Micro-nanofluidic chip and parallel enrichment detection method for 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 parallel enrichment detection method for biochemical marker molecules, wherein the chip comprises a chip body, and the chip body is provided with a first channel, a plurality of second channels, a third channel and a nanoflow channel, wherein the first channel is provided with a first liquid inlet; each of the second channels is provided with a detection area and is connected to the first channel; the third channel is connected to the second liquid inlet; the nanoflow channel has an ion selection function, one side of the nanoflow channel is connected to the third channel, and the other side is respectively connected to a plurality of second channels. The micro-nano fluidic chip of the present application can realize the simultaneous enrichment detection of dozens or even hundreds of biochemical marker molecules through the parallel second channel design, greatly reducing the cost of disease screening, and providing strong support for early screening of diseases, condition monitoring and treatment effect evaluation.

Description

Technical Field

[0001] The invention belongs to the field of micro-nano fluid and biochemical sensor technology, and specifically relates to a micro-nano fluidic chip and a parallel enrichment detection method for biochemical marker molecules. Background Art

[0002] Clinical samples such as blood contain rich biochemical information, such as disease markers such as proteins and nucleic acids, and have great potential for wide application in clinical diagnosis. Currently, related blood testing technologies, such as capillary electrophoresis and colloidal gold immunochromatography, can only detect one or a few markers per sample, and most detection technologies have low sensitivity and cannot provide enough biochemical information for clinical diagnosis, and cannot meet application needs. Summary of the invention

[0003] The purpose of the present invention is to provide a micro-nanofluidic chip and a parallel enrichment detection method for biochemical marker molecules to solve the problem in the prior art that the micro-nanofluidic chip cannot provide enough biochemical information for clinical diagnosis.

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

[0005] a first channel, wherein a first liquid inlet is provided on the first channel;

[0006] A plurality of second channels, each of which is provided with a detection zone, and each of which is connected to the first channel;

[0007] a third channel, the third channel being communicated with the second liquid inlet;

[0008] A nanoflow channel, wherein the nanoflow channel has an ion selection function, one side of the nanoflow channel is connected to the third channel, and the other side of the nanoflow channel is respectively connected to the plurality of second channels.

[0009] The micro-nanofluidic chip provided by the present invention may also have the following additional technical features:

[0010] In a specific embodiment of the present invention, the number of the third channels is two, and the two third channels are disposed on both sides of the first channel.

[0011] In a specific embodiment of the present invention, the third channel includes a third main channel and a plurality of third branch channels connected to the third main channel, and each of the third branch channels is connected to a plurality of the second channels through the nanoflow channel.

[0012] In a specific embodiment of the present invention, the two third channels are symmetrically arranged with respect to the first channel.

[0013] In a specific embodiment of the present invention, the first channel and the third channel are arranged in a curved shape.

[0014] In a specific embodiment of the present invention, the bending position of the first channel and / or the third channel is arranged in an arc shape, or

[0015] The bending positions of the first channel and / or the third channel are arranged at right angles.

[0016] In a specific embodiment of the present invention, the second channel is arranged in a straight line, a broken line and / or a curved line; and / or the characteristic size of the second channel is 5-30 um.

[0017] In a specific embodiment of the present invention, when the second channel is arranged in a zigzag shape, the second channel includes one or more folded corners, and the angle of each folded corner is between 30-150°.

[0018] In a specific embodiment of the present invention, the first channel and the third channel are stacked, and a plurality of the second channels are vertically arranged to the first channel and the third channel.

[0019] The second aspect of the present invention further provides a parallel enrichment detection method for biochemical marker molecules, which is performed using any of the micro-nanofluidic chips described above, comprising:

[0020] Adding buffer to the first liquid inlet and the second liquid inlet, standing until the buffer fills the first channel, the second channel, the third channel and the nanoflow channel, and then packaging and storing;

[0021] Injecting the sample to be tested into the first liquid inlet, and applying voltages of different magnitudes to the first liquid inlet and the second liquid inlet to form an electric field directed from the second liquid inlet to the first liquid inlet, so that the biochemical marker molecules are enriched in the second channel;

[0022] The biochemical marker molecules enriched in the detection area of ​​the second channel are subjected to sensing analysis.

[0023] The embodiment of the present application is based on the principle of nanofluids and combined with microelectronic process technology to construct a micro-nanofluidic chip for efficient enrichment of biochemical target molecules. The micro-nanofluidic chip combines the advantages of microfluidic technology for precise manipulation of microfluids and nanofluidic technology for dynamic controllable regulation of target molecules, and integrates the entire detection process including sample addition, specific separation, enrichment, and detection on the micro-nanofluidic chip, making the entire detection process miniaturized, automated, and integrated. At the same time, through the parallel second channel design, it can achieve the simultaneous enrichment and detection of dozens or even hundreds of biochemical marker molecules, thereby providing enough biochemical information for clinical diagnosis, which greatly reduces the cost of disease screening and provides strong support for early screening of diseases, disease monitoring, and treatment effect evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0026] Figure 2 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0027] Figure 3 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0028] Figure 4 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0029] Figure 5 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0030] Figure 6 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0031] Figure 7 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0032] Figure 8 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0033] Fig. 9 for Figure 2 The main view;

[0034] Fig.10 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0035] Fig.11 A schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention;

[0036] Fig.12 Schematic diagram of the structure of a micro-nanofluidic chip in one embodiment of the present invention.

[0037] Description of reference numerals:

[0038] 100-micro-nanofluidic chip;

[0039] 10-chip body, 20-second channel, 21-detection area; 30-nanoflow channel; 40-third channel, 41-second liquid inlet, 42-third main channel, 43-third branch channel; 50-first channel, 51-first liquid inlet, 52-first main channel, 53-first branch channel. DETAILED DESCRIPTION

[0040] 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.

[0041] It should be understood that the terms used herein 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 herein 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.

[0042] 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.

[0043] For ease of description, spatial relative terms may be used in the text 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, the element described as "below other elements or features" or "below other elements or features" will then 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 biochemical marker molecules.

[0049] Reference Figure 1-Figure 12 A micro-nanofluidic chip 100 provided in an embodiment of the present invention includes a chip body 10, on which a first channel 50, a plurality of second channels 20, a third channel 40 and a nanoflow channel 30 are provided, wherein the first channel 50 is provided with a first liquid inlet 51; each second channel 20 is provided with a detection area 21, and each second channel 20 is connected to the first channel 50; the third channel 40 is connected to the second liquid inlet 41; the nanoflow channel 30 has an ion selection function, one side of the nanoflow channel 30 is connected to the third channel 40, and the other side is respectively connected to the plurality of second channels 20.

[0050] The first channel 50 is provided with a first liquid inlet 51, which is used to introduce the sample to be enriched into the micro-nanofluidic chip 100 through the first liquid inlet 51, and is also used to guide the sample to be enriched to the second channel 20. The first channel 50 is usually a large channel with a size of hundreds of microns and a very low flow resistance. Specifically, the flow rate and flow rate of the sample in the first channel 50 can be accurately controlled by micro-nanofluidic technology to ensure that the sample is fully contacted and interacted in the second channel 20 after entering through the first channel 50. In addition, the first channel 50 can also adjust the concentration and pH value of the sample by introducing a buffer, a diluent or other auxiliary liquid to further optimize the enrichment effect.

[0051] The second channel 20 is connected to the micro-nano flow channel 30 and is the location where the biochemical target is finally enriched. Charged particles migrate into the second channel 20 under the drive of the electric field, but are restricted by the micro-nano flow channel 30 and cannot pass through. They will stay in the second channel 20 and accumulate, ultimately achieving high-multiple enrichment of charged particles (10 4 -10 7 To achieve simultaneous enrichment detection of multiple targets, the micro-nanofluidic chip 100 is designed with dozens or even hundreds of parallel second channels 20, each of which is connected to the third channel 40 through the micro-nano flow channel 30 to form a complete current loop.

[0052] The detection zone 21 is located in the second channel 20, usually at the tip of the second channel 20 away from the first liquid inlet 51. By modifying the probe in the detection zone 21, specific analysis of the enriched biochemical marker molecules can be achieved. The detection zone 21 can use different sensing technologies, such as surface plasmon resonance (SPR), electrochemical methods, fluorescence detection, chemiluminescence, etc., to achieve quantitative detection or qualitative analysis of the enriched marker molecules. Through the precise control of micro-nanofluidics technology, high-sensitivity and high-selectivity biosensing can be achieved in the detection zone 21.

[0053] The probe coating can be carried out by adsorption. Before the device is bonded, a probe solution with a diameter of 10-30um is added to the detection area 21 by micro-spotting, and the probe will naturally adsorb to the interface. The probe can also be coated on nanoparticles, including polystyrene particles, silica particles, and magnetic beads, and then the particle solution is spotted in the detection area 21. After the solution evaporates, the nanoparticles will be firmly adsorbed on the interface and will not easily fall off or dissolve in the aqueous solution again. Alternatively, the device bonding can be completed first, and when the actual work is in progress, the nanoparticles of the modified probe are limited to the detection area 21 by the electric field.

[0054] The third channel 40 corresponds to the second channel 20 and is located on the other side of the nanoflow channel 30. Under the action of an external bias, ion concentration polarization occurs on both sides of the nanoflow channel 30, that is, ion enrichment occurs on one side and ion depletion occurs on the other side. The third channel 40 corresponds to the ion concentration polarization depletion side. Unlike the second channel 20, the resistivity of the third channel 40 will increase many times. In order to reduce the pressure drop in the third channel 40, the third channel 40 is usually designed as a large channel, at the order of hundreds of microns, which can increase convection and diffusion, reduce the pressure drop in the third channel 40 and the interference with the enrichment process.

[0055] The nanoflow channel 30 refers to a channel array with a characteristic size at the nanometer level, and therefore has particle selectivity. When charged particles migrate to the nanoflow channel 30 with ion selectivity, they will be affected by the wall charge of the nanoflow channel 30. Normally, ions with the same electrical properties as the wall charge can pass through, while ions with opposite electrical properties to the wall charge will be blocked.

[0056] The nanoflow channel 30 is connected between the second channel 20 and the third channel 40. Therefore, when the charged particles with the opposite polarity to the wall charge of the nanoflow channel 30 migrate to the second channel 20, due to the effect of the wall charge of the nanoflow channel 30, the charged particles cannot enter the nanoflow channel 30, but will migrate along the second channel 20 to the tip of the second channel 20. At the same time, the nanoflow channel 30 will generate electroosmosis (a fluid flow phenomenon driven by an electric field, which usually only occurs at the micrometer / nanometer scale) under the drive of the external bias, so the charged particles in the second 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 second channel 20, while for low-mobility molecules, they will be flushed out of the second channel 20 by the electroosmotic flow. Through reasonable design, the enrichment of biochemical marker molecules can be achieved, and the impurity molecules are washed away by the electroosmotic flow, which ultimately achieves the enrichment and purification effect of biochemical marker molecules, avoiding cell debris and the like from entering the second channel 20 to cause blockage, or too many background protein molecules are enriched to cause protein precipitation.

[0057] During operation, a buffer solution is added through the first liquid inlet 51, and the solution is allowed to stand for 1-3 minutes. After the buffer solution fills the second channel 20 and the nanoflow channel 30 through capillary action, a buffer solution is added through the second liquid inlet 41. The buffer solution is PBS, or KCl, etc., and the ion concentration of the buffer solution is 1-10mM. After the flow channel structure in the micro-nanofluidic chip 100 is filled with the buffer solution, it can be temporarily packaged and stored with Scotch tape. During testing, the sample to be tested is added to the first liquid inlet 51 to replace the pre-added buffer solution. The first liquid inlet 51 is grounded, and the second liquid inlet 41 is connected to the positive potential / working electrode. The charged particles in the sample to be tested will migrate into the second channel 20 under the action of the electric field force. Negatively charged particles are restricted by the nanoflow channels 30 on both sides of the second channel 20, and will continue to be enriched in the second channel 20. The enrichment process is stable and not easily affected by environmental factors. At the same time, the required working voltage is low (<10V), and the in-situ enrichment of biochemical target molecules can be achieved by tens of thousands of times within 10 minutes.

[0058] Subsequently, biochemical sensing technology can be used to quantitatively detect the enriched biochemical molecules. The detection area 21 can integrate a variety of sensors, such as optical sensors, electrochemical sensors, etc., for real-time monitoring of the characteristics of the enriched biochemical molecules. These sensors can detect biochemical molecules with high sensitivity and selectivity, and convert the detection signals into electrical signals or optical signals. For optical sensors, fluorescence, absorption spectroscopy, surface plasmon resonance and other technologies can be used for detection. Electrochemical sensors can detect biochemical molecules by measuring changes in current or potential. Biosensors use the specific binding reaction of biological recognition elements (such as enzymes, antibodies, nucleic acids, etc.) with target biochemical molecules to generate specific biological signals, thereby realizing the detection of biochemical molecules.

[0059] The embodiment of the present application is based on the principle of nanofluids and combined with microelectronic process technology to construct a micro-nanofluidic chip 100 for efficient enrichment of biochemical target molecules. The micro-nanofluidic chip 100 combines the advantages of microfluidic technology for precise control of microfluids and nanofluidic technology for dynamic controllable regulation of target molecules, and integrates a whole set of detection processes including sample addition, specific separation, enrichment, and detection on the micro-nanofluidic chip 100, so that the entire detection process is miniaturized, automated, and integrated. On the one hand, the micro-nanofluidic chip 100 can realize multi-channel parallel enrichment and detection through the parallel design of multiple second channels 20, realize the simultaneous detection of multiple biochemical markers, and provide comprehensive biochemical information, which greatly reduces the cost of disease screening and provides strong support for early screening of diseases, disease monitoring, and treatment effect evaluation. On the one hand, the micro-nanofluidic chip 100 realizes accurate detection of enriched biochemical molecules through advanced biosensing technology, with high sensitivity and high selectivity. On the one hand, the micro-nanofluidic chip 100 uses micro-nanofluidic technology to achieve rapid separation and multi-channel enrichment, which can save time and improve efficiency. On the one hand, the micro-nanofluidic chip 100 is manufactured using micro-nano processing technology, which is not only low-cost and miniaturized, but also easy to use in on-site diagnostic environments.

[0060] In a specific embodiment, 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.

[0061] 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.

[0062] Optionally, the nano-flow channel 30 is configured as a nano-gap, and the nano-gap is formed by assembling nanoparticles.

[0063] 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.

[0064] 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.

[0065] In a specific embodiment, the second channel 20 is usually 100-800 um long, 5-30 um wide, and 1-5 um deep, and the specific size needs to be determined according to actual needs.

[0066] In a specific embodiment, the channel surface can be modified, and probes corresponding to biochemical marker molecules can be introduced into the second 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 second channel 20.

[0067] In one embodiment, the number of the third channels 40 is two, and the two third channels 40 are disposed on both sides of the first channel 50. By providing two third channels 40, the design flexibility of the micro-nanofluidic chip 100 can be further improved, thereby increasing the number of second channels 20 and increasing the types of separated markers.

[0068] In one embodiment, the third channel 40 includes a third main channel 42 and a plurality of third branch channels 43 connected to the third main channel 42, and each third branch channel 43 is connected to a plurality of second channels 20 through a nano-flow channel 30. By configuring the third channel 40 as a third main channel 42 and a plurality of third branch channels 43 connected to each other, and making each third branch channel 43 connected to a plurality of second channels 20, the number of second channels 20 can be further increased, thereby increasing the types of separated markers.

[0069] Optional, such as Figure 3As shown, when the number of the third channel 40 is one, the first channel 50 is optimized and includes a first main channel 52 and a plurality of first branch channels 53 connected to the first main channel 52, and the first branch channels 53 are forked between two adjacent third branch channels 43 to form a cross-finger structure. A nano-flow channel 30 is provided on one side of the third branch channel 43 adjacent to the adjacent first branch channel 53, and the nano-flow channel 30 is connected to the first branch channel 53 adjacent to the corresponding side through a plurality of second channels 20. At this time, the first liquid inlet 51 is provided at both ends of the first main channel 52 and the free end of the first branch channel 53. The second liquid inlet 41 is provided at both ends of the third main channel 42 and the free end of the third branch channel 43.

[0070] Furthermore, the first main channel 52 and the third main channel 42 may be arranged at an angle, preferably in parallel.

[0071] Furthermore, the first branch channel 53 and the third branch channel 43 may be arranged at an angle, preferably in parallel.

[0072] Furthermore, the plurality of second channels 20 connected between adjacent first branch channels 53 and third branch channels 43 may be arranged at an angle, preferably in parallel.

[0073] Optional, such as Figure 4 As shown, when the number of the third channels 40 is two and they are arranged on both sides of the first channel 50, the third branch channel 43 of one of the third channels 40 is forked between the two adjacent third branch channels 43 of the other third channel 40 to form a cross-finger structure. At this time, the first channel 50 is S-shaped and arranged along the gap between the cross-finger structures. Nanoflow channels 30 are arranged on both sides of each third branch channel 43, and each nanoflow channel 30 is connected to the first channel 50 through a plurality of second channels 20. At this time, the first liquid inlet 51 is arranged at both ends of the first channel 50, and the second liquid inlet 41 is arranged at both ends of the third main channel 42 and the free end of the third branch channel 43.

[0074] Furthermore, the two third main channels 42 may be arranged at an angle, preferably in parallel.

[0075] Furthermore, the plurality of third branch channels 43 may be arranged at an angle, preferably in parallel.

[0076] Furthermore, a portion of the first channel 50 that is in communication with the third branch channel 43 through the second channel 20 and the nano-flow channel 30 is disposed in parallel with the third branch channel 43 .

[0077] Furthermore, the plurality of second channels 20 connected between adjacent first channels 50 and third branch channels 43 may be arranged at an angle, preferably in parallel.

[0078] like Figure 2 As shown, in one embodiment, the two third channels 40 are symmetrically arranged about the first channel 50. By making the two third channels 40 symmetrical about the first channel 50, the number of second channels 20 can be doubled, so that more second channels 20 can be obtained, thereby increasing the types of separated markers.

[0079] Furthermore, the two symmetrically arranged third channels 40 may be arranged at an angle, and are preferably arranged in parallel.

[0080] The second liquid inlet 41 can be disposed at both ends of the third channel 40, or can be connected to the third channel 40 through a zigzag channel that is connected to the third channel 40 and arranged at an angle. Specifically, the zigzag channel is offset to the side away from the first channel 50 to avoid interference in liquid inlet caused by the first liquid inlet 51 and the second liquid inlet 41 being too close.

[0081] In one embodiment, the first channel 50 and the third channel 40 are arranged in a curved shape. In this case, the first liquid inlet 51 is arranged at both ends of the first channel 50, and the third liquid inlet is arranged at both ends of the third channel 40. In this way, the length of the first channel 50 and the third channel 40 can be extended by bending, thereby increasing the number of the second channels 20.

[0082] Optionally, the nano-flow channels 30 may be continuously arranged along the extension direction of the first channel 50 or the third channel 40 , or may be arranged at intervals along the extension direction of the first channel 50 or the third channel 40 .

[0083] In one embodiment, the bending position of the first channel 50 and / or the third channel 40 is set in an arc shape, or the bending position of the first channel 50 and / or the third channel 40 is set at an angle. This can further expand the design flexibility of the micro-nanofluidic chip 100, and the solution can be designed as needed.

[0084] One or more bending positions may be set on the first channel 50 and the third channel 40. The bending position may be set in an arc shape, wherein the curved arc shape may be a major arc, a minor arc or a semicircular arc. The bending position may also be set at an angle, wherein the angle ranges from 45° to 150°, such as 60°, 90°, 120°, etc. When there are multiple bending positions on the same channel, the setting methods of the multiple bending positions may be the same or different. The setting methods of the bending positions on different channels may be the same or different.

[0085] Optional, such as Figure 5 As shown, the first channel 50, the third channel 40 and the nano-flow channel 30 are each provided with two bending positions, and each bending position is arranged in a semicircular arc.

[0086] Optional, such as Figure 6As shown, the first channel 50, the third channel 40 and the nano-flow channel 30 are each provided with six bending positions, and each bending position is arranged at a right angle.

[0087] In one embodiment, the second channel 20 is arranged in a straight line, a broken line and / or a curved line. In this way, the second channel 20 can be designed according to the electroosmotic flux requirement and the enrichment performance requirement, thereby further ensuring the parallel classification and enrichment requirements of the micro-nanofluidic chip 100.

[0088] The other end of the second channel 20 relative to the detection area 21 is arranged at an angle with the first channel 50 . Specifically, the angle ranges from 30° to 90°.

[0089] One end of the detection zone 21 in the second channel 20 is connected to the nanoflow channel 30. Specifically, Figure 8 As shown, when the second channel 20 is linear, the second channel 20 is connected to the nanoflow channel 30 only through the detection zone 21. Figure 9-12 As shown, when the second channel 20 is in a zigzag or curved shape, except for the detection area 21 , part of the second channel 20 extends along the length direction of the nanoflow channel 30 and is connected to the nanoflow channel 30 .

[0090] In the same micro-nanofluidic chip 100, Fig. 9 As shown, the shapes of the plurality of second channels 20 may be the same, such as Figure 10-12 As shown, the shapes of the plurality of second channels 20 may also be different.

[0091] In one embodiment, when the second channel 20 is arranged in a folded line shape, the second channel 20 includes one or more folded corners, and the angle of each folded corner is between 30° and 150°.

[0092] Optional, such as Fig. 9 As shown, the second channel 20 has an angle, and each angle is 90°. Of course, the angle of the angle can also be any angle between 30-150°, such as 45°, 60°, 120°, 150°, etc. Fig.10 As shown, in the second channel 20 , the two channels arranged at an angle may be arranged perpendicular to or at an angle to the length direction of the first channel 50 or the nano-flow channel 30 .

[0093] In one embodiment, the width of the second channel 20 is 5-30 um, for example, the width of the second channel 20 can be 8 um, 10 um, 15 um, 20 um, 24 um, 28 um, which can be selected according to the needs. Fig.10 As shown, in the same micro-nanofluidic chip 100, the widths of multiple second channels 20 can be the same or different, and in the same second channel 20, the widths of the second channel 20 can be the same or can be set to different widths according to the broken line segments.

[0094] Figure 10-12 Eight different first channel 50 designs are shown in order to fully demonstrate various possible structural options, and it does not mean that the actual chip will contain all eight designs at the same time. In actual chips, one of the eight designs is usually selected, and then dozens to hundreds of them are arranged in parallel.

[0095] In one embodiment, the first channel 50 and the third channel 40 are stacked, and the plurality of second channels 20 are vertically arranged with respect to the first channel 50 and the third channel 40. Figure 7 As shown, in this embodiment, the first channel 50, the second channel 20, the third channel 40 and the nano-flow channel 30 are arranged in three dimensions to achieve an ultra-high-density enrichment channel array design.

[0096] The above embodiment expands the design form of the second channel 20 by expanding the shape, width, bending angle and other aspects of the second channel 20, which can further enrich the design form of the second channel 20, thereby facilitating the differentiated design of the second channel 20, thereby facilitating the second channel 20 to enrich marker molecules with different characteristics.

[0097] In a specific embodiment, the body includes a substrate and a cover plate, the nanoflow channel 30 is arranged on the substrate; the first channel 50 and the third channel 40 are arranged on the cover plate, the second channel 20 can be arranged on the substrate or on the cover plate, and the cover plate and the substrate are adapted for bonding packaging.

[0098] In order to prepare the micro-nano fluidic chip 100, the device flow channel structure is processed by MEMS (micro-electromechanical system) process, and silicon, glass, quartz and other materials can be selected as substrate materials. The nanoflow channel 30 can be made of nanopores, nanochannels, nano shallow grooves, etc. prepared by micromachining, or porous silicon, PAA, nafion, MOF and other non-micromachining nanomaterials, or nanoparticles are self-assembled to form nano gaps. The first channel 50 has strict dimensional parameters and is prepared by micromachining to accurately control its size. The first channel 50 can be processed on the substrate or on the cover material. The third channel 40 and the second channel 20 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. Finally, bonding and packaging are performed to complete the construction of the entire micro-nano fluidic chip 100. In order to reduce the preparation cost, the cover material can be selected from polymer materials such as PMMA.

[0099] The embodiment of the present invention further provides a parallel enrichment detection method for biochemical marker molecules, which is performed using any one of the micro-nanofluidic chips described above, comprising:

[0100] Adding buffer to the first liquid inlet and the second liquid inlet, standing until the buffer fills the first channel, the second channel, the third channel and the nanoflow channel, and then packaging and storing;

[0101] Injecting the sample to be tested into the first liquid inlet, and applying voltages of different magnitudes to the first liquid inlet and the second liquid inlet to form an electric field directed from the second liquid inlet to the first liquid inlet, so that the biochemical marker molecules are enriched in the second channel;

[0102] The biochemical marker molecules enriched in the detection area of ​​the second channel are subjected to sensing analysis.

[0103] Embodiment 1:

[0104] This embodiment Figure 2 Taking the micro-nanofluidic chip 100 shown as an example, the preparation process and use method of the micro-nanofluidic chip 100 in the present application are further explained.

[0105] Specifically, the substrate material of the micro-nanofluidic chip 100 is quartz glass, 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 a nano-shallow groove array, which can be prepared in a standardized manner, and the characteristic size of the nano-shallow groove is less than 50nm, meeting the ion selectivity requirements; the second channel 20 and the first channel 50 are prepared by PDMS molding.

[0106] 1. Device preparation:

[0107] 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 2um, 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 unit. Then, using etching technology such as IBE, 50nm of quartz is etched, and finally laser scribing is performed to complete the preparation of the device substrate.

[0108] 2. SU-8 photolithography to prepare a PDMS mold. The mold is designed as a SU-8 double-layer structure. The first layer structure represents the second channel 20, and the second layer structure represents the first channel 50 and the third channel 40. The second channel 20 is a bent structure array, symmetrically distributed on both sides of the first channel 50, with a total of 120. The second channel 20 has a depth of 2um, a width of 7.5um, a length of 100um+100um, and a period of 150um. The tip of the second channel 20 is disc-shaped, with a diameter of 30um and a depth of 2um, which serves as the detection area 21. The first channel 50 and the third channel 40 are 50um deep and 200um wide. After the mold is prepared, the second channel 20 and other structures are prepared by the mold reversing process. The material is PDMS, and the ratio of PDMS to curing agent is 5:1. The commonly used 10:1 is not used to enhance the hardness of PDMS and prevent collapse during the subsequent bonding process.

[0109] 3. The capture antibody is modified on 200nm polystyrene particles by covalent bonding, and then the antibody-modified nanoparticle suspension is spotted on the detection area 21 by micro-spotting, with a spot diameter of 20um. After drying, it is placed in a sealed bag for future use. If 20 markers need to be detected simultaneously, 20 types of capture antibodies can be spotted separately.

[0110] 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, place it on a 90 degree hot plate and bake for 5 to 10 minutes.

[0111] 2. Prepare the reagents required for the test

[0112] 1.1mM PBS buffer: Use 1×PBS to prepare the buffer required for enrichment detection, and dilute 1×PBS 150 times with deionized water;

[0113] 2. Blocking solution: Add 0.1% BSA and 0.05% tween-20 to 1 mM PBS buffer;

[0114] 3. Washing solution: Add 0.05% tween-20 to 1 mM PBS buffer;

[0115] 4. Prepare the samples to be tested.

[0116] 3. Enrichment Detection

[0117] During operation, the first liquid inlet 51 is sequentially added with the blocking liquid, and the solution is allowed to stand for 1-3 minutes. After the blocking liquid fills the second channel 20 and the nanoflow channel 30 through capillary action, the multiple second liquid inlets 41 are sequentially added with the blocking liquid. After the flow channel structure in the micro-nanofluidic chip 100 is filled with the blocking liquid, it is packaged with Scotch tape and stored at 4 degrees. It is sealed for about 30 minutes, and then the blocking liquid is replaced with a washing solution. Finally, the sample to be tested is added to one of the first liquid inlets 51. The two first liquid inlets 51 are grounded, and the multiple second liquid inlets 41 are connected to the positive potential / working electrode. The charged particles in the sample to be tested will be affected by the electric field force and migrate into the parallel second channel 20.

[0118] 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, the first channel is a sample channel; the first channel is a large channel, the size of which is in the order of hundreds of microns, and a first liquid inlet is provided on the first channel; A plurality of second channels, each of the second channels having a first end and a second end, the second end being provided with a detection zone, and each of the first ends being connected to the first channel; the characteristic dimension of the second channels being 5-30 μm; a third channel, wherein the characteristic size of the third channel is in the order of hundreds of microns; the third channel is connected to the second liquid inlet, and the second liquid inlet is arranged at both ends of the third channel; A nano-flow channel, wherein the nano-flow channel has an ion selection function, one end of the nano-flow channel is connected to the third channel, and the other end of the nano-flow channel is respectively connected to the second ends of the plurality of second channels; The number of the third channels is two, and the two third channels are arranged on both sides of the first channel; The first liquid inlet and the second liquid inlet are also used to connect electrodes to apply bias voltage to the micro-nanofluidic chip.

2. The micro-nanofluidic chip according to claim 1, characterized in that: The third channel includes a third main channel and a plurality of third branch channels communicating with the third main channel, and each of the third branch channels is communicated with a plurality of the second channels through the nano-flow channel.

3. The micro-nanofluidic chip according to claim 1, characterized in that: The two third channels are symmetrically arranged with respect to the first channel.

4. The micro-nanofluidic chip according to claim 1, characterized in that: The first channel and the third channel are arranged in a curved shape.

5. The micro-nanofluidic chip according to claim 4, characterized in that: The bending position of the first channel and / or the third channel is arranged in an arc shape, or The bending positions of the first channel and / or the third channel are arranged at an angle.

6. The micro-nanofluidic chip according to claim 1, characterized in that: The second channel is arranged in a straight line, a folded line and / or a curved line.

7. The micro-nanofluidic chip according to claim 6, characterized in that: When the second channel is arranged in a folded line shape, the second channel includes one or more folded corners, and the angle of each folded corner is between 30-150°.

8. The micro-nanofluidic chip according to claim 1, characterized in that: The first channel and the third channel are stacked, and the plurality of second channels are vertically arranged to the first channel and the third channel.

9. A parallel enrichment detection method for biochemical marker molecules, characterized in that: The method is accomplished by using the micro-nanofluidic chip according to any one of claims 1 to 8, comprising: Adding buffer to the first liquid inlet and the second liquid inlet, standing until the buffer fills the first channel, the second channel, the third channel and the nanoflow channel, and then packaging and storing; Injecting the sample to be tested into the first liquid inlet, and applying voltages of different magnitudes to the first liquid inlet and the second liquid inlet to form an electric field directed from the second liquid inlet to the first liquid inlet, so that the biochemical marker molecules are enriched in the second channel; The biochemical marker molecules enriched in the detection area of ​​the second channel are subjected to sensing analysis.

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