustration, in another specific embodiment of the present disclosure, multiple molecule detection units in the same molecule detection unit group can form a linear arrangement structure in sequence.

[0163] In this molecule detection chip, a molecule detection array can be provided, and one or more molecule detection unit groups can be provided in this molecule detection array to form one row or multiple rows of molecule detection unit groups.

[0164] The sample flow channels of the respective molecule detection units in each row of molecule detection units (i.e., one molecule detection unit group) can be interconnected according to their respective arrangement sequences to form a common sample flow channel in the second structural layer; the first buffer flow channels of the respective molecule detection units can also be interconnected according to their respective arrangement sequences to form a first common buffer flow channel in the third structural layer. Therefore, the respective molecule detection units in each row of molecule detection units can share the above-mentioned common sample flow channel and the above-mentioned first common buffer flow channel.

[0165] In addition, in the technical solution of the present disclosure, according to the needs of the actual application scenario, one row of molecule detection units (i.e., one molecule detection unit group) can be provided in the molecule detection array, or multiple rows of molecule detection units (i.e., multiple molecule detection unit groups) can be provided in the molecule detection array. In addition, according to the needs of the actual application scenario, the number of the common sample flow channel and / or the first common buffer flow channel in the molecule detection array can be preset.

[0166] For example, as an illustration, in a specific embodiment of the present disclosure, when the molecular detection array includes one or more groups of molecular detection units, the number of common sample channels and / or the first common buffer channels in the molecular detection array can be made equal to the number of groups of molecular detection units in the molecular detection array.

[0167] For another example, as an illustration, in another specific embodiment of the present disclosure, when the molecular detection array includes multiple groups of molecular detection units, the number of common sample channels and / or the first common buffer channels in the molecular detection array can be made less than the number of groups of molecular detection units in the molecular detection array.

[0168] For another example, as an illustration, in another specific embodiment of the present disclosure, when the molecular detection array includes one or more groups of molecular detection units, the number of the first common buffer channels in the molecular detection array can be made equal to the number of groups of molecular detection units in the molecular detection array.

[0169] For another example, as an illustration, in another specific embodiment of the present disclosure, when the molecular detection array includes multiple groups of molecular detection units, the number of the first common buffer channels in the molecular detection array can be made less than the number of groups of molecular detection units in the molecular detection array.

[0170] In the technical solution of the present disclosure, according to the needs of the actual application scenario, the above-mentioned specific embodiments can be combined accordingly, so as to obtain various different specific implementation manners, which will not be listed one by one here.

[0171] In addition, in the technical solution of the present disclosure, the number of molecular detection units in each group of molecular detection units in the molecular detection array can also be flexibly set according to the needs of the actual application scenario.

[0172] For example, as an illustration, in a specific embodiment of the present disclosure, when the molecular detection array includes multiple groups of molecular detection units, the number of molecular detection units in each group of molecular detection units can be equal or unequal.

[0173] In addition, in the technical solution of the present disclosure, a method for preparing a molecular detection chip is also proposed.

[0174] For example, as an illustration, as Figure 19 shown, in a specific embodiment of the present disclosure, the method for preparing the above-mentioned molecular detection chip may include the following steps:

[0175] Step A1, prepare a substrate and form the sensing electrodes of each molecular detection unit in the molecular detection chip on the top of the substrate.

[0176] For example, as an example, such as Figure 20 As shown, in a specific embodiment of the present disclosure, the base layer (i.e., substrate 1) can be fabricated first through semiconductor processes, and this base layer can be used to contain the circuit structures required for the device. Additionally, during the fabrication process of substrate 1, sensing electrodes 4 for each molecular detection unit in the molecular detection chip can be formed respectively on the top of substrate 1 by means of semiconductor processes. The sensing electrodes 4 can be connected to different types of circuit boards or circuit chips through different semiconductor packaging methods (for example, they can be connected to different types of circuit boards or circuit chips through metal leads disposed in substrate 1), which will not be elaborated one by one here.

[0177] Step A2: Form a first structural layer on the substrate, and form a corresponding single-hole liquid storage cavity and a bonding hole for the second buffer liquid flow channel for each molecular detection unit in the molecular detection chip in the first structural layer.

[0178] In this step, a first structural layer 2 will be formed on the substrate 1 first, and then a corresponding single-hole liquid storage cavity 103 and a bonding hole for the second buffer liquid flow channel 105 will be formed for each molecular detection unit in the molecular detection chip in this first structural layer 2.

[0179] Furthermore, in the technical solution of the present disclosure, multiple implementation manners can be used to implement the above-mentioned step A2. The following will take several specific implementation manners as examples to introduce the technical solution of the present disclosure.

[0180] For example, as an example, in a specific embodiment of the present disclosure, when the first structural layer includes a first insulating layer and a second insulating layer, the above step A2 may include the following steps:

[0181] Step A201: For each molecular detection unit in the molecular detection chip, form a liquid resistance flow channel sacrificial structure at a preset position on the substrate and the sensing electrode.

[0182] For example, as an example, such as Figure 21 As shown, in a specific embodiment of the present disclosure, for each molecular detection unit in the molecular detection chip, a liquid resistance flow channel sacrificial structure 41 can be formed at a preset position (i.e., the position where the liquid resistance flow channels 104 of each molecular detection unit need to be formed) on the substrate 1 and the sensing electrode 4 through a certain pattern transfer method (for example, electron beam lithography or laser direct writing, etc.) and / or material deposition process (for example, physical vapor deposition or chemical vapor deposition, etc.).

[0183] For example, as an example, in a specific embodiment of the present disclosure, the liquid resistance flow channel sacrificial structure 41 can be a sacrificial material such as amorphous silicon.

[0184] Step A202: Form a first insulating layer 21 on the substrate, the sensing electrode, and the sacrificial structure of the liquid resistance flow channel.

[0185] For example, as an example, as Figure 22 shown, in a specific embodiment of the present disclosure, when the first structural layer 2 includes a first insulating layer 21 and a second insulating layer 22, the first insulating layer 21 covering the substrate 1, the sensing electrode 4, and the sacrificial structure 41 of the liquid resistance flow channel can be formed by a certain material deposition process (such as physical vapor deposition or chemical vapor deposition, etc.).

[0186] Step A203: Process the first insulating layer to expose part of the sensing electrode and the sacrificial structure of the liquid resistance flow channel at a preset position.

[0187] For example, as an example, as Figure 23 shown, in a specific embodiment of the present disclosure, the first insulating layer can be processed by a certain pattern transfer method (such as photolithography, etc.) and / or a certain etching process (such as reactive ion etching, etc.) to expose part of the sensing electrode 4 and the sacrificial structure 41 of the liquid resistance flow channel at a preset position.

[0188] Step A204: Form a second insulating layer on the first insulating layer, and form corresponding single-hole liquid storage cavities and bonding holes for the second buffer liquid flow channel for each molecule detection unit in the molecule detection chip in the second insulating layer.

[0189] For example, as an example, as Figure 24 shown, in a specific embodiment of the present disclosure, the second insulating layer 22 can be formed on the first insulating layer 21 by a certain material deposition process (such as physical vapor deposition or chemical vapor deposition, etc.); then, by a certain pattern transfer method (such as photolithography, etc.) and / or a certain etching process (such as reactive ion etching, etc.), corresponding single-hole liquid storage cavities 103 and bonding holes for the second buffer liquid flow channel 105 are formed for each molecule detection unit in the molecule detection chip in the second insulating layer 22. Among them, the single-hole liquid storage cavities 103 and the bonding holes for the second buffer liquid flow channel 105 are isolated from each other and do not communicate directly. For example, the single-hole liquid storage cavities 103 correspond to the positions of the sensing electrodes 4, and the bonding holes for the second buffer liquid flow channel 105 correspond to the middle positions of the sacrificial structures 41 of the liquid resistance flow channels.

[0190] Therefore, through the above steps A201 - A204, the above-mentioned insulating layer 2 and the first structural layer 2 can be formed, and the single-hole liquid storage cavities 103 for each molecule detection unit in the molecule detection chip and the bonding holes for the second buffer liquid flow channel 105 can be formed in the first structural layer 2.

[0191] Step A3: On the substrate material for the second structural layer, form a cavity of the corresponding sample flow channel and the second buffer solution flow channel for each molecule detection unit in the molecule detection chip, thereby forming the second structural layer.

[0192] For example, as an illustration, such as Figure 25 and Figure 26 shown, in a specific embodiment of the present disclosure, through a certain pattern transfer method (such as photolithography, etc.) and / or a certain material etching process (such as reactive ion etching, etc.), on the substrate material 30 (such as materials like glass, silicon dioxide, quartz, etc.) for fabricating the second structural layer, form a cavity of the corresponding sample flow channel 102 for each molecule detection unit in the molecule detection chip, and a corresponding through-hole as the second buffer solution flow channel 105, thereby forming the second structural layer 3. Among them, the sample flow channel 102 and the second buffer solution flow channel 105 are isolated from each other and do not communicate directly with each other.

[0193] Step A4: Bond the second structural layer and the first structural layer to form a bonded structure.

[0194] For example, as an illustration, such as Figure 27 shown, in a specific embodiment of the present disclosure, when the first structural layer 2 includes a first insulating layer 21 and a second insulating layer 22, and the bottom of the second structural layer 3 is embedded in the second insulating layer 22, the second structural layer 3, the first insulating layer 21, and the second insulating layer 22 can be bonded, inserting the second buffer solution flow channel of the second structural layer into the bonding hole in the first structural layer to form a bonded structure, realizing the construction of the sample flow channel 102, the single-hole liquid storage cavity 103, and the second buffer solution flow channel 105. Among them, the bottom of the second structural layer 3 passes through the second insulating layer 22 and abuts against the top of the first insulating layer 21, while the bottom of the second buffer solution flow channel 105 can communicate with the top of the liquid resistance flow channel sacrificial structure 41, and the sample flow channel 102 is located above the single-hole liquid storage cavity 103 and communicates with the single-hole liquid storage cavity 103.

[0195] Another example, as an illustration, in another specific embodiment of the present disclosure, when the bottom of the second structural layer abuts against the top of the first structural layer (or the second insulating layer) and is not embedded in the first structural layer (or the second insulating layer), the second structural layer and the first structural layer can also be bonded, directly bonding the second structural layer on the top of the first structural layer (or the second insulating layer) to form a bonded structure, which can also realize the construction of the sample flow channel 102, the single-hole liquid storage cavity 103, and the second buffer solution flow channel 105, such that the bottom of the second buffer solution flow channel 105 can communicate with the top of the liquid resistance flow channel sacrificial structure 41, and the sample flow channel 102 is located above the single-hole liquid storage cavity 103 and communicates with the single-hole liquid storage cavity 103.

[0196] Step A5: For each molecule detection unit in the molecule detection chip, form a corresponding liquid resistance flow channel in the first structural layer.

[0197] In this step, in the first structural layer 2, a corresponding liquid resistance flow channel 104 can be formed for each molecule detection unit in the molecule detection chip.

[0198] For example, as an illustration, Figure 28 As shown, in a specific embodiment of the present disclosure, a certain etching process (such as xenon fluoride silicon etching process, etc.) can be used to remove the liquid resistance flow channel sacrificial structure 41 in each molecule detection unit to form a liquid resistance flow channel 104 connecting the single-hole liquid storage cavity 103 and the second buffer solution flow channel 105.

[0199] Step A6: On the substrate material for the third structural layer, form a corresponding first buffer solution flow channel for each molecule detection unit in the molecule detection chip to form the third structural layer.

[0200] For example, as an illustration, Figure 29 As shown, in a specific embodiment of the present disclosure, through a certain pattern transfer method (such as photolithography, etc.) and / or a certain material etching process (such as reactive ion etching, etc.), on the substrate material for fabricating the third structural layer (such as materials like glass, silicon dioxide, quartz, etc.), a corresponding first buffer solution flow channel 101 is formed for each molecule detection unit in the molecule detection chip, thereby forming the third structural layer 5.

[0201] In addition, in a specific embodiment of the present disclosure, the forming of the third structural layer may further include: on the substrate material for the third structural layer (i.e., in the third structural layer 5), forming a corresponding driving electrode 6 for each molecule detection unit in the molecule detection chip.

[0202] Step A7: Bond the third structural layer and the bonding structure.

[0203] For example, as an illustration, Figure 30 As shown, in a specific embodiment of the present disclosure, the third structural layer 5 and the bonding structure including the second structural layer 3 and the first structural layer 2 can be bonded so that the second buffer solution flow channel 105 communicates with the first buffer solution flow channel 101, thereby forming the molecule detection chip.

[0204] Therefore, through the above steps A1 - A7, the required molecule detection chip can be prepared.

[0205] In addition, in the technical solution of the present disclosure, a film layer 301 may further be formed at the connection between the single-hole liquid storage cavity 103 of each molecule detection unit and the sample flow channel 102, and nanopores 302 may be formed on the film layer 301.

[0206] For example, as an example, in a specific embodiment of the present disclosure, the method for preparing the molecule detection chip may further include:

[0207] Step A8: Form a film layer at the connection between the single-hole liquid storage cavity of each molecule detection unit and the sample flow channel, and form nanopores on the film layer.

[0208] In the technical solution of the present disclosure, the film layer 301 may be formed at the connection between the two according to the positions of the single-hole liquid storage cavity 103 and the sample flow channel 102 of each molecule detection unit; then, the corresponding nanopores 302 may be formed on the film layer 301.

[0209] In addition, in the technical solution of the present disclosure, there is also provided the use of the molecule detection unit, molecule detection chip, method for preparing the molecule detection chip, or the chip prepared by the method for preparing the molecule detection chip in any one of the above embodiments in preparing a nanopore sensor or in nanopore characterization of an analyte. The above molecule detection unit, molecule detection chip, method for preparing the molecule detection chip, and the chip prepared by the method for preparing the molecule detection chip can be widely applied to the preparation of a nanopore sensor or for nanopore characterization of an analyte, and have great application potential in the field of biological detection technology.

[0210] In summary, in the technical solution of the present disclosure, since the first buffer flow channel, the sample flow channel, and the single-hole liquid storage cavity are respectively arranged in different layers, and the first buffer flow channel is located in the third structural layer above the sample flow channel and the single-hole liquid storage cavity, the arrangement density of nanopore devices per unit area can be effectively increased, and the throughput of the nanopore sequencing device can be greatly improved, so that ultra-high-throughput nanopore gene sequencing can be achieved. In addition, since the above molecule detection unit is a stacked multi-layer structure, the structures of each layer can be separately fabricated using mature semiconductor processing techniques, and then the structures of each layer can be formed into the above multi-layer structure by bonding, thereby effectively improving the production and manufacturing efficiency.

[0211] In addition, since a multi-layer flow channel is used to construct two independent micro-channel systems, and the two systems can be interconnected by removing the sacrificial structure of the liquid resistance flow channel between them before final application, enabling the fabrication of the liquid resistance flow channel and the buffer flow channel on the same side of the substrate, such that the single-hole liquid storage cavity, the liquid resistance flow channel, the buffer flow channel, and the sample flow channel are all located on the same side of the substrate. Therefore, the liquid resistance flow channel and the buffer flow channel can be constructed on the same surface of the substrate, effectively improving the chip fabrication efficiency based on the voltage sequencing method and further enhancing the throughput of the nanopore sequencing device.

[0212] Moreover, in the technical solution of the present disclosure, since the sample flow channel and the buffer flow channel are isolated from each other, different voltages can be applied to the different liquids in the two flow channels, thus ensuring the realization of the basic principle of voltage sequencing. Additionally, the above two separated flow channels can also greatly reduce cross-contamination and leakage between samples and improve the signal-to-noise ratio of the signal.

[0213] The above are only the preferred embodiments of the present disclosure and are not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present disclosure shall be included within the scope of protection of the present disclosure.

Claims

1. A molecular detection unit, characterized in that, The molecular detection unit includes: a substrate, a first structural layer, a second structural layer, sensing electrodes, a third structural layer, a first buffer liquid flow channel, a sample flow channel, a single-hole liquid storage cavity, a liquid resistance flow channel, and a second buffer liquid flow channel; The first structural layer is disposed on the top of the substrate; The second structural layer is disposed above the first structural layer; The third structural layer is disposed above the second structural layer; The liquid resistance flow channel and the single-hole liquid storage cavity are disposed in the first structural layer; The bottom of the single-hole liquid storage cavity communicates with one end of the liquid resistance flow channel; The sample flow channel is disposed in the second structural layer, above the single-hole liquid storage cavity and communicates with the single-hole liquid storage cavity; The first buffer liquid flow channel is disposed in the third structural layer; The bottom of the second buffer liquid flow channel communicates with the other end of the liquid resistance flow channel; the top of the second buffer liquid flow channel communicates with the first buffer liquid flow channel; The sensing electrodes are disposed at the connection of the single-hole liquid storage cavity and the liquid resistance flow channel.

2. The molecular detection unit according to claim 1, wherein The molecular detection unit further includes: a membrane layer; The membrane layer is disposed at the connection of the sample flow channel and the single-hole liquid storage cavity; Nanopores are disposed on the membrane layer.

3. The molecular detection unit according to claim 1, wherein The molecular detection unit further includes: driving electrodes; The driving electrodes are disposed in the third structural layer and connected to the first buffer liquid flow channel.

4. The molecular detection unit according to claim 1, wherein The molecular detection unit further includes: two sample inlets and outlets and two buffer inlets and outlets; The two sample solution inlets and outlets communicate with the sample flow channel respectively; The two buffer inlets and outlets communicate with the first buffer liquid flow channel respectively.

5. The molecular detection unit according to claim 4, wherein, The molecular detection unit further includes: a sample flow channel interface and a buffer liquid flow channel interface; The sample flow channel interface is disposed through the sample solution inlet and outlet, and the top of the sample flow channel interface extends out from the top of the sample solution inlet and outlet; the bottom of the sample flow channel interface communicates with the sample flow channel; The buffer liquid flow channel interface is disposed through the buffer inlet and outlet, and the top of the buffer liquid flow channel interface extends out from the top of the buffer inlet and outlet; the bottom of the buffer liquid flow channel interface communicates with the first buffer liquid flow channel.

6. The molecular detection unit according to claim 5, wherein: A first driving electrode is disposed in the sample flow channel interface, and a second driving electrode is disposed in the buffer liquid flow channel interface.

7. A molecular detection chip, characterized in that, The molecular detection chip includes: a molecular detection array; The molecular detection array includes at least one group of molecular detection units; each group of molecular detection units includes a plurality of molecular detection units according to any one of claims 1 to 6; Wherein, the sample flow channels of each molecular detection unit communicate with each other to form a common sample flow channel; and / or, The first buffer liquid flow channels of each molecular detection unit communicate with each other to form a first common buffer liquid flow channel; and / or, The second buffer liquid flow channels of each molecular detection unit communicate with each other to form a second common buffer liquid flow channel.

8. The molecular detection chip according to claim 7, wherein The molecular detection chip further includes: two common sample solution inlets and outlets and / or two common buffer inlets and outlets; The two common sample solution inlets and outlets communicate with the common sample flow channel respectively; The two common buffer inlets and outlets are respectively communicated with the first common buffer channel.

9. The molecular detection chip according to claim 8, wherein The molecular detection chip further includes: a common sample channel interface and / or a common buffer channel interface; The common sample channel interface is disposed in the common sample solution inlet and outlet, and the top of the common sample channel interface extends from the top of the common sample solution inlet and outlet; the bottom of the common sample channel interface is communicated with the common sample channel. The common buffer channel interface is disposed in the common buffer inlet and outlet, and the top of the common buffer channel interface extends from the top of the common buffer inlet and outlet; the bottom of the common buffer channel interface is communicated with the first common buffer channel.

10. The molecular detection chip according to claim 9, wherein: A first common driving electrode is disposed in the common sample channel interface; A second common driving electrode is disposed in the common buffer channel interface.

11. The molecular detection chip according to claim 7, wherein: The first buffer channels in the respective molecular detection units in the molecular detection array are interconnected to form a first common buffer channel, The second buffer channels in the respective molecular detection units in the molecular detection array are respectively communicated with the first common buffer channel.

12. The molecular detection chip according to claim 7, wherein: A plurality of molecular detection units in the same molecular detection unit group are uniformly arranged around a common center.

13. The molecular detection chip according to claim 12, wherein: The shapes of the sample channels, the first buffer channels and / or the second buffer channels in the respective molecular detection units are cylindrical.

14. The molecular detection chip according to claim 7, wherein: A plurality of molecular detection units in the same molecular detection unit group form a linear arrangement in sequence.

15. The molecular detection chip according to claim 14, wherein: When the molecular detection array includes one or more molecular detection unit groups, the number of the common sample channels and / or the first common buffer channels in the molecular detection array is equal to the number of the molecular detection unit groups in the molecular detection array.

16. The molecular detection chip according to claim 14, wherein: When the molecular detection array includes a plurality of molecular detection unit groups, the number of the common sample channels and / or the first common buffer channels in the molecular detection array is less than the number of the molecular detection unit groups in the molecular detection array.

17. A method for preparing a molecular detection chip, characterized in that, The method includes: Preparing a substrate, and forming sensing electrodes of the respective molecular detection units in the molecular detection chip on the top of the substrate; Forming a first structural layer on the substrate, and forming corresponding single-hole liquid storage cavities and bonding holes for the second buffer channels for each molecular detection unit in the molecular detection chip in the first structural layer; On the substrate material for the second structural layer, forming cavities of the corresponding sample channels and second buffer channels for each molecular detection unit in the molecular detection chip to form a second structural layer; Bond the second structural layer and the first structural layer to form a bonded structure; In the first structural layer, a corresponding liquid resistance flow channel is formed for each molecule detection unit in the molecule detection chip; On the substrate material for the third structural layer, a corresponding first buffer solution flow channel is formed for each molecule detection unit in the molecule detection chip to form the third structural layer; Bond the third structural layer and the bonded structure.

18. The method according to claim 17, wherein The forming the first structural layer on the substrate and forming a corresponding single-hole liquid storage cavity and a bonding hole for the second buffer solution flow channel for each molecule detection unit in the molecule detection chip in the first structural layer includes: When the first structural layer includes a first insulating layer and a second insulating layer, for each molecule detection unit in the molecule detection chip, a liquid resistance flow channel sacrificial structure is formed at a preset position on the substrate and the sensing electrode; Form a first insulating layer on the substrate, the sensing electrode and the liquid resistance flow channel sacrificial structure; Process the first insulating layer to expose part of the sensing electrode and the liquid resistance flow channel sacrificial structure at a preset position; Form a second insulating layer on the first insulating layer, and form a corresponding single-hole liquid storage cavity and a bonding hole for the second buffer solution flow channel for each molecule detection unit in the molecule detection chip in the second insulating layer.

19. The method according to claim 17, characterized in that The method further includes: Form a film layer at the connection between the single-hole liquid storage cavity of each molecule detection unit and the sample flow channel, and form a nanopore on the film layer; and / or, The forming the third structural layer further includes: forming a corresponding driving electrode for each molecule detection unit in the molecule detection chip on the substrate material for the third structural layer.

20. Use of the molecule detection unit, the molecule detection chip, the preparation method of the molecule detection chip, or the chip prepared by the preparation method of the molecule detection chip according to any one of claims 1 to 19 in preparing a nanopore sensor or in nanopore characterization of an analyte.

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