Molecular detection unit and chip, preparation method, and use

Abstract

Disclosed in the present disclosure are a molecular detection unit and chip, a preparation method, and a use. The molecular detection unit comprises: a substrate, a first structural layer, second structural layers, sensing electrodes, third structural layers, first buffer solution flow channels, sample flow channels, single-hole liquid storage cavities, liquid resistor flow channels, and second buffer solution flow channels; the liquid resistor flow channels and the single-hole liquid storage cavities are provided in the first structural layer; the bottom of the corresponding single-hole liquid storage cavity is communicated with one end of the corresponding liquid resistor flow channel; the sample flow channels are arranged in the second structural layers and are communicated with the single-hole liquid storage cavities; the first buffer solution flow channels are provided in the third structural layers; the bottom of the corresponding second buffer solution flow channel is communicated with the other end of the corresponding liquid resistor flow channel; the top of the corresponding second buffer solution flow channel is communicated with the corresponding first buffer solution flow channel; and the corresponding sensing electrode is arranged at the position where the corresponding single-hole liquid storage cavity is communicated with the corresponding liquid resistor flow channel. The application of the present disclosure can improve the arrangement density of nanopore devices per unit area, so that the chip manufacturing efficiency can be improved, thereby improving the throughput of nanopore sequencing devices.

Description

A molecular detection unit, chip, preparation method and application Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a molecular detection unit, chip, preparation method, and application. Background Technology

[0002] Nanopore sequencing devices are devices that determine the base pair sequence of a DNA molecule by detecting changes in electrical signals generated when a biomolecule (e.g., a DNA molecule) passes through a nanopore. These devices typically consist of a thin film that isolates two liquid reservoirs and a nanopore embedded within the film. The nanopore can be a naturally occurring or artificially synthesized protein molecule with a porous structure (e.g., nanoporous proteins), or it can be created on the film using physical processing methods such as ion beams. When sequencing a biomolecule (e.g., a DNA molecule) using a nanopore sequencing device, the liquid reservoirs on either side of the film are at different potentials to drive the DNA molecule through the nanopore. As the target DNA passes through the nanopore, the electrical signals between the two liquid reservoirs are simultaneously recorded. By analyzing these signals, the DNA structure can be sequenced.

[0003] In existing technologies, nanopore devices primarily employ a method of monitoring the ionic current changes between two reservoir chambers to calculate the base pair sequence of the target DNA. Stretched DNA molecules are relatively long, requiring a certain amount of time to pass through the nanopore. Within this specific timeframe, only a small portion of the DNA molecule's base sequence remains within the nanopore. Since each base has a different effect on the magnitude of the ionic current change, the magnitude of the ionic current varies accordingly due to the changes in this small segment of the base sequence within the nanopore. Therefore, by analyzing the changes in ionic current throughout the DNA's passage through the nanopore, the base sequence of the target DNA can be calculated using appropriate computational methods.

[0004] When using ion current-based sequencing methods, nanopore devices of this type require high accuracy in ion current detection. Existing bio-nanopore sequencing devices utilize nanoporous proteins with a resistance of approximately 1 gigaohm in the sequencing environment, resulting in an ion current of about 100 picoamperes during sequencing. Therefore, variations in device structure and testing environment significantly impact the accuracy of ion current measurement, thus placing high demands on the amplification performance and stability of the device circuitry. Furthermore, the measurement of ion current is affected by the electrode area size, limiting the total number of nanopore devices per unit area and consequently restricting the throughput of commercially available nanopore devices.

[0005] In addition, in the voltage sequencing method based on the voltage divider principle in the existing technology, a voltage divider is constructed by adding a buffer channel connected to the liquid reservoir on one side of the membrane with embedded nanopores. This allows the device to obtain the resistance change in the nanopore by detecting the potential change in the liquid reservoir on one side of the membrane, and then calculate the base sequence of the target DNA by corresponding methods.

[0006] In existing technologies, liquid resistance channels and buffer solutions are typically fabricated using substrate vias. However, in the field of micro / nano fabrication, common via fabrication methods have certain requirements regarding material thickness and via geometry. Most existing nanopore sequencing devices are based on silicon wafers, with thicknesses typically ranging from several hundred micrometers, while the required pore diameters are often in the range of a few micrometers. In existing, relatively mature high aspect ratio micro / nano fabrication technologies, due to factors such as loading effects and mask layer area, only deep-hole etching with a maximum aspect ratio of 15:1 can be effectively achieved in actual production. Therefore, how to effectively fabricate such substrate via structures has become a crucial technical challenge for the fabrication and mass production of voltage-based sequencing chips. Summary of the Invention

[0007] Embodiments of this disclosure provide a molecular detection unit, a chip, a preparation method, and its applications.

[0008] In a first aspect, embodiments of this 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 channel, a sample channel, a single-well reservoir, a liquid resistance channel, and a second buffer channel.

[0009] The first structural layer is disposed on 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 chamber is connected to one end of the liquid resistance flow channel;

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

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

[0016] The bottom of the second buffer solution channel is connected to the other end of the liquid resistance channel; the top of the second buffer solution channel is connected to the first buffer solution channel.

[0017] The sensing electrode is disposed at the connection between the single-hole liquid storage chamber and the liquid resistance flow channel.

[0018] Secondly, embodiments of this disclosure provide a molecular detection chip, which includes a molecular detection array;

[0019] The molecular detection array includes at least one group of molecular detection units; each group of molecular detection units includes multiple molecular detection units as described in any one of the above-mentioned items.

[0020] The sample channels of each molecular detection unit are interconnected to form a common sample channel; and / or,

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

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

[0023] Thirdly, embodiments of this disclosure provide a method for fabricating a molecular detection chip, the method comprising:

[0024] A substrate was prepared, and sensing electrodes for each molecular detection unit in the molecular detection chip were formed on the top of the substrate.

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

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

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

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

[0029] On the substrate material used 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] Bonding is performed on the third structural layer and the bonding structure.

[0031] Fourthly, the embodiments of this disclosure provide the use of the molecular detection unit, molecular detection chip, method for preparing the molecular detection chip, or chip prepared by the method for preparing the molecular detection chip in any of the above embodiments in the preparation of nanopore sensors or in the characterization and analysis of nanopores.

[0032] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0033] The accompanying drawings are provided to better understand this solution and do not constitute a limitation of this disclosure. Wherein:

[0034] Figure 1 is a schematic diagram of the structure of a molecular detection unit in a specific embodiment of this disclosure.

[0035] Figure 2 is a schematic diagram of the AA' section of Figure 1.

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

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

[0038] Figure 5 is a schematic diagram of the structure of a molecular detection unit with a membrane layer in a specific embodiment of this disclosure.

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

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

[0041] Figure 8 is a schematic diagram of the BB' cross-section in Figure 7.

[0042] Figure 9 is a schematic diagram of the structure of a molecular detection unit with a membrane layer in another specific embodiment of this disclosure.

[0043] Figure 10 is a schematic diagram of the structure of a molecular detection array in a specific embodiment of this disclosure.

[0044] Figure 11 is a schematic diagram of the CC' section of Figure 10.

[0045] Figure 12 is a schematic diagram of the molecular detection array in another specific embodiment of this disclosure.

[0046] Figure 13 is a schematic diagram of the DD' cross-section in Figure 12.

[0047] Figure 14 is a schematic diagram of the structure of a molecular detection array with a membrane layer in another specific embodiment of this disclosure.

[0048] Figure 15 is a schematic diagram of the molecular detection array in another specific embodiment of this disclosure.

[0049] Figure 16 is a schematic diagram of the EE' section of Figure 15.

[0050] Figure 17 is a schematic diagram of the structure of a molecular detection array with a membrane layer in another specific embodiment of this disclosure.

[0051] Figure 18 is a schematic diagram of a molecular detection array in another specific embodiment of this disclosure.

[0052] Figure 19 is a schematic flowchart of the preparation method of the molecular detection chip in a specific embodiment of this disclosure.

[0053] Figure 20 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0054] Figure 21 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0055] Figure 22 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0056] Figure 23 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0057] Figure 24 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0058] Figure 25 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0059] Figure 26 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0060] Figure 27 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0061] Figure 28 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0062] Figure 29 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure.

[0063] Figure 30 is a schematic diagram of the fabrication process of the molecular detection chip in a specific embodiment of this disclosure. Detailed Implementation

[0064] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of this 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 art to which this disclosure pertains.

[0065] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this disclosure. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0066] Where there is no conflict, the embodiments and features described herein can be combined with each other.

[0067] To make the technical solutions and advantages of this disclosure clearer, the following description, in conjunction with the accompanying drawings and specific embodiments, will provide a more detailed account of this disclosure.

[0068] This disclosure presents a molecular detection unit.

[0069] As shown in Figures 1 and 2, in a specific embodiment of this 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 channel 101, a sample channel 102, a single-well liquid storage chamber 103, a liquid resistance channel 104, and a second buffer channel 105.

[0070] The first structural layer 2 is disposed on 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 flow 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 chamber 103 is connected to one end of the liquid resistance flow channel 104;

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

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

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

[0078] The sensing electrode 4 is disposed at the connection between the single-hole liquid storage chamber 103 and the liquid resistance flow channel 104.

[0079] In addition, in the technical solution disclosed herein, corresponding membrane layers and nanopores can be further set in the above-mentioned molecular detection unit according to the needs of actual application scenarios.

[0080] The membrane layer separates the single-pore liquid storage chamber from the sample channel, and the nanopores enable liquid communication between the sample channel and the single-pore liquid storage chamber.

[0081] For example, as shown in FIG5, in a specific embodiment of this disclosure, the molecular detection unit described above may further include: a membrane layer 301;

[0082] The membrane layer 301 is disposed at the connection between the sample flow channel 102 and the single-pore liquid storage chamber 103; the membrane layer 301 is provided with nanopores 302.

[0083] In addition, in the technical solution disclosed herein, different membrane layers can be formed in the molecular detection unit as needed for actual application scenarios.

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

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

[0086] Additionally, as an example, as shown in Figures 3 and 4, in a specific embodiment of this 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 top of the first insulating layer 21;

[0089] The liquid resistance 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 disclosed herein, a first insulating layer 21 and a second insulating layer 22 can be further provided in the first structural layer 2, and then the liquid resistance flow channel 104 and the single-hole liquid storage cavity 103 are respectively provided in the first insulating layer 21 and the second insulating layer 22.

[0092] In addition, in the technical solution disclosed herein, the specific positional relationship between the second structural layer 3 and the second buffer channel 105 can be set according to the needs of the actual application scenario.

[0093] For example, as shown in FIG3, in a specific embodiment of this disclosure, the second structural layer 3 may be disposed on top of the second insulating layer 22;

[0094] The second buffer solution channel 105 penetrates 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 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 example, as shown in FIG4, 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 solution channel 105 penetrates 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 and passes through the second insulating layer 22 to abut against the top of the first insulating layer 21.

[0099] Additionally, as an example, as shown in FIG5, in a specific embodiment of this disclosure, the molecular detection unit described above may further include: a driving electrode 6;

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

[0101] Before using the aforementioned molecular detection unit, considering storage and transportation requirements, buffer solutions (a polar solution containing the required electrolyte) can be injected into and filled into the sample channel 102, the first buffer solution channel 101, and the second buffer solution channel 105, respectively. Since the single-well reservoir 103 is connected to the second buffer solution channel 105 through the liquid resistance channel 104, the single-well reservoir 103 and the liquid resistance channel 104 will also be filled with buffer solutions.

[0102] When the aforementioned molecular detection unit is required, the 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 positive or negative pressure provided by an external pump. For example, when a positive pressure is used to drive the sample solution into the sample channel 102, the sample solution in the sample solution storage device (e.g., storage bottle, etc.) will enter the sample channel 102 from one end under the pressure provided by the external pump, flow through the sample channel 102 and exit from the other end of the sample channel 102, and be collected by a waste liquid collection device (e.g., waste bottle, etc.) to prevent contamination.

[0104] Under pressure, the sample solution moves within the sample channel 102 at a low Reynolds number, exhibiting a laminar flow state. Under these conditions, the sample solution will not pass through the membrane layer 301 into the single-pore reservoir 103. When there is no pressure, the membrane layer 301, acting as a thin film between the single-pore reservoir 103 and the sample channel 102, also prevents interdiffusion between the sample solution in the sample channel 102 and the buffer solution in the single-pore reservoir 103.

[0105] When the aforementioned molecular detection unit is in operation, a voltage can be applied to one side of the sample channel 102 (e.g., applying a voltage to the sample solution in the sample channel 102, denoted as V3, as shown in Figure 5), and a voltage can be applied to one side of the first buffer channel 101 (e.g., applying a voltage to the buffer solution in the first buffer channel 101 via the driving electrode 6, denoted as V2, as shown in Figure 5). Since the first buffer channel 101 can be connected to the single-well reservoir 103 via the second buffer channel 105 and the liquid resistance channel 104, the voltage V1 finally applied to the single-well reservoir 103 will have a certain difference from the voltage V2. Because different voltages can be applied to different liquids, the basic principle of voltage sequencing can be guaranteed. Therefore, only when the preset voltages V2 and V3 are applied to the buffer solution and sample solution will the electrolyte and target sample molecules pass through the nanopore 302 and the liquid resistance channel 104 under the action of the electric field force.

[0106] When DNA molecules 303 in the sample solution pass through nanopores (e.g., nanoporous proteins) 302 embedded in the membrane layer 301, the resistance R1 of the nanopores 302 can be considered a variable resistance as the DNA molecules 303 pass through, while the resistance of the liquid resistance channel 104 can be considered a fixed resistance. As the base sequence of the DNA molecules 303 passes through the nanopores 302 sequentially, the resistance R1 changes accordingly, causing a change in the voltage V1 between the two resistances. Therefore, based on the change pattern of the voltage V1 detected by the sensing electrode 4, the base arrangement of the DNA molecules 303 can be calculated, thus enabling the sequencing of the DNA molecules 303.

[0107] For example, in another specific embodiment of this disclosure, the resistance between voltages V2 and V3 is mainly composed of the resistance R1 of the nanopore-embedded membrane 301 and the resistance R2 of the liquid resistive flow channel 2. Voltage V1 is acquired and amplified by an amplification circuit 43 and transmitted and recorded in the electrical signal database of the sequencing system 44 (e.g., a computer), as shown in Figure 6. Since DNA molecules pass through the nanopores during sequencing, and the DNA base combinations passing through the nanopores at different times differ, the overall resistance R1 of the nanopore-embedded membrane 301 changes over time; that is, resistance R1 can be considered a variable resistance. Therefore, the total resistance of the nanopore-embedded membrane 301 and the current base combinations passing through the nanopores can be determined by the voltage V1 at a given time. Then, using a deep learning algorithm, the base sequence of the DNA molecule passing through the pore protein can be calculated.

[0108] In the molecular detection unit disclosed herein, the first buffer channel 101 is disposed in the third structural layer 5, the sample channel 102 is disposed in the second structural layer 3, and the single-well reservoir 103 is disposed in the first structural layer 2, forming a stacked multilayer structure. Since the first buffer channel 101, sample channel 102, and single-well reservoir 103 are disposed in different layers, and the first buffer channel 101 is located in the third structural layer 5 above the sample channel 102 and the single-well reservoir 103, the arrangement density of nanopore devices per unit area can be effectively increased (for example, the channel density of the nanopore chip can be increased to the level of 100,000 devices per square centimeter), greatly increasing the throughput of the nanopore sequencing device, thereby enabling ultra-high-throughput nanopore gene sequencing. Furthermore, since the molecular detection unit is a stacked multilayer structure, each layer can be fabricated separately using mature semiconductor processing technology, and then the layers can be bonded together to form the multilayer structure, thereby effectively improving manufacturing efficiency.

[0109] In addition, the single-well liquid storage chamber 103, liquid resistance channel 104, first buffer channel 101, second buffer channel 105 and sample channel 102 in the above molecular detection unit are all located on the same side of the substrate 1. Therefore, liquid resistance channels and buffer channels can be constructed on the same surface of the substrate, which can further improve the chip fabrication efficiency of voltage-based sequencing methods and further increase the throughput of nanopore sequencing devices.

[0110] Furthermore, in the molecular detection unit described above in this disclosure, the sample channel 102 and the first buffer channel 101 are isolated from each other. The sample solution can flow in from one end of the sample channel 102 and then flow out from the other end of the sample channel 102 without entering the first buffer channel 101. Similarly, the buffer solution can flow in from one end of the first buffer channel 101 and then flow out from the other end of the first buffer channel 101 without entering the sample channel 102.

[0111] Because the two flow channels are separate, different voltages can be applied to different liquids within each channel, thus ensuring the implementation of the basic principle of voltage sequencing. Furthermore, the separation of the two flow channels significantly reduces cross-contamination and leakage between samples, improving the signal-to-noise ratio.

[0112] Additionally, as an example, as shown in Figures 7 and 8, in a specific embodiment of this disclosure, the molecular detection unit may further include: two sample solution inlets / outlets 1021 and two buffer solution inlets / outlets 1011;

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

[0114] The two buffer inlets and outlets 1011 are respectively connected to the first buffer channel 101.

[0115] For example, as shown in Figures 7 and 8, the sample solution inlet / outlet 1021 can penetrate the third structural layer 5 and communicate with the sample flow channel 102 in the second structural layer 3; while the buffer solution inlet / outlet 1011 can be located in the third structural layer 5 and communicate with the first buffer solution flow channel 101 in the third structural layer 5.

[0116] In the aforementioned molecular detection unit, the sample channel 102 and two sample solution inlets / outlets 1021 constitute a sample microfluidic system, while the first buffer channel 101, the second buffer channel 105, and two buffer inlets / outlets 1011 constitute a buffer microfluidic system. Liquids in both microfluidic systems enter from one inlet / outlet and exit from the other, remaining isolated from each other. For example, the sample solution will only enter the sample channel 102 from one sample solution inlet / outlet 1021 and then exit from the other sample solution inlet / outlet 1021; during this process, the sample solution will not enter the buffer microfluidic system. Similarly, the buffer solution can enter the first buffer channel 101 and the second buffer channel 105 from one buffer inlet / outlet 1011 and then exit the first buffer channel 101 from the other buffer inlet / outlet 1011; during this process, the buffer solution in the buffer microfluidic system will not enter the sample microfluidic system.

[0117] Because the two microfluidic systems described above are separate, different voltages can be applied to different liquids within each system, thus ensuring the implementation of the basic principle of voltage sequencing. Furthermore, the separation of the two microfluidic systems significantly reduces cross-contamination and leakage between samples, improving the signal-to-noise ratio.

[0118] Additionally, as an example, as shown in FIG9, in a specific embodiment of this disclosure, the above-mentioned molecular detection unit may further include: a sample flow channel interface 12 and a buffer flow channel interface 11.

[0119] The sample flow channel interface 12 is inserted into the sample solution inlet / outlet 1021, with the top of the sample flow channel interface 12 extending from the top of the sample solution inlet / outlet 1021; the bottom of the sample flow channel interface 12 is connected to the sample flow channel 102.

[0120] The buffer solution channel interface 11 is inserted into the buffer solution inlet / outlet 1011, and the top of the buffer solution channel interface 11 extends out from the top of the buffer solution inlet / outlet 1011; the bottom of the buffer solution channel interface 11 is connected to the first buffer solution channel 101.

[0121] In the technical solution disclosed herein, 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 be connected to the sample solution inlet / outlet 1021, and the buffer flow channel interface 11 can be connected to the buffer inlet / outlet 1011.

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

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

[0124] In addition, in the technical solution disclosed herein, 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 one specific embodiment of this disclosure, the shape of the liquid resistance channel 104 can be straight (as shown in Figure 1), curved, arc-shaped, or circumferential.

[0126] For example, in one specific embodiment of this disclosure, the liquid resistance channel 104 may be partially curved or arc-shaped, while the other part may be straight.

[0127] In addition, a molecular detection chip is also proposed in the technical solution disclosed herein.

[0128] For example, as an example, in another specific embodiment of this 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 multiple molecular detection units; the molecular detection units can be the molecular detection units in any of the above embodiments.

[0130] 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] The molecular detection chip may include a molecular detection array, and one or more molecular detection unit groups may be set in the molecular detection array, and each molecular detection unit group may contain multiple molecular detection units.

[0134] Since the sample channels in each molecular detection unit are all located in the same structural layer (i.e., the second structural layer), the sample channels of each molecular detection unit in each molecular detection unit group can be interconnected according to their respective arrangement order, forming a common sample channel in the second structural layer; or, the sample channels of all molecular detection units in the molecular detection array can also be interconnected, forming a common sample channel in the second structural layer. Similarly, since the first buffer channels in each molecular detection unit are all located in the same structural layer (i.e., the third structural layer), the first buffer channels of each molecular detection unit in each molecular detection unit group can also be interconnected according to their respective arrangement order, forming a first common buffer channel in the third structural layer; or, the first buffer channels of all molecular detection units in the molecular detection array can also be interconnected, forming 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 order, forming a second common buffer channel in the second structural layer; or, the second buffer channels of all molecular detection units in the molecular detection array can also be interconnected, forming a second common buffer channel in the second structural layer. Therefore, each molecular detection unit in each molecular detection unit group or in the entire molecular detection array can share the aforementioned common sample channel, and / or share the aforementioned first common buffer channel, and / or share the aforementioned second common buffer channel. Thus, the required liquid (e.g., buffer solution and / or sample solution) can be injected into each molecular detection unit through the aforementioned common sample channel, or the required liquid (e.g., buffer solution) can be injected into each molecular detection unit through the aforementioned first common buffer channel.

[0135] In addition, in the technical solution disclosed herein, 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 this disclosure, a molecular detection unit group may include 2, 4, 6, 8 or more molecular detection units. In the technical solutions of this 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; therefore, they will not be listed individually here.

[0137] For example, as shown in Figure 10, in another specific embodiment of this disclosure, a molecular detection unit group may include four 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 here.

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

[0140] In other words, when all molecular detection units in a molecular detection chip share a common sample channel and a common first buffer solution channel, only two common sample solution inlets / outlets and / or two common buffer solution inlets / outlets can be set in the entire molecular detection chip. The two common sample solution inlets / outlets are connected to the shared common sample channel, and the two common buffer solution inlets / outlets are connected to the shared first common buffer solution channel, as shown in Figures 12 and 13. Therefore, it is equivalent to all molecular detection units in the molecular detection array sharing two common sample solution inlets / outlets and / or two common buffer solution inlets / outlets. When the above-mentioned molecular detection array is needed, buffer solution or sample solution can be injected into the sample channel of each molecular detection unit from one common sample solution inlet / outlet and flow out of the sample channel from the other common sample solution inlet / outlet; buffer solution can be injected into the first buffer solution channel and the second buffer solution channel of each molecular detection unit from one common buffer solution inlet / outlet and flow out of the first buffer solution channel from the other common buffer solution inlet / outlet.

[0141] Additionally, as an example, as shown in FIG14, in a specific embodiment of this disclosure, the molecular detection chip may further include: a common sample flow channel interface 501 and / or a common buffer flow channel interface 502.

[0142] The common sample flow channel interface 501 is inserted into the common sample solution inlet / outlet 51, with the top of the common sample flow channel interface 501 extending from the top of the common sample solution inlet / outlet 51; the bottom of the common sample flow channel interface 501 is connected to the common sample flow channel.

[0143] The common buffer solution channel interface 502 is inserted into the common buffer solution inlet / outlet 52, with the top of the common buffer solution channel interface 502 extending from the top of the common buffer solution inlet / outlet 52; the bottom of the common buffer solution channel interface 502 is connected to the first common buffer solution channel.

[0144] In the technical solution disclosed herein, 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 be connected to the common sample solution inlet / outlet 51, and the common buffer solution flow channel interface 502 can be connected to the common buffer solution inlet / outlet 52.

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

[0146] At this time, the aforementioned 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 for the driving electrode to be connected. Thus, the corresponding liquid can be injected into the common sample solution inlet / outlet 51 or allowed 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 common sample flow channel and the liquid in 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 allowed to flow out of the common buffer solution inlet / outlet 52 through the aforementioned common buffer solution flow channel interface 502, and a corresponding voltage V2 can be applied to the first common buffer solution flow channel and the liquid in the first buffer solution flow channel through the second common driving electrode in the common buffer solution flow channel interface 502.

[0147] Furthermore, in the technical solution disclosed herein, the second buffer channels in each molecular detection unit of the molecular detection array can be independent of each other or directly interconnected.

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

[0149] For example, in another specific embodiment of this disclosure, the first buffer channels in each molecular detection unit of 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 molecular detection unit are independently connected to the first common buffer channel. In this case, the second buffer channel in each molecular detection unit is equivalent to a hollow needle or tube extending downwards from the first common buffer channel, penetrating the second structural layer and inserted into the liquid resistance channel of each molecular detection unit, thereby connecting the liquid resistance channel of each molecular detection unit to the first common buffer channel.

[0150] Furthermore, in the technical solutions disclosed herein, multiple molecular detection units in a molecular detection unit group can be arranged in various different ways.

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

[0152] For example, in another specific embodiment of this disclosure, multiple molecular detection units in the same molecular detection unit group can be arranged in a circle or polygon (e.g., quadrilateral, pentagon, hexagon, etc.) around a common center.

[0153] Additionally, as an example, in another specific embodiment of this disclosure, when multiple molecular detection units in the same molecular detection unit group are uniformly arranged around a common center (e.g., 6 molecular detection units are uniformly arranged in a hexagon, as shown in Figures 15 and 18), the multiple molecular detection units in the same molecular detection unit group can share a first buffer channel and a second buffer channel.

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

[0155] The arrangements shown in Figures 15 and 18 can form molecular detection unit groups through hexagonal close-packing. At the same time, a vertical second buffer channel or a second common buffer channel can be set at the center of the molecular detection unit group, thereby saving the total volume occupied by the second buffer channel or the second common buffer channel and further increasing the arrangement density of nanoporous devices per unit area.

[0156] Furthermore, in the technical solution disclosed herein, since each molecular detection unit is provided with a liquid resistance channel 104, a dummy via 61 can be introduced in the array design of the molecular detection chip to achieve array uniformity. Of course, the aforementioned dummy via may not be introduced.

[0157] In addition, in the technical solution disclosed herein, the sample channels in each molecular detection unit of the molecular detection array can be independent of each other and connected to form a common sample channel through corresponding channels; or they can be directly connected to each other, with the sample channels in each molecular detection unit directly serving as part of the common sample channel.

[0158] For example, as shown in Figures 15 and 18, in another specific embodiment of this disclosure, the sample channels in each molecular detection unit can be cylindrical. Therefore, the sample channels in each molecular detection unit are independent of each other, and the sample channels are connected to each other as a common sample channel through corresponding channels (not shown in the figures) provided in the second structural layer. The shape of the sample channels described above is a non-parallel channel design. The cylindrical sample channels actually form a wide channel array of columns, rather than a series of parallel narrow channels, thereby simplifying the introduction of sample solution into the sample channels.

[0159] In addition, in the technical solution disclosed herein, the first buffer channels in each molecular detection unit of the molecular detection array can be independent of each other and connected to form a first common buffer channel through corresponding channels; or they can be directly connected to each other, with the first buffer channels in each molecular detection unit directly serving as part of the first common buffer channel.

[0160] For example, as shown in Figures 15 and 16, in another specific embodiment of this disclosure, the first buffer channel and the second buffer channel in each molecular detection unit may also be cylindrical. Multiple molecular detection units in the same molecular detection unit group share a first buffer channel and a second buffer channel; or, the first buffer channels of each molecular detection unit in the same molecular detection unit group are interconnected to form a first common buffer channel, and the second buffer channels of each molecular detection unit in the same molecular detection unit group are interconnected to form a second common buffer channel.

[0161] For example, in another specific embodiment of this disclosure, the shape of the first buffer solution channel in each molecular detection unit can also be cylindrical. The first buffer solution channels in each molecular detection unit are independent of each other, and the first buffer solution channels are connected to each other through corresponding channels (not shown in the figure) disposed in the third structural layer to form a first common buffer solution channel. The shape of this first buffer solution channel is also a non-parallel channel design, rather than a series of parallel narrow channels, which simplifies the introduction of buffer solution into the first buffer solution channel.

[0162] Additionally, as an example, in another specific embodiment of this disclosure, multiple molecular detection units in the same molecular detection unit group can be arranged in a linear array in sequence.

[0163] In this molecular detection chip, a molecular detection array can be set up, and one or more molecular detection unit groups can be set up in the molecular detection array to form a row or multiple molecular detection unit groups.

[0164] In each row of molecular detection units (i.e., a group of molecular detection units), the sample channels of each molecular detection unit can be interconnected according to their respective arrangement order, forming a common sample channel in the second structural layer; the first buffer channels of each molecular detection unit can also be interconnected according to their respective arrangement order, forming a first common buffer channel in the third structural layer. Therefore, each molecular detection unit in each row of molecular detection units can share the aforementioned common sample channel and the aforementioned first common buffer channel.

[0165] Furthermore, in the technical solution disclosed herein, a single row of molecular detection units (i.e., a group of molecular detection units) can be set in the molecular detection array according to the needs of the actual application scenario, or multiple rows of molecular detection units (i.e., multiple groups of molecular detection units) can be set in the molecular detection array. In addition, the number of common sample channels and / or first common buffer channels in the molecular detection array can be preset according to the needs of the actual application scenario.

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

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

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

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

[0170] In the technical solutions disclosed herein, the above specific embodiments can be combined in accordance with the needs of actual application scenarios to obtain various different specific implementation methods, which will not be listed one by one here.

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

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

[0173] In addition, the present disclosure also proposes a method for fabricating a molecular detection chip.

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

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

[0176] For example, as shown in FIG20, in a specific embodiment of this disclosure, a substrate layer (i.e., substrate 1) can be fabricated first using semiconductor processes. This substrate layer can be used to contain the circuit structure required for the device. Furthermore, during the fabrication of substrate 1, sensing electrodes 4 for each molecular detection unit in the molecular detection chip can be formed on the top of substrate 1 using semiconductor processes. These sensing electrodes 4 can be connected to different types of circuit boards or circuit chips through different semiconductor packaging methods (e.g., 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 further here.

[0177] Step A2: A first structural layer is formed on the substrate, and a corresponding single-well liquid reservoir and a bonding hole for the second buffer flow channel are formed in the first structural layer for each molecular detection unit in the molecular detection chip.

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

[0179] Furthermore, step A2 described above can be implemented using various methods in the technical solution disclosed herein. The following will use several specific implementation methods as examples to illustrate the technical solution of this disclosure.

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

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

[0182] For example, as shown in FIG21, in a specific embodiment of the present disclosure, for each molecular detection unit in the molecular detection chip, a liquid resistance channel sacrificial structure 41 can be formed at a predetermined position on the substrate 1 and the sensing electrode 4 (i.e., at the position where the liquid resistance channel 104 of each molecular detection unit needs to be formed) by a certain patterning transfer method (e.g., electron beam exposure or laser direct writing) and / or material deposition process (e.g., physical vapor deposition or chemical vapor deposition).

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

[0184] Step A202: A first insulating layer 21 is formed on the substrate, sensing electrode and liquid resistance channel sacrificial structure.

[0185] For example, as shown in FIG22, in a specific embodiment of this disclosure, when the first structural layer 2 includes a first insulating layer 21 and a second insulating layer 22, the first insulating layer 21 can be formed first by a certain material deposition process (e.g., physical vapor deposition or chemical vapor deposition, etc.) covering the substrate 1, the sensing electrode 4 and the liquid resistance channel sacrificial structure 41.

[0186] Step A203: The first insulating layer is processed to expose part of the sensing electrode and the sacrificial structure of the liquid resistance channel at a preset location.

[0187] For example, as shown in FIG23, in a specific embodiment of this disclosure, the first insulating layer can be processed by a certain pattern transfer method (e.g., photolithography) and / or a certain etching process (e.g., reactive ion etching) to expose part of the sensing electrode 4 and the liquid resistance channel sacrificial structure 41 at a preset location.

[0188] Step A204: A second insulating layer is formed on the first insulating layer, and a corresponding single-well liquid reservoir and a bonding hole for the second buffer solution channel are formed in the second insulating layer for each molecular detection unit in the molecular detection chip.

[0189] For example, as shown in FIG24, in a specific embodiment of this disclosure, a second insulating layer 22 can be formed on the first insulating layer 21 by a material deposition process (e.g., physical vapor deposition or chemical vapor deposition); then, by a pattern transfer method (e.g., photolithography) and / or an etching process (e.g., reactive ion etching), a corresponding single-hole liquid reservoir 103 and a bonding hole for the second buffer solution channel 105 are formed in the second insulating layer 22 for each molecular detection unit in the molecular detection chip. The single-hole liquid reservoir 103 and the bonding hole for the second buffer solution channel 105 are isolated from each other and are not directly connected. For example, the single-hole liquid reservoir 103 corresponds to the position of the sensing electrode 4, while the bonding hole for the second buffer solution channel 105 corresponds to the middle position of the liquid resistance channel sacrificial structure 41.

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

[0191] Step A3: On the substrate material used for the second structural layer, a cavity for a corresponding sample flow channel and a second buffer flow channel are formed for each molecular detection unit in the molecular detection chip, thus forming the second structural layer.

[0192] For example, as shown in Figures 25 and 26, in a specific embodiment of this disclosure, a corresponding sample channel 102 cavity and a corresponding through-hole as a second buffer solution channel 105 can be formed for each molecular detection unit in the molecular detection chip on the substrate material 30 (e.g., glass, silicon dioxide, quartz, etc.) used to fabricate the second structural layer, by means of a pattern transfer method (e.g., photolithography) and / or a material etching process (e.g., reactive ion etching, etc.), thereby forming the second structural layer 3. The sample channel 102 and the second buffer solution channel 105 are isolated from each other and are not directly connected.

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

[0194] For example, as shown in FIG27, in a specific embodiment of this 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 together. The second buffer solution channel of the second structural layer is inserted into the bonding hole in the first structural layer to form a bonded structure, thereby realizing the construction of the sample channel 102, the single-well reservoir 103, and the second buffer solution channel 105. 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 channel 105 can communicate with the top of the liquid resistance channel sacrificial structure 41. The sample channel 102 is located above and communicates with the single-well reservoir 103.

[0195] For example, in another specific embodiment of this 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, with the second structural layer directly bonded to the top of the first structural layer (or the second insulating layer) to form a bonded structure. This can also realize the construction of the sample channel 102, the single-well liquid storage cavity 103, and the second buffer solution channel 105, so that the bottom of the second buffer solution channel 105 can communicate with the top of the liquid resistance channel sacrificial structure 41, and the sample channel 102 is located above the single-well liquid storage cavity 103 and communicates with the single-well liquid storage cavity 103.

[0196] Step A5: In the first structural layer, a corresponding liquid resistance flow channel is formed for each molecular detection unit in the molecular detection chip.

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

[0198] For example, as shown in FIG28, in a specific embodiment of this disclosure, a certain etching process (e.g., silicon fluoride etching process, etc.) can be used to remove the liquid resistance channel sacrificial structure 41 in each molecular detection unit to form a liquid resistance channel 104 connecting the single-hole reservoir 103 and the second buffer channel 105.

[0199] Step A6: On the substrate material used 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.

[0200] For example, as shown in FIG29, in a specific embodiment of this disclosure, a corresponding first buffer channel 101 can be formed for each molecular detection unit in the molecular detection chip on a substrate material (e.g., glass, silicon dioxide, quartz, etc.) used to fabricate the third structural layer by a certain pattern transfer method (e.g., photolithography, etc.) and / or a certain material etching process (e.g., reactive ion etching, etc.), thereby forming the third structural layer 5.

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

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

[0203] For example, as shown in FIG30, in a specific embodiment of this disclosure, the third structural layer 5 and the above-mentioned bonding structure including the second structural layer 3 and the first structural layer 2 can be bonded together, so that the second buffer channel 105 is connected to the first buffer channel 101, thereby forming a molecular detection chip.

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

[0205] In addition, in the technical solution disclosed herein, a membrane layer 301 can be further formed at the connection between the single-hole liquid storage cavity 103 of each molecular detection unit and the sample flow channel 102, and a nanopore 302 can be formed on the membrane layer 301.

[0206] For example, as an example, in a specific embodiment of this disclosure, the method for fabricating the molecular detection chip may further include:

[0207] Step A8: A membrane layer is formed at the connection between the single-pore liquid storage chamber and the sample flow channel of each molecular detection unit, and nanopores are formed on the membrane layer.

[0208] In the technical solution disclosed herein, a membrane layer 301 can be formed at the connection between the single-pore liquid storage chamber 103 and the sample flow channel 102 of each molecular detection unit, according to their respective positions; then, a corresponding nanopore 302 is formed on the membrane layer 301.

[0209] Furthermore, this disclosure also proposes the use of the molecular detection unit, molecular detection chip, method for preparing the molecular detection chip, or chip prepared by the method described above in the fabrication of nanopore sensors or in the characterization and analysis of nanopores. The molecular detection unit, molecular detection chip, method for preparing the molecular detection chip, and chip prepared by the method described above can be widely used in the fabrication of nanopore sensors or in the characterization and analysis of nanopores, and have great application potential in the field of biodetection technology.

[0210] In summary, in the technical solution disclosed herein, since the first buffer channel, sample channel, and single-well reservoir are respectively disposed in different layers, and the first buffer channel is located in the third structural layer above the sample channel and single-well reservoir, the arrangement density of nanopore devices per unit area can be effectively increased, greatly improving the throughput of nanopore sequencing devices, thereby enabling ultra-high-throughput nanopore gene sequencing. Furthermore, since the aforementioned molecular detection unit is a stacked multilayer structure, each layer can be fabricated separately using mature semiconductor processing technology, and then the layers can be bonded together to form the aforementioned multilayer structure, thereby effectively improving manufacturing efficiency.

[0211] Furthermore, by employing multi-layered channels to construct two independent microchannel systems, and by removing the sacrificial structure of the liquid resistance channel between these two systems before final application, interconnection can be achieved. This allows the fabrication of the liquid resistance channel and buffer channel on the same side of the substrate, ensuring that the single-well reservoir, liquid resistance channel, buffer channel, and sample channel are all located on the same side of the substrate. Therefore, liquid resistance channels and buffer channels can be constructed on the same surface of the substrate, effectively improving the chip fabrication efficiency of voltage-based sequencing methods and further enhancing the throughput of nanopore sequencing devices.

[0212] Furthermore, in the technical solution disclosed herein, since the sample channel and the buffer channel are isolated from each other, different voltages can be applied to the different liquids in the two channels, thereby ensuring the realization of the basic principle of voltage sequencing. In addition, the aforementioned separation of the two channels can significantly reduce cross-contamination and leakage between samples, improving the signal-to-noise ratio.

[0213] The above description is merely a preferred embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this 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, a sensing electrode, a third structural layer, a first buffer channel, a sample channel, a single-well reservoir, a liquid resistance channel, and a second buffer channel; The first structural layer is disposed on 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 chamber is connected to one end of the liquid resistance flow channel; The sample flow channel is disposed in the second structural layer, located above the single-hole liquid storage cavity and communicating with the single-hole liquid storage cavity; The first buffer solution channel is disposed in the third structural layer; The bottom of the second buffer solution channel is connected to the other end of the liquid resistance channel; the top of the second buffer solution channel is connected to the first buffer solution channel. The sensing electrode is disposed at the connection between the single-hole liquid storage chamber and the liquid resistance flow channel.

2. The molecular detection unit according to claim 1, characterized in that, The molecular detection unit further includes: a membrane layer; The membrane layer is disposed at the connection between the sample flow channel and the single-hole liquid storage chamber; The membrane layer has nanopores.

3. The molecular detection unit according to claim 1, characterized in that, The molecular detection unit further includes: a driving electrode; The driving electrode is disposed in the third structural layer and connected to the first buffer solution channel.

4. The molecular detection unit according to claim 1, characterized in that, The molecular detection unit further includes: two sample inlet / outlet and two buffer inlet / outlet; The two sample solution inlets and outlets are respectively connected to the sample flow channel; The two buffer inlets and outlets are respectively connected to the first buffer channel.

5. The molecular detection unit according to claim 4, characterized in that, The molecular detection unit further includes: a sample flow channel interface and a buffer flow channel interface; The sample flow channel interface is inserted into the sample solution inlet / outlet, with the top of the sample flow channel interface extending from the top of the sample solution inlet / outlet; the bottom of the sample flow channel interface is connected to the sample flow channel. The buffer solution channel interface is inserted into the buffer solution inlet / outlet, with the top of the buffer solution channel interface extending from the top of the buffer solution inlet / outlet; the bottom of the buffer solution channel interface is connected to the first buffer solution channel.

6. The molecular detection unit according to claim 5, characterized in that: A first driving electrode is provided in the sample flow channel interface, and a second driving electrode is provided in the buffer 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 as described in any one of claims 1 to 6; The sample channels of each molecular detection unit are interconnected to form a common sample channel; and / or, The first buffer channels of each molecular detection unit are interconnected to form a first common buffer channel; and / or, The second buffer channels of each molecular detection unit are interconnected to form a second common buffer channel.

8. The molecular detection chip according to claim 7, characterized in that, The molecular detection chip also includes: two common sample solution inlets and / or two common buffer solution inlets and / or outlets; The two common sample solution inlets and outlets are respectively connected to the common sample flow channel; The two common buffer inlets and outlets are respectively connected to the first common buffer channel.

9. The molecular detection chip according to claim 8, characterized in that, The molecular detection chip also includes: a common sample flow channel interface and / or a common buffer flow channel interface; The common sample flow channel interface is inserted into the common sample solution inlet and outlet, with the top of the common sample flow channel interface extending from the top of the common sample solution inlet and outlet; the bottom of the common sample flow channel interface is connected to the common sample flow channel. The common buffer solution channel interface is inserted into the common buffer solution inlet and outlet, with the top of the common buffer solution channel interface extending from the top of the common buffer solution inlet and outlet; the bottom of the common buffer solution channel interface is connected to the first common buffer solution channel.

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

11. The molecular detection chip according to claim 7, characterized in that: The first buffer channels in each molecular detection unit of the molecular detection array are interconnected to form a first common buffer channel. The second buffer channel in each molecular detection unit of the molecular detection array is connected to the first common buffer channel.

12. The molecular detection chip according to claim 7, characterized in that: Multiple molecular detection units in the same molecular detection unit group are evenly arranged around a common center.

13. The molecular detection chip according to claim 12, characterized in that: The sample channel, first buffer channel, and / or second buffer channel in each molecular detection unit are cylindrical in shape.

14. The molecular detection chip according to claim 7, characterized in that: Multiple molecular detection units in the same molecular detection unit group are arranged in a linear array in sequence.

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

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

17. A method for fabricating a molecular detection chip, characterized in that, The method includes: A substrate was prepared, and sensing electrodes for each molecular detection unit in the molecular detection chip were formed on the top of the substrate. A first structural layer is formed on the substrate, and a corresponding single-well liquid reservoir and a bonding hole for the second buffer flow channel are formed in the first structural layer for each molecular detection unit in the molecular detection chip. On the substrate material used for the second structural layer, a cavity for a corresponding sample flow channel and a second buffer flow channel are formed for each molecular detection unit in the molecular detection chip, thus forming the second structural layer; The second structural layer and the first structural layer are bonded together to form a bonded structure; In the first structural layer, a corresponding liquid resistance flow channel is formed for each molecular detection unit in the molecular detection chip; On the substrate material used 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. Bonding is performed on the third structural layer and the bonding structure.

18. The method according to claim 17, characterized in that, The step of forming a first structural layer on the substrate, and forming a corresponding single-well liquid reservoir and a bonding hole for the second buffer flow channel for each molecular detection unit in the molecular 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 molecular detection unit in the molecular detection chip, a liquid resistance channel sacrificial structure is formed at a preset position on the substrate and the sensing electrode. A first insulating layer is formed on the substrate, sensing electrode, and liquid resistance channel sacrificial structure; The first insulating layer is processed to expose part of the sensing electrode and the sacrificial structure of the liquid resistance flow channel at a preset location; A second insulating layer is formed on the first insulating layer, and a corresponding single-well reservoir and a bonding hole for the second buffer solution channel are formed in the second insulating layer for each molecular detection unit in the molecular detection chip.

19. The method according to claim 17, characterized in that, The method also includes: A membrane layer is formed at the connection between the single-pore reservoir of each molecular detection unit and the sample flow channel, and nanopores are formed on the membrane layer; and / or, The formation of the third structural layer further includes: forming a corresponding driving electrode for each molecular detection unit in the molecular detection chip on the substrate material used for the third structural layer.

20. The use of the molecular detection unit, the molecular detection chip, the method for preparing the molecular detection chip, or the chip prepared by the method for preparing the molecular detection chip in the preparation of nanopore sensors or in the characterization and analysis of nanopores.

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