CN117531553A - Micro-nanofluidic chip and parallel enrichment detection method of biochemical marker molecules - Google Patents

Abstract

The invention belongs to the technical field of micro-nano fluidics and biochemical sensing, and specifically relates to a micro-nano fluidic chip and a parallel enrichment detection method of biochemical marker molecules. The chip includes a chip body, and the chip body is provided with a third One 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 The first channel is connected; the third channel is connected with the second liquid inlet; the nanoflow channel has an ion selection function, one side of the nanoflow channel is connected with the third channel, and the other side is connected with a plurality of third channels respectively. The two channels are connected. The micro-nanofluidic chip of this application can realize the simultaneous enrichment and detection of dozens or even hundreds of biochemical marker molecules through the parallel second channel design, which greatly reduces the cost of disease screening and provides early screening and disease monitoring for diseases. and provide strong support for treatment effectiveness evaluation.

Description

Micro-nanofluidic chip and parallel enrichment detection method of biochemical marker molecules

Technical field

The invention belongs to the technical fields of micro-nano fluidics and biochemical sensing, and specifically relates to a micro-nano fluidic chip and a parallel enrichment detection method of biochemical marker molecules.

Background technique

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

Contents of the invention

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

The invention provides a micro-nanofluidic chip, which includes a chip body, and the chip body is provided with:

A first channel, the first channel is provided with a first liquid inlet;

A plurality of second channels, each second channel is provided with a detection area, and each second channel is connected to the first channel;

a third channel, the third channel is connected with the second liquid inlet;

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

The micro-nanofluidic chip provided by the present invention can also have the following additional technical features:

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

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 passes through the nanoflow channel. Connected to a plurality of second channels.

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

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

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

The bending position of the first channel and/or the third channel is arranged at a right angle.

In a specific embodiment of the present invention, the second channel is arranged in a linear, zigzag and/or curved shape; and/or the characteristic size of the second channel is 5-30um.

In a specific embodiment of the present invention, 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°.

In a specific embodiment of the present invention, the first channel and the third channel are arranged in a stack, and a plurality of the second channels are arranged perpendicularly to the first channel and the third channel.

The second aspect of the present invention also provides a parallel enrichment and detection method of biochemical marker molecules, which is completed by using any of the above-mentioned micro-nanofluidic chips, including:

Add the buffer solution to the first injection port and the second injection port, and let it stand until the buffer solution fills the first channel, the second channel, the third channel and the nanoflow channel, and then it is packaged and stored;

Inject the sample to be tested into the first injection port, and at the same time load voltages of different sizes on the first and second injection ports to form an electric field directed from the second injection port to the first injection port, so that the biochemical markers Molecules are enriched in the second channel;

Sensing analysis is performed on the biochemical marker molecules enriched in the detection area of the second channel.

The embodiment of this application constructs a micro-nanofluidic chip for efficient enrichment of biochemical target molecules based on the principle of nanofluidics and combined with microelectronics technology. This micro-nanofluidic chip combines the advantages of microfluidic technology for precise control of microfluids and nanofluidic technology for dynamically controllable regulation of target molecules. It integrates the entire detection process including sample addition, specific separation, enrichment, and detection. On the micro-nanofluidic chip, the entire detection process is miniaturized, automated and integrated. At the same time, through the parallel second channel design, the simultaneous enrichment and detection of dozens or even hundreds of biochemical marker molecules can be achieved, thereby providing clinical diagnosis. Sufficient biochemical information greatly reduces the cost of disease screening and provides strong support for early screening of diseases, disease monitoring and treatment effect evaluation.

Description of drawings

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description The drawings illustrate some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 2 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 3 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 4 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 5 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 6 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 7 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 8 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 9 is a front view of Figure 2;

Figure 10 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 11 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention;

Figure 12 is a schematic structural diagram of a micro-nanofluidic chip in one embodiment of the present invention.

Explanation of reference symbols:

100-Micro-nanofluidic chip;

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-the first channel, 51-the first liquid inlet, 52-the first main channel, 53-the first branch channel.

Detailed ways

Exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a thorough understanding of the invention, and to fully convey the scope of the invention to those skilled in the art.

It is to be understood that the terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The terms "comprises", "includes", "contains" and "having" are inclusive and thus indicate the presence of stated features, steps, operations, elements and/or parts but do not exclude the presence or addition of one or Various other features, steps, operations, elements, components, and/or combinations thereof. The method steps, procedures, and operations described herein are not to be construed as requiring that they be performed in the particular order described or illustrated, unless an order of performance is expressly indicated. It should also be understood that additional or alternative steps may be used.

Although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections shall not be referred to as restricted by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Terms such as "first," "second," and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the example embodiments.

For ease of description, spatially relative terms may be used herein to describe the relationship of one element or feature to another element or feature as shown in the figures. These relative terms, such as "inner", "outer", "inner" ”, “outside”, “below”, “below”, “above”, “above”, etc. Such spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" or "beneath" the other elements or features. Features above". Thus, the example term "below" may include an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

Driven by an external bias voltage, charged particles will migrate directionally through electrophoresis. When charged particles migrate to a nanostructure with particle selectivity, they will be affected by the charge on the wall of the nanostructure. Normally, ions with the same electrical charge as the wall charge can pass through, but ions with the opposite electrical charge as the wall charge will be hindered. This phenomenon is unique to nano-sized fluids and is called ion selectivity. When the thickness of the electric double layer on the inner surface of the nanostructure is close to or larger than the characteristic size of the nanostructure, the nanostructure will produce ion selectivity.

Below the scale of hundreds of nanometers, the flow channel structure has a very high surface-to-body ratio, and the physical phenomena produced will be different from the field of microfluidics. At this scale, the role played by the flow channel surface will be dominant. Among them, the most important physical phenomena need to be considered. It's the electric double layer. When a solid substance comes into contact with a liquid, due to the dissociation of surface molecular groups, the solid surface will have a layer of fixed charge. The ions in the solution will be affected by the Coulomb force of the fixed charge on the solid surface, resulting in repulsion of same ions and counter ions in the solid. Surface convergence to maintain electrically neutral conditions at the solid surface. This will form a shielding area in the solution close to the solid surface. This area is mainly composed of counterions. We call this area an electric double layer.

In microfluidic research, the size of the flow channel is on the order of microns, and the influence of the electric double layer can be completely ignored. However, in the field of nanofluidics, the characteristic length of the flow channel is no longer large enough to ignore the electric double layer. The physical quantity that usually characterizes the characteristic thickness of the electric double layer is the Debye length λD. For a 1mM KCl solution, the Debye length λD has reached 10nm. This value is close to the characteristic length of the nanochannel, and even larger than the characteristic size of the nanochannel in many cases. Therefore, for the nanochannel, the Debye length is approximately equal to or larger than the channel. Characteristic size, the liquid in the channel is charged, and the concentration of counter ions is much higher than that of the same ions. This also makes the nanochannel ion selective, allowing only counterions to pass through.

Electric double layers and ion selectivity are phenomena unique to the field of nanofluidics and are often related to the size of the nanostructure and the charge density on the inner surface. The nanostructure size and internal surface charge density are determined by the material's own characteristics and processing parameters, and are fixed once the micro-nanofluidic device is prepared.

Based on this, an embodiment of the present invention proposes a micro-nanofluidic chip 100 that utilizes the above characteristics of nanostructures to enrich biochemical marker molecules.

Referring to Figures 1-12, a micro-nanofluidic chip 100 provided by an embodiment of the present invention includes a chip body 10. The chip body 10 is provided with a first channel 50, a plurality of second channels 20, a third channel 40 and a nanofluidic chip. Flow channel 30, 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 with the first channel 50; the third The 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 connected to the plurality of second channels 20 respectively.

The first channel 50 is provided with a first liquid inlet 51 for introducing the sample to be enriched into the micro-nanofluidic chip 100 through the first liquid inlet 51 and for guiding the sample to be enriched to the second channel 20 . The first channel 50 is usually a large channel, with dimensions on the order of hundreds of microns, and has very low flow resistance. Specifically, the flow rate and flow rate of the sample in the first channel 50 can be precisely controlled through micro-nanofluidic technology to ensure that the sample is fully contacted and interacted with 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 buffer, diluent or other auxiliary liquids to further optimize the enrichment effect.

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 driven by the electric field, but are restricted by the micro-nano flow channel 30 and cannot pass through, so they will stay in the second channel 20 and accumulate, ultimately achieving high-fold enrichment of charged particles ( 10 ⁴ -10 ⁷ times enriched). In order to achieve simultaneous enrichment detection of multiple targets, dozens or even hundreds of parallel second channels 20 are designed on the micro-nanofluidic chip 100. Each second channel 20 passes through the micro-nanofluidic channel 30 and the third channel 40. connected to form a complete current loop.

The detection area 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 area 21, specific analysis of the enriched biochemical marker molecules can be achieved. The detection area 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 enriched marker molecules. Through precise control of micro-nanofluidic technology, high sensitivity and high selectivity biosensing can be achieved in the detection zone 21.

The probe coating can be carried out by adsorption. Before device bonding, a probe solution with a diameter of 10-30um is dropped in the detection area 21 by micro-spotting, and the probe will naturally adsorb to the interface. The probe can also be coated on nanoparticles first, including polystyrene particles, silica particles, and magnetic beads, and then the particle solution is spotted on the detection area 21. After the solution evaporates to dryness, the nanoparticles will be firmly adsorbed on the interface. It is not easy to fall off or redissolve in aqueous solution. Alternatively, the device bonding may be completed first, and then during actual work, the nanoparticles of the modified probe may be limited to the detection area 21 through an electric field.

The third channel 40 corresponds to the second channel 20 and is located on the other side of the nanofluid channel 30 . Under the action of an external bias voltage, ion concentration polarization will occur 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. . Different from the second channel 20, the resistivity of the third channel 40 will increase many times. In order to reduce the voltage drop in the third channel 40, the third channel 40 is usually designed as a large channel, in the order of hundreds of microns, which can increase Large convection and diffusion reduce the pressure drop in the third channel 40 and interfere with the enrichment process.

The nanoflow channel 30 refers to a channel array with a characteristic size on the nanometer scale, and therefore has particle selectivity. When the charged particles migrate to the nanoflow channel 30 with ion selectivity, they will be affected by the charge on the wall of the nanoflow channel 30 , usually ions with the same electrical charge as the wall can pass through, but ions with the opposite electrical charge as the wall will be blocked.

The nanoflow channel 30 is connected between the second channel 20 and the third channel 40. Therefore, when the charged particles with opposite polarity to the wall charge of the nanoflow channel 30 migrate to the second channel 20, they are affected by the charge on the wall of the nanoflow channel 30. As a result, the charged particles cannot enter the nanofluid channel 30 , but will migrate along the second channel 20 to the tip of the second channel 20 . At the same time, the nanofluid channel 30 will generate electroosmotic flow (a fluid flow phenomenon driven by an electric field, which usually only occurs at the micron/nano scale) driven by an external bias voltage. Therefore, the charged particles in the second channel 20 are simultaneously subject to the electroosmotic flow drag force. and the effect of 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 and prevents cell debris and the like from entering the second channel 20 to cause blockage or overflow. Protein precipitation occurs due to the enrichment of multiple background protein molecules.

When working, add the buffer solution through the first liquid inlet 51 and let it stand for 1-3 minutes. After the buffer liquid fills the second channel 20 and the nanoflow channel 30 through capillary action, add the buffer liquid through the second liquid inlet 41. The buffer is PBS, or KCl, etc., and the buffer ion concentration is 1-10mM. After the flow channel structure in the micro-nanofluidic chip 100 is filled with buffer solution, it can be temporarily sealed 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. The first liquid inlet 51 is connected to the ground, and the second liquid inlet 41 is connected to the positive potential/working electrode. The charged particles in the sample to be measured will migrate into the second channel 20 under the action of the electric field force. The 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 operating voltage is low (<10V), and it can achieve tens of thousands of times in-situ enrichment of biochemical target molecules within 10 minutes.

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 enriched biochemical molecules. These sensors can detect biochemical molecules with high sensitivity and selectivity, and convert detection signals into electrical or optical signals. For optical sensors, techniques such as fluorescence, absorption spectroscopy, and surface plasmon resonance can be used for detection. Electrochemical sensors can detect biochemical molecules by measuring changes in current or potential. Biosensors use specific binding reactions between biological recognition elements (such as enzymes, antibodies, nucleic acids, etc.) and target biochemical molecules to generate specific biological signals, thereby realizing the detection of biochemical molecules.

The embodiment of the present application constructs a micro-nanofluidic chip 100 for efficient enrichment of biochemical target molecules based on the principle of nanofluidics and combined with microelectronic process technology. The micro-nanofluidic chip 100 combines the microfluidic technology with microfluidic control of microfluids. With the advantages of precise control and nanofluidic technology for dynamic controllable regulation of target molecules, the entire detection process including sample addition, specific separation, enrichment, and detection is integrated on the micro-nanofluidic chip 100, making the entire detection process miniaturized and Automation and integration. On the one hand, the micro-nanofluidic chip 100 is designed in parallel through multiple second channels 20 to achieve multi-channel parallel enrichment and detection, realize simultaneous detection of multiple biochemical markers, and provide comprehensive biochemical information, which greatly reduces the risk of disease. Screening costs provide strong support for early disease screening, condition monitoring and treatment effect evaluation. On the one hand, the micro-nanofluidic chip 100 achieves precise detection of enriched biochemical molecules through advanced biosensing technology, with high sensitivity and 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-nano fluidic chip 100 is prepared using micro-nano processing technology, which is low-cost and miniaturized, and can be easily applied in on-site diagnostic environments.

In a specific embodiment, the nanofluid channel 30 forms multiple microchannels with a characteristic size less than 100 nm by arranging the following structures: a nanohole array, a nanochannel array, a nanoshallow groove array, and a nanoslit.

Optionally, the nanofluidic channel 30 is configured as a nanohole array, a nanochannel array, and a nanoshallow groove array. The nanohole array, nanochannel array, and nanoshallow groove array can be formed by micromachining the chip body 10, or they can be formed. Non-micromachined nanomaterials such as porous silicon, PAA, nafion, and MOF with nanohole arrays, nanochannel arrays, and nanoshallow groove arrays are stacked on the chip body 10 to form.

Optionally, the nanoflow channel 30 is set as a nanogap, and the nanogap is formed by assembling nanoparticles.

Optionally, the nanoflow channel 30 is configured as a nanohole array, a nanochannel array, or a nanoshallow groove array filled with nanomaterials, where the filling material can be nafion, and the nanohole array, nanochannel array, or nanoshallow groove array can be It is formed by micro-machining, or it can be formed by non-micro-machining nanomaterials such as porous silicon and PAA.

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

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

In a specific embodiment, the channel surface can be modified, and a probe corresponding to the biochemical marker molecule can be introduced into the second channel 20 to specifically identify and capture the enriched biochemical marker. For example, by specifically labeling a target biochemical molecule with a nucleic acid aptamer or a nucleic acid-modified antibody, the charge-to-body ratio can be much greater than the background protein and other impurities, so that only a small amount of the target biochemical molecule and its charge-to-body ratio can be achieved. The molecules are enriched into the second channel 20.

In one embodiment, the number of the third channels 40 is two, and the two third channels 40 are located on both sides of the first channel 50 . By setting 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 thereby increasing the types of separated markers.

In one embodiment, the third channel 40 includes a third main channel 42 and a plurality of third branch channels 43 connected with the third main channel 42. Each third branch channel 43 communicates with a plurality of second channels through the nanoflow channel 30. 20 connected. By configuring the third channel 40 to be a connected third main channel 42 and a plurality of third branch channels 43, 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. quantity, thereby increasing the types of separated markers.

Optionally, as shown in FIG. 3 , 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 third channels connected to the first main channel 52 . A branch channel 53 is formed, and the first branch channel 53 is forked between two adjacent third branch channels 43 to form an interdigitated interdigitated structure. The third branch channel 43 is provided with a flow channel 30 on a side adjacent to the adjacent first branch channel 53. The flow channel 30 is connected to the adjacent first branch channel 53 on 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 .

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

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

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

Optionally, as shown in FIG. 4 , 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 forks into the other third channel. An interdigitated interdigitated structure is formed between two adjacent third branch channels 43 of 40. At this time, the first channel 50 is in an S shape and is arranged along the gap between the interdigitated structures. Nanoflow channels 30 are provided 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 provided at both ends of the first channel 50 , and 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 .

Furthermore, the two third main channels 42 can be arranged at an angle, preferably in parallel.

Furthermore, the plurality of third branch channels 43 can be arranged at angles, preferably in parallel.

Furthermore, the portion of the first channel 50 that communicates with the third branch channel 43 through the second channel 20 and the nanoflow channel 30 is arranged in parallel with the third branch channel 43 .

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

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

Furthermore, the two symmetrically arranged third channels 40 may be arranged at an angle, preferably in parallel.

The second liquid inlet 41 can be provided at both ends of the third channel 40 , or can be connected to the third channel 40 through a folded line channel that is connected to the third channel 40 and arranged at an angle. Specifically, the folded channel is shifted away from the first channel 50 to avoid liquid inlet interference caused by the first liquid inlet 51 and the second liquid inlet 41 being too close to each other.

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

Optionally, the nanofluid 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 .

In one embodiment, the bending positions of the first channel 50 and/or the third channel 40 are arranged in an arc shape, or the bending positions of the first channel 50 and/or the third channel 40 are arranged in an included angle. This can further expand the design flexibility of the micro-nano fluidic chip 100, and the solution can be designed on demand.

One or more bending positions may be provided on the first channel 50 and the third channel 40 . The bending position can be set in an arc shape, in which the bending arc shape can be a superior arc, a minor arc or a semi-circular arc. The bending position can also be set in an included angle, where the included angle ranges from 45° to 150°, specifically as follows 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 bending positions on different channels can be set in the same way or in different ways.

Optionally, as shown in FIG. 5 , the first channel 50 , the third channel 40 and the nanofluid channel 30 are each provided with two bending positions, and each bending position is arranged in a semicircular arc.

Optionally, as shown in FIG. 6 , the first channel 50 , the third channel 40 and the nanofluid channel 30 are each provided with six bending positions, and each bending position is arranged at a right angle.

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

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

One end of the detection area 21 in the second channel 20 is connected to the nanofluid channel 30. Specifically, as shown in Figure 8, when the second channel 20 is linear, the second channel 20 is only connected to the nanofluid channel 30 through the detection area 21. . As shown in FIGS. 9-12 , 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 with the nanoflow channel 30 .

In the same micro-nanofluidic chip 100, as shown in FIG. 9, the shapes of the plurality of second channels 20 may be the same. As shown in FIGS. 10-12, the shapes of the plurality of second channels 20 may also be different.

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 30-150°.

Optionally, as shown in FIG. 9 , the second channel 20 has a folding angle, and the angle of each folding angle is 90°. Of course, the angle of the folding angle can also be any angle between 30° and 150°, such as 45°, 60°. °, 120°, 150°, etc. As shown in FIG. 10 , in the second channel 20 , the two channels arranged at an angle may be arranged perpendicularly or at an angle to the length direction of the first channel 50 or the nanoflow channel 30 .

In one embodiment, the width of the second channel 20 is 5-30um. For example, the width of the second channel 20 can be 8um, 10um, 15um, 20um, 24um, or 28um, which can be selected according to needs. As shown in FIG. 10 , in the same micro-nanofluidic chip 100 , the widths of the plurality of second channels 20 can be the same or different, and in the same second channel 20 , the widths of the second channels 20 can be the same, or can be based on Polyline segments are set to different widths.

Figures 10-12 all show 8 different first channel 50 designs. This is to fully demonstrate various possible structural options, and does not mean that the actual application chip will contain all 8 designs at the same time. In actual chips, one of these eight designs is usually selected, and then dozens to hundreds of wires are arranged in parallel.

In one embodiment, the first channel 50 and the third channel 40 are arranged in a stack, and the plurality of second channels 20 are arranged perpendicularly to the first channel 50 and the third channel 40 . As shown in FIG. 7 , this embodiment achieves an ultra-high-density enrichment channel array design by arranging the first channel 50 , the second channel 20 , the third channel 40 and the nanoflow channel 30 in a three-dimensional arrangement.

The above embodiment expands the design form of the second channel 20 in terms of the shape, width, and bending angle 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. This facilitates enrichment of marker molecules with different characteristics in the second channel 20 .

In a specific embodiment, the body includes a substrate and a cover plate, the nanofluid channel 30 is provided on the substrate; the first channel 50 and the third channel 40 are provided on the cover plate, and the second channel 20 can be provided on the substrate, It can also be provided on the cover plate, and the cover plate and the substrate are adapted for bonding and packaging.

In order to prepare the micro-nanofluidic chip 100, the device flow channel structure is processed using MEMS (micro-electromechanical systems) technology, and materials such as silicon, glass, and quartz can be selected as substrate materials. The nanofluid channel 30 can use nanopores, nanochannels, nanoshallow grooves, etc. prepared by micromachining, or can choose nanomaterials prepared by non-micromachining such as porous silicon, PAA, nafion, MOF, or use nanoparticles to self-assemble to form nanogaps. . The first channel 50 has strict dimensional parameters and is prepared by micro-machining to precisely control its size. The first channel 50 can be processed on the substrate or on the cover material. The size of the third channel 40 and the second channel 20 are relatively large, usually in the order of hundreds of microns, and their dimensional accuracy is not strictly required, and they are usually processed on the cover plate. Finally, bonding and packaging are completed to complete the construction of the entire micro-nanofluidic chip 100. In order to reduce the preparation cost, polymer materials such as PMMA can be selected as the cover plate material.

Embodiments of the present invention also provide a method for parallel enrichment and detection of biochemical marker molecules, which is accomplished using any one of the above micro-nanofluidic chips, including:

Add the buffer solution to the first injection port and the second injection port, and let it stand until the buffer solution fills the first channel, the second channel, the third channel and the nanoflow channel, and then seals and saves;

Inject the sample to be tested into the first injection port, and at the same time load voltages of different sizes on the first and second injection ports to form an electric field directed from the second injection port to the first injection port, so that the biochemical markers Molecules are enriched in the second channel;

Sensing analysis is performed on the biochemical marker molecules enriched in the detection area of the second channel.

Example 1:

The micro-nanofluidic chip 100 shown in FIG. 2 of this embodiment is taken as an example to further illustrate the preparation process and usage method of the micro-nanofluidic chip 100 in this application.

Specifically, the substrate material of the micro-nanofluidic chip 100 is quartz glass, which has the advantage of being self-insulating and does not require additional insulation processing steps. It can be micro-processed using MEMS technology; the nanofluidic channel 30 uses a nano-shallow groove array and can be prepared in a standardized manner. , the characteristic size of the nano-shallow groove is less than 50nm, which meets the ion selectivity requirements; the second channel 20 and the first channel 50 are prepared by flipping the PDMS mold.

1. Device preparation:

1. Prepare a 100-nanometer shallow groove array on a quartz glass substrate through stepper photolithography. 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 nanometer shallow groove array is located at Then use etching technology, such as IBE, to etch the quartz to 50nm, and finally laser scribe to complete the preparation of the device substrate.

2. Use SU-8 photolithography to prepare a PDMS flip 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 an array of bent structures, symmetrically distributed on both sides of the first channel 50, with a total of 120 channels. 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, serving as the detection area 21. The first channel 50 and the third channel 40 have a depth of 50um and a width of 200um. After the mold preparation is completed, the mold flipping process is used to prepare the second channel 20 and other structures. The material is PDMS. 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 subsequent bonding. collapse during the process.

3. Use covalent bonding to modify the capture antibody on 200nm polystyrene particles, and then use micro-spotting to spot the modified antibody nanoparticle suspension in the detection area 21. The spotting diameter 20um, wait for it to dry and then put it in a sealed bag for later use. If you need to detect 20 markers at the same time, 20 types of capture antibodies can be spotted separately.

4. The bonding surface of PDMS and quartz substrate is treated with oxygen plasma. Equipment: oxygen plasma cleaning machine (PDC-MG), power 150w, time 30s, and then aligned and bonded under a microscope. In order to enhance the bonding strength, it can be baked on a 90 degree hot plate for 5 to 10 minutes.

2. Preparation of reagents required for testing

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

2. Blocking solution: Add 0.1% BSA and 0.05% tween-20 to 1mMPBS buffer;

3. Washing solution: Add 0.05% tween-20 to 1mMPBS buffer;

4. Prepare the sample to be tested.

3. Enrichment detection

During operation, the sealing liquid is added to the first liquid inlet 51 in sequence and left for 1-3 minutes. After the sealing liquid fills the second channel 20 and the nanoflow channel 30 through capillary action, multiple second liquid inlets 41 are added to the sealing liquid in sequence. liquid. The flow channel structure in the micro-nanofluidic chip 100 is filled with sealing liquid and then sealed with Scotch tape and stored at 4 degrees. Block for about 30 minutes, then replace the blocking solution with 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 connected to the ground, and the plurality of second liquid inlets 41 are connected to the positive potential/working electrode. The charged particles in the sample to be measured will be affected by the electric field force and migrate into the parallel second channel 20 .

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention. scope.

Claims (10)

1. A micro-nanofluidic chip, characterized in that it includes a chip body, and the chip body is provided with: A first channel, the first channel is provided with a first liquid inlet; A plurality of second channels, each second channel is provided with a detection area, and each second channel is connected to the first channel; a third channel, the third channel is connected with the second liquid inlet; A nanoflow channel has an ion selection function. One side of the nanoflow channel is connected to the third channel, and the other side is connected to a plurality of second channels. 2. The micro-nanofluidic chip according to claim 1, wherein the number of the third channels is two, and the two third channels are respectively located on both sides of the first channel. 3. The micro-nanofluidic chip according to claim 1 or 2, characterized in that the third channel includes a third main channel and a plurality of third branch channels connected to the third main channel, each The third branch channel is connected to a plurality of second channels through the nanoflow channel. 4. The micro-nanofluidic chip according to claim 2, wherein the two third channels are arranged symmetrically with respect to the first channel. 5. The micro-nanofluidic chip according to claim 1 or 2, characterized in that the first channel and the third channel are arranged in a curved shape. 6. The micro-nanofluidic chip according to claim 5, 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 included angle. 7. The micro-nanofluidic chip according to claim 1, characterized in that the second channel is arranged in a linear, zigzag and/or curved shape; and/or the characteristic size of the second channel is 5 -30um. 8. The micro-nanofluidic chip according to claim 7, characterized in that when the second channel is arranged in a folded line, the second channel includes one or more folded corners, and the angle of each folded corner is between Between 30-150°. 9. The micro-nanofluidic chip according to claim 1, wherein the first channel and the third channel are stacked, and a plurality of the second channels are connected with the first channel and the third channel. The third channel is set vertically. 10. A parallel enrichment detection method of biochemical marker molecules, characterized in that it is completed using the micro-nanofluidic chip according to any one of claims 1 to 9, including: Add the buffer solution to the first injection port and the second injection port, and let it stand until the buffer solution fills the first channel, the second channel, the third channel and the nanoflow channel, and then it is packaged and stored; Inject the sample to be tested into the first injection port, and at the same time load voltages of different sizes on the first and second injection ports to form an electric field directed from the second injection port to the first injection port, so that the biochemical markers Molecules are enriched in the second channel; Sensing analysis is performed on the biochemical marker molecules enriched in the detection area of the second channel.