A micro-nanofluidic chip and a method for separating biochemical marker molecules

Abstract

The present invention belongs to the field of micro-nano fluid and biochemical sensor technology, and specifically relates to a micro-nano fluidic chip and a method for separating biochemical marker molecules using the micro-nano fluidic chip. The micro-nano fluidic chip includes a chip body, and the chip body is provided with a first channel, a second channel and a nano-flow channel, the first channel is connected between the first injection port and the downstream analysis system; the second channel is connected to the second injection port; the nano-flow channel has an ion selection function and is connected between the first channel and the second channel. Through the above structure, it is possible to achieve non-blocking continuous separation of biochemical marker molecules in complex samples, achieve purification of biochemical marker molecules, eliminate interference from background components, improve analytical performance and detection sensitivity, have the advantages of low detection cost, fast response time, and low sample and reagent consumption, and is suitable for clinical medical diagnosis and other fields, and helps to improve the diagnostic accuracy and treatment effect of diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of micro-nano fluid and biochemical sensing, and specifically relates to a micro-nano fluidic chip and a method for separating biochemical marker molecules using the micro-nano fluidic chip. Background Art

[0002] In clinical medical diagnosis, clinical samples such as blood contain rich biochemical information, which is crucial for the diagnosis and treatment of diseases. Micro-nanofluidic devices can integrate the detection of biochemical markers onto a chip, and only microliter samples are needed to achieve detection. However, the background components in clinical samples are complex, which will seriously interfere with the analysis process of micro-nanofluidic chips, especially the sensing of trace biochemical marker molecules. In the related technology, clinical samples are separated and purified by using extraction, magnetic bead enrichment and other processes. Among them, extraction uses the difference in solubility of the target biochemical molecules in the two phases, and magnetic bead enrichment is to modify the capture antibody on the magnetic beads to specifically identify and capture the target biochemical molecules, and then wash them to achieve the separation and purification of the target biochemical molecules. However, the above methods have problems such as low efficiency and inability to process low-volume samples, and cannot meet actual needs. Summary of the invention

[0003] The purpose of the present invention is to provide a micro-nanofluidic chip and a method for separating biochemical marker molecules using the micro-nanofluidic chip, so as to solve the problems of low separation efficiency of biochemical marker molecules and inability to process low volume samples in the prior art.

[0004] The present invention provides a micro-nanofluidic chip, comprising a chip body, wherein the chip body is provided with:

[0005] A first channel, wherein the first channel is connected between the first injection port and a downstream analysis system;

[0006] a second channel, the second channel being in communication with the second liquid injection port;

[0007] The nanoflow channel has an ion selection function and is connected between the first channel and the second channel.

[0008] The micro-nanofluidic chip provided by the present invention may also have the following additional technical features:

[0009] In a specific embodiment of the present invention, the nanoflow channel forms a plurality of microchannels with a characteristic size less than 100 nm by setting the following structures: a nanohole array, a nanochannel array, a nano shallow groove array, and a nano gap.

[0010] In one embodiment of the present invention, the nanohole array, nanochannel array or nano shallow groove array is formed by micromachining;

[0011] The nano gap is formed by filling nano materials.

[0012] In a specific embodiment of the present invention, the first channel is a straight channel, a gradient channel, a bipolar channel, a tertiary channel, a bending channel or a corrugated channel; and / or

[0013] The characteristic size of the first channel is 1-20 μm.

[0014] In a specific embodiment of the present invention, a third channel is further provided on the chip body, the first liquid injection port is provided at the end of the third channel, and the middle portion is connected to the first channel.

[0015] In a specific embodiment of the present invention, a fourth channel is provided on the chip body, and the fourth channel is connected between the first channel and the downstream analysis system.

[0016] In a specific embodiment of the present invention, the other end of the fourth channel relative to the first channel is a sealed end, a nanopore is provided on one side of the sealed end, and the sealed end is connected to the downstream analysis system through the nanopore; or

[0017] The other end of the fourth channel relative to the first channel is an open end, and the open end is communicated with the downstream analysis system.

[0018] In a specific embodiment of the present invention, the chip body includes a substrate and a cover plate bonded and packaged with the substrate; the nanoflow channel is arranged on the substrate, and the first channel, the second channel and the third channel are arranged on the substrate or the cover plate.

[0019] The present invention also provides a method for separating biochemical marker molecules, which is accomplished using any one of the micro-nanofluidic chips described above, comprising:

[0020] Adding buffer to the first injection port and the second injection port, leaving the buffer to stand until the first channel, the second channel and the nanoflow channel are filled with the buffer, and then packaging and storing;

[0021] The sample to be tested is injected into the first injection port, and voltages of different magnitudes are simultaneously applied to the first injection port and the second injection port to form an electric field directed from the second injection port to the first injection port, so that the biochemical marker molecules are purified and separated in the first channel and migrated to the downstream analysis system.

[0022] In a specific embodiment of the present invention, the downstream analysis system includes an immunoassay system, a nanopore sequencing analysis system or a flow analysis system.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The micro-nanofluidic chip provided by the present application is based on the principle of micro-nanofluidics, applies a large-scale nanofluidic channel array, and cleverly utilizes the characteristics of its high electroosmotic flux and the synergistic effect of electroosmotic flow and electrophoresis, which can realize the separation of biochemical marker molecules in complex clinical samples based on charge-to-body ratio, and the separation process is continuous and can be integrated into the downstream analysis system, which has great application value. The micro-nanofluidic chip disclosed in the present invention does not involve any mechanical moving components, and does not rely on auxiliary consumables such as magnetic beads, and can realize the non-blocking continuous separation of biochemical marker molecules in complex samples, realize the purification of biochemical marker molecules, eliminate the interference of background components, and can realize the efficient separation and detection sensitivity of biochemical marker molecules. It has the advantages of low detection cost, small sample and reagent consumption, etc., has great application value, and can help improve the diagnostic accuracy and treatment effect of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention is shown;

[0027] Figure 2 Several different forms of downstream analysis systems in the present invention are shown;

[0028] Figure 3 A schematic diagram of separation of biochemical marker molecules in a micro-nanofluidic chip in one embodiment of the present invention is shown;

[0029] Figure 4 A schematic diagram of separation and analysis of biochemical marker molecules in a micro-nanofluidic chip in one embodiment of the present invention is shown.

[0030] Description of reference numerals:

[0031] 100-micro-nanofluidic chip;

[0032] 10 - chip body, 20 - first channel, 21 - detection area; 30 - nanoflow channel; 40 - second channel, 41 - second liquid injection port; 50 - third channel, 51 - first liquid injection port, 60 - fourth channel. DETAILED DESCRIPTION

[0033] The exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0034] It should be understood that the terms used in the text are only for the purpose of describing specific example embodiments, and are not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms "one", "an" and "said" as used in the text may also be meant to include plural forms. The terms "include", "comprise", "contain", and "have" are inclusive, and therefore specify the existence of stated features, steps, operations, elements and / or parts, but do not exclude the existence or addition of one or more other features, steps, operations, elements, parts, and / or combinations thereof. The method steps, processes, and operations described herein are not interpreted as necessarily requiring them to be performed in the specific order described or illustrated, unless the execution order is clearly indicated. It should also be understood that additional or alternative steps may be used.

[0035] Although the terms first, second, third, etc. can be used in the text to describe multiple elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms can only be used to distinguish an element, component, region, layer or section from another region, layer or section. Unless the context clearly indicates, terms such as "first", "second" and other numerical terms do not imply order or sequence when used in the text. Therefore, the first element, component, region, layer or section discussed below can be referred to as the second element, component, region, layer or section without departing from the teaching of the example embodiments.

[0036] For ease of description, spatial relative terms may be used herein to describe the relationship of one element or feature relative to another element or feature as shown in the figure, such as "inside", "outside", "inner side", "outer side", "below", "below", "above", "above", etc. Such spatial relative terms are intended to include different orientations of the device in use or operation in addition to the orientation depicted in the figure. For example, if the device in the figure is turned over, then the elements described as "below other elements or features" or "below other elements or features" will subsequently be oriented as "above other elements or features" or "above other elements or features". Therefore, the example term "below..." can include both upper and lower orientations. The device can be oriented otherwise (rotated 90 degrees or in other directions) and the spatial relative descriptors used in the text are interpreted accordingly.

[0037] Driven by an external bias, charged particles will migrate in a directional manner through electrophoresis. When charged particles migrate to a nanostructure with particle selectivity, they will be affected by the charge on the nanostructure wall. Usually, ions with the same charge as the wall can pass through, while ions with the opposite charge will be blocked. This phenomenon is unique to nano-sized fluids and is called ion selectivity. When the thickness of the double electric layer on the inner surface of the nanostructure is close to or greater than the characteristic size of the nanostructure, the nanostructure will produce ion selectivity.

[0038] Below the scale of 100 nanometers, the flow channel structure has a very high surface-to-volume ratio, and the physical phenomena produced will be different from those in the microfluidics field. At this scale, the role played by the flow channel surface will be dominant, and the most important physical phenomenon to consider is the double electrical layer. When a solid substance comes into contact with a liquid, due to the dissociation of surface molecular clusters, the solid surface will carry a layer of fixed charge, and the ions in the solution will be subject to the Coulomb force of the fixed charge on the solid surface, resulting in common ion repulsion and counter ions convergence on the solid surface to maintain the electrical neutrality condition on the solid surface. This will form a shielding area where the solution is close to the solid surface, which is mainly composed of counter ions. We call this area a double electrical layer.

[0039] In microfluidics research, the flow channel size is in the micrometer range, and the influence of the double layer can be completely ignored. However, in the field of nanofluids, the characteristic length of the flow channel is not large enough to ignore the double layer. The physical quantity that usually characterizes the characteristic thickness of the double layer is the Debye length λD. For a 1mM KCl solution, the Debye length λD has reached 10nm, which is close to the characteristic length of the nanochannel, and even greater than the characteristic size of the nanochannel in many cases. Therefore, for nanochannels, the Debye length is approximately equal to or greater than the characteristic size of the channel, the liquid in the channel is charged, and the concentration of counterions is much higher than that of the same ions. This also makes the nanochannel ion-selective, allowing only counterions to pass through.

[0040] Double layer and ion selectivity are phenomena unique to nanofluidics and are usually related to the size of the nanostructure and the internal surface charge density. The size and internal surface charge density of the nanostructure are determined by the material properties and processing parameters, and are fixed once the micro-nanofluidic device is prepared.

[0041] Based on this, an embodiment of the present invention proposes a micro-nanofluidic chip 100, which utilizes the above-mentioned characteristics of the nanostructure to enrich and separate biochemical marker molecules.

[0042] Figure 1 , Figure 3 , Figure 4 The three-dimensional structure diagram of the micro-nanofluidic chip 100 provided in the embodiment of the present application is shown. The micro-nanofluidic chip 100 includes a chip body 10. Specifically, the chip body 10 is provided with a first channel 20, a second channel 40 and a nano-flow channel 30, wherein the first channel 20 is connected between the first injection port 51 and the downstream analysis system; the second channel 40 is connected to the second injection port 41; the nano-flow channel 30 has an ion selection function and is connected between the first channel 20 and the second channel 40.

[0043] The first injection port 51 and the second injection port 51 are used to add liquid to the micro-nano fluidic chip 100. The liquid can be an auxiliary liquid, such as a buffer, a blocking liquid, a washing liquid, an osmotic solution, or a sample solution. The liquid in the first injection port 51 can flow to the first channel 20, and the liquid in the second injection port 41 can flow to the second channel 40. The first injection port 51 and the second injection port 41 are also used to connect electrodes to apply a bias voltage to the micro-nano fluidic chip 100.

[0044] The nanoflow channel 30 refers to a channel array with a characteristic size at the nanometer level, and therefore has particle selectivity. When a charged particle migrates to the nanoflow channel 30 with ion selectivity, it will be affected by the wall charge of the nanoflow channel 30. Generally, ions with the same electrical properties as the wall charge can pass through, while ions with the opposite electrical properties to the wall charge will be blocked. The nanoflow channel 30 is connected between the first channel 20 and the second channel 40. Therefore, when a charged particle with the opposite polarity to the wall charge of the nanoflow channel 30 migrates to the first channel 20, due to the effect of the wall charge of the nanoflow channel 30, the charged particle cannot enter the nanoflow channel 30, but will migrate along the first channel 20 to the tip of the first channel 20. At the same time, the nanoflow channel 30 will generate electroosmosis (a fluid flow phenomenon driven by an electric field, usually only occurring at the micrometer / nanometer scale) under the drive of an external bias, so the charged particles in the first channel 20 are simultaneously affected by the electroosmotic drag force and the electric field force. For high mobility molecules, they will be enriched in the first channel 20, while for low mobility molecules, they will be flushed out of the first channel 20 by the electroosmotic flow. Through reasonable design, the biochemical marker molecules can be enriched, and the impurity molecules are washed away by the electroosmotic flow, ultimately achieving the effect of enriching and purifying the biochemical marker molecules and eliminating the interference of background components.

[0045] Since the first channel 20 is connected to the downstream analysis system, the biochemical marker molecules enriched in the first channel 20 can further migrate to the downstream analysis system along with the first channel 20 and continue to be analyzed in the downstream analysis system.

[0046] When used, biochemical molecules will accumulate and enrich in the first channel 20 under the ion selectivity of the nanoflow channel 30, and will be affected by the synergistic effect of the electric field force and the electroosmotic flow drag. Electroosmotic flow will be generated in the nanoflow channel 30, and will flow into the first channel 20, and finally flow to the third flow channel. While the charged particles are affected by the electric field force and the particle selective structure enrichment, they are also affected by the flow drag. The particles that are affected by the flow drag greater than the electric field force will be repelled and cannot enter the first channel 20, and the particles that are affected by the electric field force greater than the flow drag will remain in the first channel 20 and flow along the first channel 20 to the downstream analysis system.

[0047] The micro-nanofluidic chip 100 provided in the embodiment of the present application, without involving any mechanical moving components and auxiliary consumables such as magnetic beads, is based on the principle of micro-nanofluidics, applies a large-scale nanoflow channel 30 array, and cleverly utilizes the characteristics of its high electroosmotic flux and the synergistic effect of electroosmotic flow and electrophoresis, so as to achieve the separation of biochemical marker molecules based on charge-to-body ratio in complex clinical samples, and the separation process is continuous and can be integrated into the downstream analysis system, which has great application value. That is, the micro-nanofluidic chip 100 provided in the present application is based on micro-nanofluidic technology to achieve efficient separation and purification of biochemical target molecules, improve analytical performance and detection sensitivity; at the same time, it can also achieve specific labeling of biochemical target molecules through specific labeling methods, improve separation and purification effects; and integrate separation and enrichment functions into one chip, simplifying the experimental operation process and reducing sample consumption. In addition, the above-mentioned micro-nanofluidic chip 100 and its enrichment and separation method are suitable for clinical medical diagnosis and other fields, which helps to improve the diagnostic accuracy and treatment effect of diseases.

[0048] In a specific embodiment, the nanoflow channel 30 forms a plurality of microchannels with a characteristic size less than 100 nm by setting the following structures: a nanohole array, a nanochannel array, a nano shallow groove array, and a nano gap.

[0049] Optionally, the nanoflow channel 30 is configured as a nanopore array, a nanochannel array, or a nano-shallow groove array. The nanopore array, the nanochannel array, and the nano-shallow groove array can be formed by micromachining the chip body 10, or by stacking non-micromachining nanomaterials such as porous silicon, PAA, nafion, MOF, etc. having nanopore arrays, nanochannel arrays, and nano-shallow groove arrays on the chip body 10.

[0050] Optionally, the nano-flow channel 30 is configured as a nano-gap, and the nano-gap is formed by assembling nanoparticles.

[0051] Optionally, the nanoflow channel 30 is configured to be a nanopore array, a nanochannel array, or a nano shallow groove array filled with nanomaterials, wherein the filling material may be nafion, and the nanopore array, nanochannel array, and nano shallow groove array may be formed by micro-fabrication, or may be formed by non-micro-fabrication nanomaterials such as porous silicon and PAA.

[0052] The characteristic size of the nanochannel 30 is less than 100 nm, where the characteristic size may refer to the width and depth of the nano-shallow groove or nanochannel, or the diameter of the nanopore. Preferably, in order to ensure the ion selectivity of the nanochannel 30 and avoid leakage, the characteristic size of the nanochannel 30 is preferably less than 50 nm.

[0053] In a specific embodiment, the first channel 20 is a straight channel, a gradient channel, a bipolar channel, a tertiary channel, a bending channel or a corrugated channel.

[0054] In a specific embodiment, the characteristic size of the first channel 20 is 1-20 μm.

[0055] The first channel 20 is connected to the nanoflow channel 30 and the downstream separation system, and is the location where the biochemical marker molecules are separated. High-mobility charged particles migrate into the first channel 20 driven by the electric field, but cannot pass through the nanoflow channel 30 due to the restriction of the nanoflow channel 30, but migrate to the top position of the first channel 20 and flow to the downstream analysis system. Low-mobility charged particles are affected by the drag force generated by the electroosmotic flow at the entrance of the first channel 20 and cannot enter the first channel 20, thereby achieving the separation and purification of high-mobility molecules, and can be coupled to the downstream analysis system for real-time analysis.

[0056] In order to achieve different separation requirements, the separation channels have different designs. The characteristic scale of the first channel 20 is between 1-20μm, where the characteristic size can refer to the width and depth of the first channel 20. For example, the characteristic size can be 2μm, 5μm, 8μm, 10μm, 15μm or 18μm. The specific size needs to be determined according to the needs. The shape of the first channel 20 mainly includes a straight channel, a gradient channel, a bipolar channel, a tertiary channel, a curved channel and an arbitrary waveform channel. It is found through experiments that the shape and size of the first channel 20 have a great influence on the separation effect and determine the value of the critical mobility.

[0057] In a specific embodiment, the channel surface can be modified, and probes corresponding to biochemical marker molecules can be introduced into the first channel 20 to specifically identify and capture the enriched biochemical markers. For example, by specifically labeling the target biochemical molecule with a nucleic acid aptamer or a nucleic acid-modified antibody, the charge-to-body ratio can be much larger than that of background proteins and other impurities, so that only the target biochemical molecule and a small amount of molecules with a larger charge-to-body ratio can be enriched into the first channel 20.

[0058] In a specific embodiment, a third channel 50 is further provided on the chip body 10 , and the third channel 50 is connected between the first channel 20 and the first liquid injection port 51 .

[0059] The third channel 50 is a sample channel, which is used to guide the sample to be enriched from the first injection port 51 to the first channel 20. The third channel 50 is usually a large channel with a size of hundreds of microns and very low flow resistance and resistance. The flow rate and flow rate of the sample solution can be accurately controlled by micro-nanofluidics technology to ensure that the sample solution is fully contacted and interacted in the first channel 20. In addition, the third channel 50 can also adjust the concentration and pH value of the sample by introducing a buffer, diluent or other auxiliary liquid to further optimize the enrichment effect.

[0060] In a specific embodiment, the third channel 50 is arranged in a V-shape, wherein the V-shaped tip of the third channel 50 is connected to the first channel 20, and each of its open ends is connected to a first liquid injection port 51. When used, a sample is added to the third channel 50 through a first liquid injection port 51, and the impurity molecules move to another first liquid injection port 51 under the electric field force and electroosmotic drag force.

[0061] In a specific embodiment, the characteristic size of the second channel 40 is on the order of hundreds of microns.

[0062] The second channel 40 corresponds to the first channel 20 and is located on the other side of the nanoflow channel 30. Under the action of an external bias, ion concentration polarization will occur on both sides of the micro-nano flow, that is, ion enrichment occurs on one side and ion depletion occurs on the other side. The second channel 40 corresponds to the ion concentration polarization depletion side. Unlike the first channel 20, the resistivity of the second channel 40 will increase many times. Since the focus of this application is in the first channel 20, in order to reduce the pressure drop in the second channel 40, the second channel 40 is usually designed as a large channel, preferably in the order of hundreds of microns, which can increase convection and diffusion, reduce the pressure drop in the second channel 40 and the interference with the enrichment process.

[0063] There are two second liquid injection ports 41 , which are disposed on both sides of the second channel 40 .

[0064] In a specific embodiment, the diameter of the first liquid injection port 51 and the second liquid injection port 41 are approximately 1-2 mm, and the depth is 2-7 mm. Liquid can be added directly into the first liquid injection port 51 and the second liquid injection port 41, or liquid can be added to the first liquid injection port 51 and the second liquid injection port 41 through an external hose.

[0065] In a specific embodiment, a fourth channel 60 is disposed on the chip body 10 , and the fourth channel 60 is connected between the first channel 20 and the downstream analysis system.

[0066] The fourth channel 60 is connected to the first channel 20, and can be a closed microgroove structure or a flow channel structure. The target biochemical molecules are separated and purified in the first channel 20 and migrate to the top of the first channel 20 away from the third channel 50 and enter the fourth channel 60, and finally migrate to the downstream analysis system through the fourth channel 60.

[0067] In one embodiment, the other end of the fourth channel 60 relative to the first channel 20 is a sealed end, that is, a closed microgroove structure. The fourth channel 60 is provided with a nanopore on one side of the sealed end and is connected to a downstream analysis system through the nanopore.

[0068] In one embodiment, the other end of the fourth channel 60 relative to the first channel 20 is an open end, that is, a flow channel structure, and is connected to a downstream analysis system through the end of the flow channel structure.

[0069] like Figure 2 As shown, in one embodiment, the downstream analysis system includes an immunoassay system, a nanopore sequencing analysis system, or a flow analysis system.

[0070] In a specific embodiment, the body includes a substrate and a cover plate bonded and packaged with the substrate, the nanoflow channel 30 is disposed on the substrate, and the first channel 20, the second channel 40, the third channel 50 and the fourth channel 60 are disposed on the substrate or the cover plate.

[0071] MEMS (micro-electromechanical system) can be used to process the flow channel system of the micro-nano fluidic chip 100. According to the observation requirements and process difficulty, materials such as silicon, glass, and quartz can be selected as substrate materials. The nanoflow channel 30 can be made of nanopores, nanochannels, nano shallow grooves, etc. prepared by micromachining, or nanomaterials such as porous silicon, PAA, nafion, MOF, etc. prepared by non-micromachining, or nanoparticles can be self-assembled to form nano gaps, and ion selective materials such as nafion can be partially filled in nanopores, nano shallow grooves, PAA, and porous silicon structures to enhance the ion selectivity of the nanoflow channel 30. The first channel 20 has strict dimensional parameters and is prepared by micromachining to accurately control its size. The first channel 20 can be processed on the substrate or on the cover material. The third channel 50 and the second channel 40 are relatively large in size, usually in the order of hundreds of microns, and the dimensional accuracy requirements are not strict, and they are usually processed on the cover material. Finally, the bonding package is completed to complete the construction of the entire micro-nano fluidic device. The packaging of the micro-nanofluidic chip 100 has different options depending on the application scenario. For scientific research, the substrate is bonded to PDMS, which is a mature method and a simple process. For product use, low-cost, scalable material packaging should be used. Specifically, the cover plate can be made of high molecular polymers such as polyimide (PI), polymethyl methacrylate (PMMA), polycarbonate (PC), and polyethylene terephthalate (PET).

[0072] The embodiment of the present application further provides a biochemical marker molecule sensing method, which is implemented using any one of the micro-nanofluidic chips 100 described above, comprising:

[0073] Add buffer to the first injection port 51 and the second injection port 41, and let it stand until the buffer fills the first channel 20, the second channel 40 and the nano-flow channel 30, and then package and store;

[0074] The sample to be tested is injected into the first injection port 51, and voltages of different magnitudes are simultaneously applied to the first injection port 51 and the second injection port 41 to form an electric field directed from the second injection port 41 to the first injection port 51, so that the biochemical marker molecules are purified and separated in the first channel 20 and migrated to the downstream analysis system.

[0075] The embodiment of the present application integrates the entire detection process including sample addition, specific separation, enrichment, detection, etc. on the micro-nanofluidic chip 100, and realizes the connection between the units in the detection process through electric drive, without involving any mechanical moving components. Through the construction of local molecular potential barriers and potential wells, under the synergistic effect of electroosmotic flow and electric field, the target marker molecules will be quickly separated and enriched from complex clinical samples to the first channel at a high multiple, and guided to the downstream analysis system through the first channel.

[0076] During operation, the flow channel of the entire micro-nanofluidic chip 100 is filled with buffer, and then the sample to be separated and purified is added to the first injection port 51, the second injection port 41 is connected to the positive potential / working electrode, the working voltage is between 5-100V, and the first injection port 51 is grounded. The charged particles in the sample will be affected by the electric field force, and the negatively charged particles will migrate into the first channel 20 under the action of the electric field force. At this time, they are restricted by the nanoflow channel 30 and will flow along the first channel 20 to the fourth channel 60, or continue to enrich in the first channel 20. During operation, electroosmotic flow will be generated in the nanoflow channel 30 and merged into the first channel 20. The charged particles in the first channel 20 are affected by the electric field force and the enrichment of the nanoflow channel 30, and are also affected by the flow drag. When the flow drag is greater than the electric field force, the particles will be discharged from the first channel 20, or cannot enter the first channel 20. Based on this principle, the separation of target biochemical molecules and background impurities is achieved. The separation process depends on the mobility of the charged particles. In actual samples, there are different substances such as nucleic acid molecules, protein molecules, cell fragments, vesicles, etc. The mobility of different types of substances varies greatly, and separation can be achieved without assistance, such as separation of nucleic acids and proteins, or separation of proteins and cell fragments, etc. In order to simply achieve the separation of substances of the same category, specific labeling can be used to improve the mobility of target biochemical molecules, such as using nucleic acid aptamers to specifically label target proteins, or using nucleic acid-modified antibodies to specifically label target proteins. The mobility of the labeled target protein is greatly improved, which is greatly different from most background proteins. At this time, by reasonably designing the flow drag, it can be achieved that only the labeled target protein and molecules with a larger charge-to-body ratio than it are separated into the first channel 20 and the fourth channel 60, while the background protein impurities remain in the third channel 50.

[0077] Electroosmotic flow refers to the flow of liquid through porous materials, capillaries, membranes, microchannels or any other fluid conduits caused by an applied bias. It is caused by the electric field force on the net mobile charge in the solution. The chemical equilibrium between the solid surface and the electrolyte solution usually leads to the existence of a net fixed charge at the interface, forming a double layer near the interface. When the electric field acts on the double layer, the net charge in the double layer is pulled and moved by the electric field force, thereby causing the fluid to flow. The effect of electroosmotic flow is most significant in nanochannels. When the thickness of the double layer is much smaller than the characteristic length scale of the channel, the effect of electroosmotic flow can be ignored.

[0078] Specific labeling of biochemical target molecules is achieved by using nucleic acid aptamers or nucleic acid-modified antibodies to specifically bind to biochemical target molecules. Due to the complexity of background impurities in actual samples, nucleic acid aptamers / nucleic acid-modified antibodies are used for specific labeling to enhance the difference between target molecules and background impurities. This difference is mainly reflected in the charge-to-body ratio.

[0079] Embodiment 1:

[0080] This embodiment uses Figure 3 The micro-nanofluidic chip 100 shown in the figure is used as an example to further illustrate the micro-nanofluidic chip 100 and its separation process in the present application. Interleukin 6 (IL-6) is the most typical cytokine related to inflammation. It plays an important role in host defense by regulating immune and inflammatory responses. Figure 3 The micro-nanofluidic chip 100 shown can successfully extract IL-6 from the third channel 50 through the first channel 20 to the fourth channel 60. This process has the effect of separating, purifying and enriching IL-6.

[0081] The overall material of the micro-nanofluidic chip 100 is quartz glass and polydimethylsiloxane (PDMS), which has the advantage of being self-insulated and does not require additional insulation treatment steps, and can be micro-processed using MEMS technology; the nanoflow channel 30 uses nano-shallow grooves, which are prepared on a quartz substrate by stepper lithography and can be prepared in a standardized manner. The characteristic size of the nano-shallow grooves is less than 50nm, meeting the requirements of ion selectivity; the first channel 20, the fourth channel 60 and the third channel 50 are prepared by PDMS molding.

[0082] 1. Device preparation:

[0083] 1. Through stepper lithography, a shallow groove array of 100 nanometers is prepared on a quartz glass substrate. The line width is 400nm, the length is 3mm, the period is 2μm, the number of arrays is 2000, and the size of each substrate is 15mm*15mm. The nano shallow groove array is located in the center of the substrate. Then, using etching technology such as IBE, the substrate is etched 50nm, and finally laser scribing is performed to complete the preparation of the device substrate.

[0084] 2. SU-8 photolithography prepares PDMS mold, the mold is designed as a SU-8 double-layer structure, the first layer structure corresponds to the first channel 20, the depth is 1 μm, the first channel 20 is a gradient channel, the length is 200 μm, the width at the entrance is 10 μm, the width at the exit is 5 μm, the structure is as follows Figure 3 The second layer structure corresponds to the third channel 50 and the fourth channel 60, with a height of 30 μm and a width of 100 μm.

[0085] 3. Based on the SU-8 double-layer mold, the first channel 20, the fourth channel 60 and the third channel 50 were prepared by the PDMS molding process. The ratio of PDMS to curing agent was 5:1. The commonly used 10:1 was not used in order to enhance the hardness of PDMS and prevent collapse in the subsequent bonding process.

[0086] 4. Treat the bonding surface of PDMS and quartz substrate with oxygen plasma, equipment: oxygen plasma cleaning machine (PDC-MG), power 150w, time 30s, then align the bonding under a microscope. To enhance the bonding strength, bake on a 90 degree hot plate for 5 to 10 minutes.

[0087] 2. Prepare the reagents required for the test

[0088] 1.1mM PBS buffer: Use 1×PBS to prepare the buffer required for enrichment detection, and dilute 1×PBS 150 times with deionized water;

[0089] 2. Blocking solution: Add 0.1% BSA and 0.05% tween-20 to 1 mM PBS buffer;

[0090] 3. Washing solution: Add 0.05% tween-20 to 1 mM PBS buffer;

[0091] 4. Prepare the samples to be tested.

[0092] 3. Separation and purification

[0093] During operation, the two first injection ports 51 are sequentially filled with blocking liquid, and the solution is left to stand for 1-3 minutes. After the blocking liquid fills the first channel 20 and the nanoflow channel 30 by capillary action, the second injection port 41 is filled with blocking liquid. After the flow channel structure in the micro-nanofluidic chip 100 is filled with blocking liquid, it is sealed with Scotch tape and stored at 4 degrees. The blocking is carried out for about 10 minutes, and then the blocking liquid is replaced with a washing solution.

[0094] Finally, the sample to be tested is added to one of the first injection ports 51. The plurality of second injection ports 41 are connected to the positive potential / working electrode, and the two first injection ports 51 are grounded. By adjusting the appropriate external bias voltage, the charged particles in the sample to be tested will be affected by the electric field force and migrate into the separation channel, move toward the tip of the separation channel, and finally flow to the downstream channel.

[0095] After the micro-nanofluidic chip 100 is prepared, there is a fixed critical mobility. Charged particles above the critical mobility will be separated, and charged particles below the critical mobility will continue to remain in the third channel 50. Therefore, the above method can achieve the separation of multiple substances from the third channel 50 to the fourth channel 60 at the same time, not only IL-6. In order to enhance the difference between IL-6 and background components, nucleic acid-modified IL-6 monoclonal antibody is added before sample addition, and the nucleic acid sequence is ATCGTACGTTGACAGCTAGCTAGGCTAATCGTACGTTGA. This method will enhance the mobility of the assembly, thereby greatly reducing the components separated to the fourth channel 60 by appropriately increasing the critical mobility of the device. Figure 3 The difference between specifically labeled IL-6 and background proteins is shown, with the high mobility IL-6-antibody complex separated to the fourth channel 60 while the relatively low mobility background proteins remain in the third channel 50.

[0096] Embodiment 2:

[0097] Nanopore sequencing technology is an innovative gene sequencing method that directly reads DNA or RNA molecules by using nanoscale biological pores or solid-state pores. In this process, the tiny current changes when the DNA or RNA molecules pass through the pores are detected and used to determine the base sequence. Compared with other sequencing technologies, nanopore sequencing has the advantages of fast speed, low cost and no need for pre-amplification of samples, so it has great potential in fast and real-time gene analysis. However, the nanopore is easily blocked during the sequencing process. For this reason, the present application integrates a nanopore structure in the fourth channel 60 of the micro-nanofluidic chip 100 to form a nanopore sequencing chip, such as Figure 4 As shown, the separated and purified nucleic acid is transported to the nanopore for sequencing via the first channel 20 and the fourth channel 60, which can greatly avoid the problem of clogging of the nanopore. Generally, the mobility of nucleic acid is higher than that of background impurities such as protein molecules and cell debris, so the separation and purification of nucleic acid can be achieved without special sample treatment. At the same time, in addition to separating and purifying the target, the micro-nanofluidic chip 100 of the present invention can also achieve a certain degree of enrichment, which can greatly improve the efficiency of nanopore sequencing.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A micro-nanofluidic chip, characterized in that: The chip body comprises: A first channel, wherein the first channel is connected between the first injection port and the downstream analysis system; the characteristic size of the first channel is 1-20 μm; A second channel, the characteristic size of which is in the order of hundreds of microns; the second channel is connected to a second liquid injection port, and the second liquid injection port is arranged on both sides of the second channel; a nano-flow channel, the nano-flow channel having an ion selection function and connected between the first channel and the second channel; The chip body is also provided with a third channel, which is a sample channel. The end of the third channel is provided with the first liquid injection port, and the middle part of the third channel is connected with the first channel. The third channel is a large channel, and its size is in the order of hundreds of microns. The first injection port and the second injection port are also used to connect electrodes, so as to apply bias voltage to the micro-nanofluidic chip; The chip body is also provided with a fourth channel, and the fourth channel is connected between the first channel and the downstream analysis system.

2. The micro-nanofluidic chip according to claim 1, characterized in that: The third channel is arranged in a V shape, wherein the V-shaped tip of the third channel is communicated with the first channel, and each open end is connected with a first liquid injection port.

3. The micro-nanofluidic chip according to claim 1, characterized in that: The nano-flow channel forms a plurality of micro-channels with a characteristic size less than 100 nm by arranging the following structures: a nano-hole array, a nano-channel array, a nano-shallow groove array or a nano-gap.

4. The micro-nanofluidic chip according to claim 3, characterized in that: The nanohole array, nanochannel array or nano shallow groove array is formed by a micromachining method; The nano gap is formed by filling nano materials.

5. The micro-nanofluidic chip according to claim 1, characterized in that: The first channel is a straight channel, a gradient channel, a bipolar channel, a tertiary channel, a curved channel or a corrugated channel.

6. The micro-nanofluidic chip according to claim 1, characterized in that: The other end of the fourth channel relative to the first channel is a sealed end, a nanopore is provided on one side of the sealed end, and the sealed end is connected to the downstream analysis system through the nanopore; or, the other end of the fourth channel relative to the first channel is an open end, and the open end is connected to the downstream analysis system.

7. The micro-nanofluidic chip according to claim 1, characterized in that: The chip body comprises a substrate and a cover plate bonded and packaged with the substrate; the nanoflow channel is arranged on the substrate, and the first channel, the second channel and the third channel are arranged on the substrate or the cover plate.

8. A method for separating biochemical marker molecules, characterized in that: The method is accomplished by using the micro-nanofluidic chip according to any one of claims 1 to 7, comprising: Adding buffer to the first injection port and the second injection port, leaving the buffer to stand until the first channel, the second channel and the nanoflow channel are filled with the buffer, and then packaging and storing; The sample to be tested is injected into the first injection port, and voltages of different magnitudes are simultaneously applied to the first injection port and the second injection port to form an electric field directed from the second injection port to the first injection port, so that the biochemical marker molecules are purified and separated in the first channel and migrated to the downstream analysis system.

9. The method for separating biochemical marker molecules according to claim 8, characterized in that: The downstream analysis system includes an immunoassay system, a nanopore sequencing analysis system or a flow analysis system.

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