Micro-nanofluidic substrate, chip, preparation method and system

Abstract

A micro / nanofluidic substrate, chip, fabrication method, and system are disclosed. The micro / nanofluidic substrate includes: a substrate (9); an electrode layer (10) located on the substrate (9), the electrode layer (10) including a first electrode (3), a second electrode (4), and a control electrode (5); and a film layer (11) located on the electrode layer (10) away from the substrate (9), the film layer (11) including a groove layer (21), a nanochannel (6), and microchannels (1, 2). The groove layer (21) includes a first groove (12), the nanochannel (6) is located in the first groove (12), the orthographic projection of the nanochannel (6) on the substrate (9) at least partially overlaps with the orthographic projection of the control electrode (5) on the substrate (9), and the microchannels (1, 2) are connected to the nanochannel (6). The microchannels (1, 2) include a first microchannel (1) and a second microchannel (2), the first microchannel (1) is connected to the first electrode (3), and the second microchannel (2) is connected to the second electrode (4).

Description

Technical Field

[0001] This disclosure relates to the field of biochemical detection, and more specifically, to a micro / nanofluidic substrate, chip, preparation method, and system. Background Technology

[0002] Microfluidics and nanofluidics is an emerging and cutting-edge interdisciplinary field involving physics, chemistry, engineering, materials science, and biology. Microfluidic chips have attracted significant attention from academia and industry due to their numerous advantages, including miniaturization, integration, automation, portability, high throughput, low cost, low power consumption, and high precision, as well as their wide range of applications. Unlike microfluidic chips, microfluidic chips contain even smaller nanostructures and primarily manipulate biological or chemical samples through nanochannel structures to achieve their chip functions.

[0003] In nanopore sequencing, the charge changes caused by bases are mainly characterized by current fluctuation parameters, including the amplitude and duration of current fluctuations. However, due to the rapid movement of individual DNA molecules in the nanopore, the amplitude and duration of current fluctuations caused by differences in base size and structure are usually small and short, and they are easily affected by external current noise, ultimately resulting in insufficient detection accuracy. This undoubtedly severely limits the development of nanopore sequencing technology. Summary of the Invention

[0004] In view of this, embodiments of the present disclosure provide a micro / nanofluidic substrate, a chip, a fabrication method, and a system.

[0005] According to one aspect of this disclosure, a micro / nanofluidic substrate is provided, comprising:

[0006] Base;

[0007] An electrode layer located on the substrate, the electrode layer including a first electrode, a second electrode, and a control electrode; and

[0008] A film layer located on the electrode layer away from the substrate, the film layer comprising a grooved layer, nanochannels, and microchannels, wherein the grooved layer includes a first groove, the nanochannels are located within the first groove, the orthographic projection of the nanochannels on the substrate at least partially coincides with the orthographic projection of the control electrode on the substrate, and the microchannels are connected to the nanochannels.

[0009] The micron channel includes a first micron channel and a second micron channel. The orthographic projection of the first micron channel on the substrate is located on the first side of the orthographic projection of the nanochannel on the substrate. The first micron channel is connected to the first electrode. The orthographic projection of the second micron channel on the substrate is located on the second side of the orthographic projection of the nanochannel on the substrate. The second micron channel is connected to the second electrode. The first side and the second side are opposite sides of the orthographic projection of the nanochannel on the substrate.

[0010] For example, according to an embodiment of this disclosure, the aspect ratio of the first groove is >0.3.

[0011] For example, according to an embodiment of the present disclosure, the substrate further includes a ground electrode, which serves as a reference electrode for the first electrode, the second electrode, and the control electrode. The ground electrode is located on the side of the film layer away from the substrate, and the orthographic projection of the ground electrode on the substrate at least partially overlaps with the orthographic projection of the control electrode on the substrate.

[0012] For example, according to an embodiment of this disclosure, a second material layer is filled between the first electrode, the second electrode, and the control electrode.

[0013] For example, according to an embodiment of the present disclosure, the substrate includes a first electrode, a second electrode, a control electrode, and M nanochannels, wherein M ≥ 1.

[0014] For example, according to an embodiment of the present disclosure, the substrate includes M first electrodes, M second electrodes, 1 control electrode, and M nanochannels, wherein M ≥ 2.

[0015] For example, according to an embodiment of the present disclosure, the substrate includes one first electrode, one second electrode, M control electrodes and M nanochannels, wherein M ≥ 2.

[0016] For example, according to an embodiment of the present disclosure, the substrate includes M first electrodes, M second electrodes, M control electrodes, and M nanochannels, wherein M ≥ 2.

[0017] Another aspect of this disclosure provides a micro / nanofluidic chip, the chip comprising the aforementioned substrate.

[0018] For example, according to an embodiment of this disclosure, the chip further includes:

[0019] A cover plate located above the substrate; and

[0020] A bonding layer located above the substrate, the bonding layer being used to bond the substrate and the cover plate.

[0021] The cover plate includes:

[0022] The second groove, the orthographic projection of the second groove on the substrate covers the orthographic projection of the microchannel on the substrate;

[0023] The inlet is used to add the sample to be tested; and

[0024] The liquid outlet is used to export the sample after the test is completed.

[0025] Another aspect of this disclosure provides a method for fabricating a micro / nanofluidic substrate, comprising:

[0026] A metal film is formed on the substrate;

[0027] The metal film layer is etched to obtain a first electrode, a second electrode, and a control electrode, forming an electrode layer;

[0028] A groove layer including a first groove is prepared on the surface of the electrode layer;

[0029] A first material layer is deposited within the first groove using a ballistic deposition method to form nanochannels; and

[0030] The first material layer is etched in a direction perpendicular to the nanochannel to obtain a microchannel.

[0031] For example, according to an embodiment of this disclosure, prior to the step of preparing a groove layer including a first groove on the surface of the electrode layer, the method further includes:

[0032] A second material layer is deposited on the electrode layer to prevent the control electrode from being connected to the nanochannel or the microchannel.

[0033] For example, according to an embodiment of this disclosure, in the step of depositing a first material layer in the first groove using a ballistic deposition method, the aspect ratio of the first groove is >0.3, and the thickness of the deposited first material layer is 600 nm.

[0034] For example, according to embodiments of this disclosure, the method further includes: fabricating a ground electrode above the nanochannel.

[0035] Another aspect of this disclosure provides a method for fabricating a micro / nanofluidic chip, comprising:

[0036] The substrate is prepared according to the method described above;

[0037] A bonding layer is deposited on the substrate;

[0038] The bonding layer is etched in a direction perpendicular to the nanochannels to form microchannels; and

[0039] The bonding layer is bonded to the cover plate.

[0040] Another aspect of this disclosure provides a micro / nanofluidic system, including the aforementioned micro / nanofluidic chip and power supply.

[0041] The micro / nano fluidic substrate, chip, fabrication method, and system disclosed herein can improve detection accuracy. Attached Figure Description

[0042] The features and advantages of this disclosure will become more apparent from a detailed description of exemplary embodiments thereof with reference to the accompanying drawings.

[0043] Figure 1 A top view of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown schematically;

[0044] Figure 2 A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a first connection method is shown in which electrodes on the substrate are connected to a bonding region;

[0045] Figure 3 A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a second connection method is shown in which electrodes on the substrate are connected to the bonding region;

[0046] Figure 4 A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a third connection method is shown for connecting electrodes on the substrate to the bonding region;

[0047] Figure 5 A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a fourth connection method is shown for connecting electrodes on the substrate to the bonding region;

[0048] Figure 6 A top view of a micro / nanofluidic chip according to an embodiment of the present disclosure is shown schematically.

[0049] Figure 7A A schematic cross-sectional view of a micro / nanofluidic chip according to an embodiment of the present disclosure is shown in the direction perpendicular to the nanochannels;

[0050] Figure 7B The illustration shows a cross-sectional view of a micro / nanofluidic chip according to an embodiment of the present disclosure, parallel to the nanochannel direction;

[0051] Figure 8 A schematic flowchart illustrating the fabrication process of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown.

[0052] Figure 9AThis schematic diagram illustrates a pre-formation pattern of nanochannels according to an embodiment of the present disclosure.

[0053] Figure 9B A schematic diagram illustrating the formation of nanochannels according to an embodiment of the present disclosure is shown.

[0054] Figure 10 A schematic SEM image of nanochannels according to an embodiment of the present disclosure is shown;

[0055] Figures 11A-11E The diagrams illustrate cross-sectional views of the micro / nanofluidic substrate formed after some steps of the fabrication method of the micro / nanofluidic substrate according to embodiments of the present disclosure are performed, in the direction perpendicular to the nanochannels.

[0056] Figures 12A-12I The diagrams illustrate cross-sectional views of the micro / nanofluidic substrate formed after some steps of the fabrication method of the micro / nanofluidic substrate according to embodiments of the present disclosure are performed, in a direction parallel to the nanochannels.

[0057] Figure 13 A schematic flowchart illustrating the fabrication process of a micro / nanofluidic chip according to an embodiment of the present disclosure is shown. Detailed Implementation

[0058] The embodiments of the present disclosure will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the disclosure. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present disclosure for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of the present disclosure.

[0059] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0060] When using expressions such as "at least one of A, B, or C," it should generally be interpreted in accordance with the meaning commonly understood by those skilled in the art (e.g., "a system having at least one of A, B, or C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, features defined with "first" or "second" may explicitly or implicitly include one or more of the stated features.

[0061] Ballistic deposition refers to a deposition method in which different deposition rates are applied at different deposition locations. The deposition rate at different deposition locations can be adjusted according to the actual situation to obtain a specific deposition film shape.

[0062] Microfluidics and nanofluidics is an emerging and cutting-edge interdisciplinary field involving physics, chemistry, engineering, materials science, and biology. Microfluidic chips have attracted significant attention from academia and industry due to their numerous advantages, including miniaturization, integration, automation, portability, high throughput, low cost, low power consumption, and high precision, as well as their wide range of applications. Unlike microfluidic chips, microfluidic chips contain even smaller nanostructures and primarily manipulate biological or chemical samples through nanochannel structures to achieve chip functionality. However, the high difficulty and cost of fabricating nanochannel structures severely restricts the development of microfluidics technology and industry; therefore, to date, no commercially available products based on microfluidic chips have emerged.

[0063] Currently reported sequencing methods include:

[0064] First-generation sequencing (Sanger sequencing), also known as human genome sequencing, uses the target DNA as a template for DNA amplification. Simultaneously, a terminating nucleotide (ddNTP) interferes with this process, resulting in a large number of DNA fragments with the same starting point but different ending points. These fragments are then aligned by length using electrophoresis and sequentially passed through a laser window. Different DNA fragments produce different fluorescence at their ending points, and the DNA sequence can be obtained by analyzing the fluorescence color and the length of the DNA fragments. This method offers advantages such as long read lengths, short sequencing time, and high accuracy, but it suffers from low throughput and high cost.

[0065] Next-generation sequencing (NGS sequencing), also known as high-throughput sequencing, involves first modifying the ends of DNA fragments and adding adapters. Then, it uses PCR or other methods to amplify the DNA fragments using primers complementary to the adapters to construct a library. Finally, it uses the slight difference in the reaction system potential to perform sequencing. This method allows sequencing to occur simultaneously with synthesis and overcomes the low throughput and high cost of first-generation sequencing. However, this method generally results in shorter read lengths and longer sequencing times. Furthermore, the amplification process is time-consuming and information is easily lost.

[0066] Third-generation sequencing, also known as single-molecule or nanopore sequencing, addresses the limitations of second-generation sequencing technology, which suffers from short read lengths and long processing times. While third-generation sequencing aims for longer read lengths and shorter processing times, it is not yet fully mature, and there are significant differences between various sequencers and their sequencing principles. For example, Pacific Biosciences uses a microscope to detect the fluorescence released after the reaction of fluorescently labeled dNTPs during PCR DNA synthesis. Oxford University's sequencer, which has not yet entered production, detects nucleotides that "fall" into the micropores during DNA cleavage by exonucleases. In addition, nanopore sequencing is becoming a hot research topic. This method detects the sequence of DNA molecules by detecting the different current changes caused by the size and structure of the four bases (A, T, C, and G) as the DNA passes through nanopores. This method can sequence single DNA or RNA molecules without PCR amplification or labeling, offering relatively low-cost genotyping, high test mobility, and the potential for rapid sample processing, while also providing real-time results. In nanopore sequencing, the charge changes caused by bases are mainly characterized by current fluctuation parameters, including the current fluctuation amplitude ΔI and the current fluctuation duration Δt. However, under normal circumstances, the current fluctuation amplitude caused by the differences in base size and structure is small and the duration is short due to the fast speed of the movement of individual DNA molecules in the nanopore. Moreover, it is easily affected by external current noise, which undoubtedly severely limits the development of nanopore sequencing technology.

[0067] Therefore, embodiments of this disclosure propose a micro / nanofluidic substrate. This substrate, through the combined action of a first electrode, a second electrode, and a control electrode with the ion selectivity of nanochannels, achieves the function of slowing down the translocation of biomolecules, overcoming the problem of insufficient detection accuracy caused by the excessively rapid movement of biomolecules in traditional nanopore sequencing methods. At the same time, by integrating a chip made using this substrate with nanopores or other single-molecule sequencing units, large-scale high-throughput sequencing can be achieved.

[0068] Figure 1 A top view of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown schematically.

[0069] like Figure 1As shown, the micro / nanofluidic substrate of this disclosure includes a substrate, an electrode layer, and a film layer. Exemplarily, the substrate can be a glass substrate, or other substrates can be selected according to actual conditions; this disclosure does not limit this. The electrode layer is located on the substrate and includes a first electrode 3, a second electrode 4, and a control electrode 5. The orthogonal projection of the first electrode 3 onto the substrate is located on a first side of the orthogonal projection of the nanochannel 6 onto the substrate, and the orthogonal projection of the second electrode 4 onto the substrate is located on a second side of the orthogonal projection of the nanochannel 6 onto the substrate. The first side and the second side are opposite sides of the orthogonal projection of the nanochannel onto the substrate. The orthogonal projection of the control electrode 5 onto the substrate is located between the orthogonal projections of the first electrode 3 and the second electrode 4 onto the substrate. The film layer 11 is located on the electrode layer 10 (e.g., ...). Figure 12F As shown), the film layer has a multilayer structure. The film layer 11 includes a grooved layer 21, nanochannels 6, a first micron channel 1, and a second micron channel 2. The grooved layer 21 includes a first groove 12 (as shown). Figure 11E As shown, the first groove 12 is used to accommodate the nanochannel 6, which is located within the first groove 12. The nanochannel 6 is formed by a first material layer 19 within the first groove 12. It should be noted that the first material layer 19 includes an oxide formed from silicon and oxygen, such as silicon dioxide, etc., and the embodiments disclosed herein are not limited to this. The orthographic projection of the nanochannel 6 on the substrate at least partially coincides with the orthographic projection of the control electrode 5 on the substrate, and the microchannel is connected to the nanochannel 6. The microchannel includes a first microchannel 1 and a second microchannel 2. The orthographic projection of the first microchannel 1 on the substrate is located on a first side of the orthographic projection of the nanochannel 6 on the substrate and is connected to the first electrode 3. The orthographic projection of the second microchannel 2 on the substrate is located on a second side of the orthographic projection of the nanochannel 6 on the substrate and is connected to the second electrode 4. The first side and the second side are opposite sides of the orthographic projection of the nanochannel on the substrate.

[0070] In this embodiment of the disclosure, the orthogonal projections of the first micron channel 1 and the second micron channel 2 onto the substrate are perpendicular to the orthogonal projections of the nanochannel 6 onto the substrate, so as to facilitate the flow of the sample to be tested into the nanochannel 6.

[0071] like Figure 1 As shown, in this embodiment of the present disclosure, each chip includes multiple nanochannels 6 (horizontal, vertical, horizontal). Figure 1Only four are schematically shown in the diagram: the first micrometer channel 1 and the second micrometer channel 2 (vertical direction), wherein the nanochannels are perpendicular to the micrometer channels. For example, the length of nanochannel 6 is 40 μm, the width is 10 nm, and the depth is 60 nm, and the spacing between two adjacent nanochannels 6 is 1 mm. For example, the length of the first micrometer channel 1 and the second micrometer channel 2 is 5 mm, and the width and depth are both 50 μm.

[0072] In the embodiments of this disclosure, the aspect ratio of the first groove 12 is >0.3, wherein the groove layer 21 can be prepared by methods such as nanoimprinting or EBL.

[0073] In embodiments of this disclosure, the substrate further includes a ground electrode 13, which serves as a reference electrode for the first electrode 3, the second electrode 4, and the control electrode 5. The voltage applied to the first electrode 3, the second electrode 4, and the control electrode 5 is adjusted based on the potential of the ground electrode 13. The ground electrode 13 is located on the side of the film layer 11 away from the substrate 9 (e.g., ...). Figure 12F and 12G As shown), the orthographic projection of the ground electrode 13 onto the substrate at least partially overlaps with the orthographic projection of the control electrode 5 onto the substrate (e.g., Figure 1 As shown, the size of the ground electrode 13 can be the same as or smaller than that of the control electrode 5, as long as the ground electrode 13 is not connected to the micron channel.

[0074] In embodiments of this disclosure, a second material layer 14 (such as...) is filled between the first electrode 3, the second electrode 4, and the control electrode 5. Figure 7B As shown), the second material layer 14 is used to prevent the control electrode 5 from connecting with the nanochannel 6 or the microchannel. Direct contact between the control electrode 5 and the sample solution in the microchannel or nanochannel will cause chip failure. Furthermore, in forming the first groove 12 (as shown...), Figure 7B As shown, there will be etching gaps. The second material layer 14 can fill the gaps caused by etching, effectively avoiding the loss of imprint or EBL pattern due to etching gaps.

[0075] It should be noted that the second material layer 14 includes an oxide formed from silicon and oxygen, such as silicon dioxide, etc., and the embodiments disclosed herein do not limit this.

[0076] Since most biomolecules are negatively charged, this disclosure provides two modes for mitigating the translocation of negatively charged biomolecules, taking the mitigation of translocation of negatively charged biomolecules as an example.

[0077] In Mode 1, a negative voltage is applied to the first micrometer channel 1 (first electrode 3) on the left and a positive voltage is applied to the second micrometer channel 2 (second electrode 4) on the right, creating an electric field between the two channels. This provides the electrophoretic force driving the movement of biomolecules. Simultaneously, a negative voltage is applied to the control electrode 5, and the ground electrode 13 is grounded. At this time, the control electrode 5 attracts the positive charges in the sample solution and the negative charges on the surface of the nanochannel 6 to form a double charge layer, inducing electroosmotic flow directed to the left. Since the biomolecules are negatively charged, they will move to the right under the drive of the electrophoretic force, while being dragged to the left by the frictional resistance between the water and themselves. Therefore, the translocation of biomolecules will be slowed down under the combined effect of the frictional resistance between the water and themselves and the resistance of electroosmotic flow.

[0078] In Mode 2, a negative voltage is applied to the left first microchannel 1 (first electrode 3), and a positive voltage is applied to the right second microchannel 2 (second electrode 4), forming an electric field between the two microchannels to provide the electrophoretic force driving the movement of biomolecules. Simultaneously, a positive voltage is applied to the control electrode 5, and the ground electrode 13 is grounded. When the concentration of the biological sample solution is high, although the induced electroosmotic flow direction is the same as the direction of biomolecule movement, the electroosmotic flow will be suppressed due to the thin double charge layer, and its promoting effect on biomolecule translocation is very weak. Initially, in the microchannel, biomolecules move to the right under the influence of electrophoretic force, and are also subject to frictional resistance between themselves and the sample solution. When biomolecules move into the nanochannel 6, and the distance between them and the nanochannel wall is less than the Debye length, the biomolecules will be attracted and fixed to the nanochannel wall by the electrostatic effect generated by the electrodes. At this time, they will also be subject to frictional resistance between themselves and the nanochannel wall. When thermal activation overcomes the electrostatic force, biomolecules are temporarily released from the nanochannel surface, then attracted by electrostatics and re-fixed, repeating this cycle of "fixation" → "release" → "fixation" → "release"... The biomolecules move to the right in a "viscosine" manner. The translocation is slowed by the combined effects of the frictional resistance between the biomolecules and the biological sample solution, and the frictional resistance between the biomolecules and the nanochannel walls.

[0079] In the embodiments of this disclosure, the combined effect of the first electrode, the second electrode, the control electrode, and the ion selectivity of the nanochannel achieves the function of mitigating biomolecular translocation. This overcomes the shortcomings of traditional nanopore sequencing methods, which suffer from insufficient amplitude and duration of current fluctuations caused by the rapid movement of biomolecules and are susceptible to external noise. It has the effect of mitigating biomolecular translocation and can greatly improve detection accuracy.

[0080] The following will combine Figures 2 to 5 The substrate according to embodiments of the present disclosure will be described in detail.

[0081] In embodiments of this disclosure, the substrate includes a first electrode, a second electrode, a control electrode, and M nanochannels, wherein M ≥ 1.

[0082] Figure 2 A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a first connection method is shown in which electrodes on the substrate are connected to a bonding region.

[0083] like Figure 2 As shown, the first electrode 3, the second electrode 4, the control electrode 5, and the ground electrode 13 are led out to the bonding region 20 (i.e., the bonding region) via wires. The orthographic projection of the bonding region 20 onto the substrate is located on the third side of the orthographic projection of the nanochannel 6 onto the substrate, and the third side is adjacent to the first side of the orthographic projection of the nanochannel 6 onto the substrate. By applying different voltages to the first electrode 3 and the second electrode 4, an electric field is formed between the two micrometer channels, providing the electrophoretic force to drive the movement of biomolecules. By grounding the ground electrode 13 and adjusting the charge on the nanochannel wall by applying different voltages to the control electrode 5, the translocation of biomolecules with different charges and quantities can be mitigated. After this substrate is fabricated into a chip, it can be integrated with other single-molecule sequencing units to achieve large-scale high-throughput sequencing.

[0084] In embodiments of this disclosure, the substrate includes M first electrodes, M second electrodes, 1 control electrode, and M nanochannels, wherein M ≥ 2.

[0085] Figure 3 A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a second connection method is shown in which electrodes on the substrate are connected to the bonding region.

[0086] like Figure 3 As shown, in this embodiment, both the first electrode 3 and the second electrode 4 are divided into multiple ( Figure 3 (Only four are schematically shown in the diagram), the number of which is the same as the number of nanochannels 6. The first electrode 3, second electrode 4, control electrode 5, and ground electrode 13 are led out to the bonding region 20 (i.e., the bonding region) via wires. By applying different electrical properties and voltages to different first electrodes 3 and second electrodes 4, the electrophoretic direction and size can be changed, thereby enabling the simultaneous slowing of translocation of biomolecules of different sizes and structures in multiple nanochannels 6. After this substrate is fabricated into a chip, it can be integrated with other single-molecule sequencing units to achieve simultaneous sequencing of multiple biomolecules.

[0087] In embodiments of this disclosure, the substrate includes one first electrode, one second electrode, M control electrodes, and M nanochannels, wherein M ≥ 2.

[0088] Figure 4A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a third connection method is shown for connecting electrodes on the substrate to the bonding region.

[0089] like Figure 4 As shown, in this embodiment, the control electrode 5 is divided into multiple ( Figure 4 (Only four are schematically shown in the diagram), the same number as the six nanochannels. The first electrode 3, second electrode 4, control electrode 5, and ground electrode 13 are led to the bonding region 20 (i.e., the bonding region) via wires. By applying different electrical charges and voltages to different control electrodes 5, the walls of multiple nanochannels can be charged with different electrical charges or concentrations, thereby simultaneously slowing the translocation of biomolecules of different sizes and structures within multiple nanochannels. After fabricating this substrate into a chip, it can be integrated with other single-molecule sequencing units to achieve simultaneous sequencing of multiple biomolecules.

[0090] In embodiments of this disclosure, the substrate includes M first electrodes, M second electrodes, M control electrodes, and M nanochannels, wherein M ≥ 2.

[0091] Figure 5 A schematic diagram of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown, wherein a fourth connection method is shown in which electrodes on the substrate are connected to the bonding region.

[0092] like Figure 5 As shown, in this embodiment, the control electrode 5, the first electrode 3, and the second electrode 4 are all divided into multiple ( Figure 5 (Only four are schematically shown in the diagram), the same number as the six nanochannels. The first electrode 3, second electrode 4, control electrode 5, and ground electrode 13 are led out to the bonding region 20 (i.e., the bonding region) via wires. By applying different electrical charges and voltages to different control electrodes 5, different first electrodes 3, and different second electrodes 4, the electrophoretic direction and size can be changed, causing the walls of multiple nanochannels to carry different electrical charges or concentrations. This allows for the simultaneous slowing of translocation of biomolecules of different sizes and structures within multiple nanochannels. After fabricating this substrate into a chip, it can be integrated with other single-molecule sequencing units to achieve simultaneous sequencing of multiple biomolecules.

[0093] The substrate of this disclosure can simultaneously mitigate the translocation of biomolecules of different sizes and structures by changing the electrode structures of the control electrode, the first electrode, and the second electrode, and by applying different voltages. After this substrate is fabricated into a chip, it can be integrated with other single-molecule sequencing units to simultaneously sequence multiple biomolecules.

[0094] Figure 6 A top view of a micro / nanofluidic chip according to an embodiment of the present disclosure is shown schematically.

[0095] like Figure 6 As shown, embodiments of this disclosure provide a micro / nanofluidic chip, the chip including the aforementioned substrate. The micro / nanofluidic chip according to embodiments of this disclosure includes: a first electrode 3, a second electrode 4, a control electrode 5, a nanochannel 6, a first microchannel 1, a second microchannel 2, an inlet 7, and an outlet 8. The inlet 7 is used to add the sample to be tested, and the outlet 8 is used to remove the sample after testing. The inlet 7 or the outlet 8 can be connected to a conduit for sample injection or sample removal and collection. The control electrode 5 is connected to an external power supply via internal chip wiring. After bonding and encapsulating the chip substrate with a cover plate, a micro / nanofluidic chip for slowing down the movement of biomolecules is obtained. During the use of this chip, if the electric field at the ends of the first and second electrodes is small (tens of kilovolts per meter), that is, the electric field required to drive the movement of biomolecules is small, applying a positive control electrode voltage helps to slow down the translocation of biomolecules. For example, Vg = 9V can reduce the translocation speed of biomolecules by an order of magnitude. If the electric field at the ends of the first and second electrodes is large (several thousand kilovolts per meter), applying a negative control electrode voltage helps to slow down the translocation of biomolecules. For example, Vg = -0.25V can reduce the translocation speed of biomolecules by an order of magnitude. The specific voltage value needs to be adjusted according to the actual situation, and the embodiments disclosed herein do not limit this.

[0096] Figure 7A The illustration shows a cross-sectional view of a micro / nanofluidic chip according to an embodiment of the present disclosure, perpendicular to the nanochannel direction. Figure 7B The illustration shows a cross-sectional view of a micro / nanofluidic chip according to an embodiment of the present disclosure, parallel to the nanochannel direction.

[0097] like Figure 7A and Figure 7B As shown, in an embodiment of this disclosure, the chip further includes: a cover plate 15 located above the substrate; and a bonding layer 16 located above the substrate, the bonding layer 16 being used to bond the substrate and the cover plate 15, wherein the cover plate 15 includes: a second groove 17, the orthographic projection of the second groove 17 on the substrate 9 covering the orthographic projection of the microchannel on the substrate 9.

[0098] It should be noted that the material of the bonding layer 16 includes oxides formed from silicon and oxygen, such as silicon dioxide, etc., and the embodiments of this disclosure are not limited thereto. The cover plate 15 can be a glass or PDMS cover plate, and the embodiments of this disclosure are not limited thereto.

[0099] like Figure 7AAs shown, the micro / nanofluidic chip is rectangular, for example, with dimensions of 70×40mm. Its main structure from bottom to top consists of: substrate 9, electrode layer 10, groove layer 21 prepared by nanoimprint adhesive or EBL adhesive, nanochannel 6, ground electrode 13, and cover plate 15. The first groove 12 used to prepare the nanochannel 6 can be any shape, such as rectangular or semi-circular (a rectangle is used as an example in Figure 7). The first electrode 3, second electrode 4, control electrode 5, and ground electrode 13 can be prepared by methods such as sputtering, ALD Dep metal, CVD deposited metal, or deposited a-Si ELA crystallization followed by doping, metal evaporation, or spraying conductive polymers such as PEDOT and then curing. They are connected to an external power supply through internal chip wiring.

[0100] like Figure 7B As shown, the main structure of the micro / nanofluidic chip, from bottom to top, consists of: a substrate 9, a control electrode 5 and a first electrode 3 and a second electrode 4 fabricated on the same mask, a groove layer 21 fabricated with nanoimprint adhesive or EBL adhesive, nanochannels 6, a ground electrode 13, and a cover plate 15. Similarly, the first electrode 3 and the second electrode 4 are connected to an external power supply via internal chip wiring. The first microchannel 1 and the second microchannel 2 are fabricated by dry etching and are perpendicular to the nanochannel 6. Furthermore, the cover plate 15 has a second groove 17 (non-penetrating) at the same position as the microchannels on the chip substrate. For example, the second groove 17 has a depth and width of 70 μm and a length of 120 μm. The cover plate 15 also has an inlet 7 and an outlet 8 (penetrating the cover plate) for connecting to external conduits. Figure 6 As shown in the figure, for example, the diameter of the liquid inlet 7 and the liquid outlet 8 is 3 mm.

[0101] The micro / nanofluidic chip of this disclosure can slow down biomolecular translocation and improve the sequencing accuracy of nanopores. At the same time, after integrating the micro / nanofluidic chip with nanopores or other single-molecule sequencing units, large-scale high-throughput sequencing can be achieved, which is of great significance to the development of fields such as biomedicine, drug diagnosis, environmental monitoring and molecular biology.

[0102] Furthermore, the micro / nanofluidic chip of this disclosure can also simultaneously slow down the translocation of biomolecules of different sizes and structures by changing the electrode structure of the control electrode, the first electrode, and the second electrode, and by applying different voltages. When integrated with other single-molecule sequencing units, it can simultaneously sequence multiple biomolecules.

[0103] Figure 8 A schematic flowchart illustrating the fabrication process of a micro / nanofluidic substrate according to an embodiment of the present disclosure is shown.

[0104] like Figure 8 As shown, the fabrication method of the micro / nano fluidic substrate according to this disclosure includes the following steps:

[0105] In operation S801: a metal film layer 18 is formed on the substrate;

[0106] In operation S802: the metal film layer 18 is etched to obtain the first electrode 3, the second electrode 4 and the control electrode 5, forming the electrode layer 10;

[0107] In operation S803: a groove layer 21 including a first groove 12 is formed on the surface of the electrode layer 10;

[0108] In operation S804: a first material layer 19 is deposited in the first groove 12 using a ballistic deposition method to form a nanochannel 6;

[0109] In operation S805: the first material layer 19 is etched in a direction perpendicular to the nanochannel 6 to obtain a microchannel.

[0110] It should be noted that the first material layer 19 includes an oxide formed from silicon and oxygen, such as silicon dioxide, etc., and the embodiments disclosed herein do not limit this.

[0111] like Figure 7A and Figure 7B As shown in the embodiments of this disclosure, before the step of preparing the groove layer 21 including the first groove 12 on the surface of the electrode layer 10, the method further includes: depositing a second material layer 14 on the electrode layer 10 to prevent the control electrode 5 from communicating with the nanochannel 6 or the microchannel. The second material layer 14 includes an oxide formed from silicon and oxygen, such as silicon dioxide, etc., and the embodiments of this disclosure are not limited thereto.

[0112] It should be noted that in this embodiment, the first material layer 19 used to form the nanochannel 6 is deposited by ballistic deposition rather than by CVD. This is because the deposition rate of the film layer in ballistic deposition is different inside the substrate groove and at the opening, which will eventually seal the nanochannel. In contrast, PECVD is isotropic deposition, and the deposition rate of the film layer inside the substrate groove and at the opening is the same, which will eventually form a pit and make it impossible to obtain the nanochannel required by the chip.

[0113] Figure 9A A schematic diagram of the nanochannels prior to formation according to an embodiment of the present disclosure is shown. Figure 9B A schematic diagram illustrating the formation of nanochannels according to an embodiment of the present disclosure is shown.

[0114] like Figure 9AAs shown, when depositing a film on a grooved substrate using ballistic deposition, the deposition rate differs at different locations inside and outside the groove. The deposition rate at a specific point on the substrate is determined by the area of ​​the target material that can be deposited at that point. It can be approximated that the deposition rate is proportional to the effective angle between that point and the target. Therefore, for a groove structure with a suitable aspect ratio, before deposition ( Figure 9A The relationship between the longitudinal deposition rates of points A to E is P. A ≈P B >P C >P E >P D The magnitude relationship of the lateral deposition rates is H B >H C >H D As deposition time increases, the effective angles (deposition rates) between points A and B and the target material remain almost constant, while the effective angles (deposition rates) between positions C to E and the target material gradually decrease, as follows: Figure 9B As shown. Since the deposition rate at point B is always significantly faster than the deposition rate at other locations inside the groove, the material deposited on the groove will eventually seal to form a nanochannel.

[0115] Figure 10 A nanochannel SEM image is schematically shown according to an embodiment of the present disclosure.

[0116] like Figure 10 As shown, the final deposited nanochannels can be observed. Whether the grooves can eventually close to form nanochannels depends on their aspect ratio and the thickness of the first material layer. Extensive experimental verification has shown that when the thickness of the first material layer is 600 nm, a groove aspect ratio greater than 0.3 makes it easier to form nanochannels. Furthermore, when the grooves are circular, droplet-shaped nanochannels with high throughput and better uniformity can be obtained.

[0117] In the embodiments of this disclosure, in the step of depositing a first material layer in the first groove using a ballistic deposition method, the aspect ratio of the first groove is >0.3, and the thickness of the deposited first material layer is 600 nm.

[0118] This disclosure utilizes ballistic deposition to prepare nanochannels, which is simple, low-cost, has good uniformity, strong stability and liquid control capabilities, and is suitable for large-scale mass production.

[0119] like Figure 7A and Figure 7B As shown in the embodiments of this disclosure, the fabrication method of the micro / nanofluidic substrate further includes: fabricating a ground electrode 13 above the nanochannel 6. The ground electrode 13 can be prepared by spraying a conductive polymer such as PEDOT onto the nanochannel 6 and then curing it, or it can be prepared by other methods. The embodiments of this disclosure do not limit this method.

[0120] Figures 11A-11E The diagrams illustrate cross-sectional views of the micro / nanofluidic substrate formed after some steps of the fabrication method of the micro / nanofluidic substrate according to embodiments of the present disclosure are performed, in the direction perpendicular to the nanochannels. Figures 12A-12I The diagrams illustrate cross-sectional views of the micro / nanofluidic substrate formed after some steps of the fabrication method of the micro / nanofluidic substrate according to embodiments of the present disclosure are performed, in a direction parallel to the nanochannels.

[0121] like Figures 11A-11E and Figures 12A-12I As shown, the specific steps for fabricating the micro / nano fluidic substrate in this embodiment include S1-S5:

[0122] Step S1:

[0123] S1-1: First, a metal film layer 18 is prepared on the substrate 9 by methods such as sputter or ALD Dep metal, CVD deposition of metal or deposition of a-Si ELA crystallization followed by doping, metal evaporation or spraying of conductive polymers such as PEDOT followed by curing (this disclosure takes Cu with a sputter dep thickness of 500 nm as an example).

[0124] S1-2: The metal film layer 18 is etched to form three electrodes: control electrode 5, first electrode 3, and second electrode 4 (the electrode size depends on the nanochannel and its spacing and the microchannel size; in this disclosure, control electrode 5 is 4 mm long and 30 μm wide, and first electrode 3 and second electrode 4 are both 4 mm long and 10 μm wide).

[0125] S1-3: To prevent chip failure caused by contact between the control electrode and the sample solution in the microchannel during chip operation, a second material layer 14 is deposited by CVD or ALD to fill the steps generated by etching. At the same time, this step can also prevent the imprint or EBL pattern loss caused by the presence of discontinuity during the preparation of the groove structure necessary for forming the nanochannel.

[0126] Step S2: A groove layer 21 including a first groove 12 is prepared on the surface of the electrode layer 10 by means of nanoimprinting or EBL (this disclosure takes nanoimprinting as an example, with an imprinting adhesive thickness of 2 μm). The bottom of the first groove 12 of the groove layer 21 can be of different shapes (this disclosure takes a rectangle as an example, with a groove width of 30 nm and a depth of 90 nm).

[0127] Step S3:

[0128] S3-1: A 600nm first material layer 19 is deposited by ballistic deposition to form nanochannels 6.

[0129] S3-2: The first micron channel 1 and the second micron channel 2 (the width and depth of the micron channel are both 50 μm) are prepared in the vertical direction of the nanochannel 6 by dry etching. The film layer 11 includes the first micron channel 1, the second micron channel 2, the nanochannel 6 and the groove layer 21.

[0130] Step S4: After spraying conductive polymers such as PEDOT onto the nanochannel 6, the ground electrode 13 is prepared by curing.

[0131] Step S5:

[0132] S5-1: Deposit bonding layer 16 by CVD or ALD (this disclosure takes CVD deposition of 600nm SiO2 as an example).

[0133] S5-2: Etch the bonding layer 16 to form the first micron channel 1 and the second micron channel 2. At this point, the chip substrate fabrication is complete.

[0134] Figure 13 A schematic flowchart illustrating the fabrication process of a micro / nanofluidic chip according to an embodiment of the present disclosure is shown.

[0135] like Figure 13 As shown, the fabrication method of the micro / nano fluidic chip according to this disclosure includes the following steps:

[0136] S1301: Fabricate a micro / nano fluidic substrate according to the above method;

[0137] S1302: Deposit a bonding layer on the substrate;

[0138] S1303: Etch the bonding layer in a direction perpendicular to the nanochannel to form a microchannel. Since the microchannel on the substrate is covered by the bonding layer during the deposition of the bonding layer, it is necessary to etch the bonding layer to re-etch out the microchannel.

[0139] S1304: Bond the bonding layer to the cover plate to obtain a micro / nano fluid control chip.

[0140] This disclosure provides a micro / nanofluidic system, including the aforementioned micro / nanofluidic chip and power supply. The micro / nanofluidic chip further includes internal wires, and the first electrode, the second electrode, and the control electrode are connected to the power supply through the internal wires, reducing the complexity of the chip-power supply connection.

[0141] Those skilled in the art will understand that the features described in the various embodiments and / or claims of this disclosure can be combined and / or combined in various ways, even if such combinations or combinations are not explicitly described in this disclosure. In particular, the features described in the various embodiments and / or claims of this disclosure can be combined and / or combined in various ways without departing from the spirit and teachings of this disclosure. All such combinations and / or combinations fall within the scope of this disclosure.

[0142] The embodiments of this disclosure have been described above. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. Although various embodiments have been described above, this does not mean that the measures in the various embodiments cannot be used advantageously in combination. The scope of this disclosure is defined by the appended claims and their equivalents. Various substitutions and modifications can be made by those skilled in the art without departing from the scope of this disclosure, and all such substitutions and modifications should fall within the scope of this disclosure.

Claims

1. A micro / nano fluidic substrate, characterized in that, include: Base; An electrode layer located on the substrate, the electrode layer including a first electrode, a second electrode, and a control electrode; as well as A film layer located on the electrode layer away from the substrate, the film layer comprising a grooved layer, nanochannels, and microchannels, wherein the grooved layer includes a first groove, the nanochannels are located within the first groove, the orthographic projection of the nanochannels on the substrate at least partially coincides with the orthographic projection of the control electrode on the substrate, and the microchannels are connected to the nanochannels. The micron channel includes a first micron channel and a second micron channel. The orthographic projection of the first micron channel on the substrate is located on the first side of the orthographic projection of the nanochannel on the substrate. The first micron channel is connected to the first electrode. The orthographic projection of the second micron channel on the substrate is located on the second side of the orthographic projection of the nanochannel on the substrate. The second micron channel is connected to the second electrode. The first side and the second side are opposite sides of the orthographic projection of the nanochannel on the substrate.

2. The substrate according to claim 1, characterized in that, The aspect ratio of the first groove is >0.

3.

3. The substrate according to claim 1, characterized in that, The substrate further includes a ground electrode, which serves as a reference electrode for the first electrode, the second electrode, and the control electrode. The ground electrode is located on the side of the film layer away from the substrate, and the orthographic projection of the ground electrode on the substrate at least partially overlaps with the orthographic projection of the control electrode on the substrate.

4. The substrate according to claim 3, characterized in that, A second material layer is filled between the first electrode, the second electrode, and the control electrode.

5. The substrate according to claim 1, characterized in that, The substrate includes a first electrode, a second electrode, a control electrode, and M nanochannels, wherein M ≥ 1.

6. The substrate according to claim 1, characterized in that, The substrate includes M first electrodes, M second electrodes, 1 control electrode, and M nanochannels, wherein M ≥ 2.

7. The substrate according to claim 1, characterized in that, The substrate includes one first electrode, one second electrode, M control electrodes, and M nanochannels, wherein M ≥ 2.

8. The substrate according to claim 1, characterized in that, The substrate includes M first electrodes, M second electrodes, M control electrodes, and M nanochannels, wherein M ≥ 2.

9. A micro / nano fluidic chip, characterized in that, The chip includes: The substrate according to any one of claims 1 to 8.

10. The chip according to claim 9, characterized in that, The chip also includes: A cover plate located above the substrate; and A bonding layer located above the substrate, the bonding layer being used to bond the substrate and the cover plate. The cover plate includes: The second groove, the orthographic projection of the second groove on the substrate covers the orthographic projection of the microchannel on the substrate; The inlet is used to add the sample to be tested; and The liquid outlet is used to export the sample after the test is completed.

11. A method for fabricating a micro / nanofluidic substrate, characterized in that, include: A metal film is formed on the substrate; The metal film layer is etched to obtain a first electrode, a second electrode, and a control electrode, forming an electrode layer; A groove layer including a first groove is prepared on the surface of the electrode layer; A first material layer is deposited in the first groove using a ballistic deposition method to form a nanochannel, wherein the orthographic projection of the nanochannel on the substrate at least partially coincides with the orthographic projection of the control electrode on the substrate; as well as The first material layer is etched in a direction perpendicular to the nanochannel to obtain a microchannel. The microchannel is connected to the nanochannel and includes a first microchannel and a second microchannel. The orthographic projection of the first microchannel on the substrate is located on a first side of the orthographic projection of the nanochannel on the substrate. The first microchannel is connected to the first electrode. The orthographic projection of the second microchannel on the substrate is located on a second side of the orthographic projection of the nanochannel on the substrate. The second microchannel is connected to the second electrode. The first side and the second side are opposite sides of the orthographic projection of the nanochannel on the substrate.

12. The method according to claim 11, characterized in that, Before the step of preparing a groove layer including a first groove on the surface of the electrode layer, the method further includes: A second material layer is deposited on the electrode layer to prevent the control electrode from being connected to the nanochannel or the microchannel.

13. The method according to claim 11, characterized in that, In the step of depositing a first material layer in the first groove using a ballistic deposition method, the aspect ratio of the first groove is >0.3, and the thickness of the deposited first material layer is 600 nm.

14. The method according to claim 13, characterized in that, The method further includes: A ground electrode is fabricated above the nanochannel.

15. A method for fabricating a micro / nano fluidic chip, characterized in that, include: The substrate is prepared according to any one of claims 11 to 14; A bonding layer is deposited on the substrate; The bonding layer is etched in a direction perpendicular to the nanochannel to form a microchannel; as well as The bonding layer is bonded to the cover plate.

16. A micro / nanofluidic system, characterized in that, Includes the micro / nano fluidic chip as described in any one of claims 9 to 10.

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