CN117504955B - A gate-controlled micro-nanofluidic device and a method for preparing the same - Google Patents

Abstract

The invention belongs to the technical field of micro-nano fluid and biochemical sensing, and particularly relates to a grid-control micro-nano fluid control device and a preparation method thereof, wherein the grid-control micro-nano fluid control device comprises a chip body, a first channel, a second channel, a nano-flow channel and a grid-control electrode are arranged on the chip body, and the first channel is communicated with a first liquid inlet; the second channel is communicated with the second liquid inlet; the nano-flow channel has an ion selection function and is communicated between the first channel and the second channel; the grid control electrode is arranged at intervals with the nano-flow channel and is suitable for adjusting the ion selection function of the nano-flow channel. The grid-control micro-nano fluidic device provided by the invention has an adjustable enrichment function, and can flexibly change the enrichment mode according to different working conditions and requirements, so as to meet different application requirements.

Description

A gate-controlled micro-nanofluidic device and a method for preparing the same

Technical Field

The invention belongs to the field of micro-nano fluid and biochemical sensing technology, and particularly relates to a gate-controlled micro-nano fluidic device and a preparation method thereof.

Background Art

Micro-nanofluidic devices, also known as Lab on a Chip, can analyze and detect different biochemical molecules through the design and modification of micro-nano flow channel structures. However, once the existing micro-nanofluidic devices are prepared, their functions are fixed and cannot be flexibly adapted to different needs, especially in the field of biochemical molecule enrichment detection, such as capillary electrophoresis chips and nanosieve arrays, which have relatively single functions.

Summary of the invention

The object of the present invention is to provide a gate-controlled micro-nanofluidic device and a preparation method thereof, so as to solve the problem of single function of the gate-controlled micro-nanofluidic device in the prior art.

A first aspect of the present invention provides a gate-controlled micro-nanofluidic device, comprising a chip body, wherein the chip body is provided with:

a first channel, the first channel being in communication with the first liquid inlet;

a second channel, the second channel being in communication with the second liquid inlet;

a nano-flow channel, the nano-flow channel having an ion selection function and connected between the first channel and the second channel;

A gate-controlled electrode is arranged at a distance from the nanoflow channel and is suitable for adjusting the ion selection function of the nanoflow channel.

The gate-controlled micro-nanofluidic device provided by the present invention may also have the following additional technical features:

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

In a specific embodiment of the present invention, the gate-controlled electrode is a single electrode, a plurality of parallel electrodes or an interdigitated electrode, the length direction of the single electrode, the plurality of parallel electrodes or the interdigitated electrode is perpendicular to the flow direction of the nanoflow channel, and is arranged above or below the nanoflow channel; or

The gate-controlled electrode is tubular and is sleeved on the outside of the nanochannel; or

The gate-controlled electrodes are parallel multi-electrodes, which are parallel to the flow direction of the nano-flow channel and are arranged at intervals between adjacent nano-channels or adjacent nano-shallow grooves.

In a specific embodiment of the present invention, the chip body further includes an insulating layer, and the insulating layer is spaced between the gate-controlled electrode and the nanocurrent channel.

In a specific embodiment of the present invention, the thickness of the insulating layer is 0.5-20 um.

In a specific embodiment of the present invention, the chip body further includes a third channel; the first liquid inlet is provided at the end of the third channel, and the middle of the third channel is connected to the first channel.

In a specific embodiment of the present invention, a detection area is provided on the first channel, or the first channel is connected to a downstream analysis system.

In a specific embodiment of the present invention, there are multiple nanoflow channels, the multiple nanoflow channels are arranged at intervals along the length direction of the first channel, and the gate-controlled electrode is provided on at least one of the nanoflow channels.

The second aspect of the present invention further provides a method for preparing a gate-controlled micro-nanofluidic device, which is used to prepare any of the gate-controlled micro-nanofluidic devices described above, comprising:

S1: Execute step a, step b and step c;

S2: execute step d;

S3: Execute step e; wherein,

Step a: patterning a gate-controlled electrode on the surface of a substrate and etching it to form, sputtering a gate-controlled electrode material onto the surface of the substrate and peeling off the gate-controlled electrode material formed on the surface of the substrate to obtain a surface gate-controlled electrode;

Step b: growing an insulating layer on the surface of the substrate;

Step c: patterning the nanofluidic channel on the substrate surface and etching it into shape;

Step d: etching the first channel, the second channel, and the third channel on the substrate surface or the cover plate, and etching a gate-controlled electrode terminal on the substrate surface;

Step e: Bonding the substrate to the cover package.

In a specific embodiment of the present invention, in S1, step a→step b→step c are performed in sequence, or step c→step b→step a are performed in sequence, or step c→step a→step b are performed in sequence.

The gate-controlled micro-nanofluidic device provided in the embodiment of the present invention has an adjustable enrichment function, and can flexibly change the enrichment mode according to different working conditions and requirements, so as to meet different application requirements. At the same time, by utilizing the regulating function of the gate-controlled electrode, an effect that cannot be achieved under non-gate control conditions can be achieved, and the enrichment multiple can be increased to a greater extent, and a higher separation resolution can be achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG1 is a schematic diagram of the structure of a gate-controlled micro-nanofluidic device according to an embodiment of the present invention;

2A-2E are schematic diagrams of the structures of different gate-controlled electrodes in the gate-controlled micro-nanofluidic device of the present invention;

FIG3 is a schematic diagram showing the regulation principle of the gate-controlled electrode on the nanocurrent channel;

FIG4 is a schematic diagram of the separation and purification process of the gate-controlled micro-nanofluidic device of the present invention;

FIG5 is a schematic diagram of the separation and purification process of the gate-controlled micro-nanofluidic device of the present invention;

FIG6 is a schematic diagram of the structure of a gate-controlled micro-nanofluidic device in another embodiment of the present invention;

FIG7 is a schematic diagram of the structure of a gate-controlled micro-nanofluidic device in another embodiment of the present invention;

FIG8 is a schematic diagram of the structure of a gate-controlled micro-nanofluidic device in another embodiment of the present invention;

9 is a schematic structural diagram of a gate-controlled micro-nanofluidic device in another embodiment of the present invention;

FIG10 is a schematic diagram of the structure of a gate-controlled micro-nanofluidic device in another embodiment of the present invention;

FIG. 11 is a schematic diagram of the structure of a gate-controlled micro-nanofluidic device in another embodiment of the present invention.

100-gate-controlled micro-nanofluidic devices;

10 - chip body, 20 - first channel, 21 - detection area; 30 - nanoflow channel; 40 - second channel, 41 - second liquid inlet; 50 - third channel, 51 - first liquid inlet, 60 - gate-controlled electrode, 70 - insulating layer.

DETAILED DESCRIPTION

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.

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

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.

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

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.

Below the scale of 100 nanometers, the flow channel structure has a very high surface-to-volume ratio, and the resulting physical phenomena 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.

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.

Double electric layer and ion selectivity are phenomena unique to the field of nanofluids, and are usually related to the size of the nanostructure and the charge density on the inner surface. The size of the nanostructure and the charge density on the inner surface are determined by the material properties and processing parameters, and are fixed once the gate-controlled micro-nanofluidic device 100 is prepared. Of course, the charge density of the nanochannel can also be changed by modifying the inner surface of the nanochannel by diffusion, but the diffusion process in the nanostructure is very slow and very inapplicable. Therefore, the gate-controlled micro-nanofluidic device 100 in the related art can no longer flexibly adapt to current needs.

In order to solve the above technical problems, an embodiment of the present invention proposes a gate-controlled micro-nanofluidic device 100 .

As shown in Figures 1-11, the gate-controlled micro-nanofluidic device 100 proposed in an embodiment of the present invention includes a chip body 10, on which are provided a first channel 20, a second channel 40, a nanoflow channel 30 and a gate-controlled electrode 60, wherein the first channel 20 is connected to the first liquid inlet 51; the second channel 40 is connected to the second liquid inlet 41; the nanoflow channel 30 has an ion selection function and is connected between the first channel 20 and the second channel 40; the gate-controlled electrode 60 is spaced apart from the nanoflow channel 30, and is suitable for adjusting the ion selection function of the nanoflow channel 30.

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

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

The gate-controlled electrode 60 refers to a control electrode used to control the nanoflow channel 30. The gate-controlled electrode 60 can adjust the surface potential of the nanoflow channel 30 to achieve the effect of regulating ion selectivity. Specifically, the gate-controlled electrode 60 controls the zeta potential of the inner surface of the nanoflow channel 30 by applying a gate voltage, which is equivalent to completing the charge modification of the inner surface of the nanoflow channel 30 in a disguised manner. At the same time, the gate-controlled electrode 60 can also adjust the speed and direction of the electroosmotic flow in the nanoflow channel 30, that is, to control the enrichment pattern of the biochemical marker molecules in the first channel 20 by controlling the electroosmotic flow flux. Electroosmotic flow refers to the flow of liquid through porous materials, capillaries, membranes, microchannels or any other fluid conduits caused by an applied bias. It is caused by the electric field force of the electric field in the solution on the net mobile charge. The size of the electroosmotic flow is positively correlated with the zeta potential of the inner surface of the nanoflow channel 30. Therefore, increasing the gate voltage can enhance the electroosmotic flow, and changing the gate voltage polarity can change the direction of the electroosmotic flow.

When applied, under the effect of the ion selectivity of the nanoflow channel 30, the biochemical molecules will accumulate and enrich in the first channel 20, and will be affected by the synergistic effect of the electric field force and the electroosmotic drag force. Electroosmotic flow will be generated in the nanoflow channel 30, and will flow into the first channel 20, and finally flow to the third flow channel. While the charged particles are affected by the electric field force and the particle selective structure enrichment, they are also affected by the flow drag force. The particles that are affected by the flow drag force greater than the electric field force will be repelled and cannot enter the first channel 20, and the particles that are affected by the electric field force greater than the flow drag force will remain in the first channel 20 and continue to enrich, and finally reach equilibrium. As shown in Figure 4, when two kinds of particles A and B with high and low mobility are added to the first liquid inlet 51 at the same time, A and B will be enriched in the first channel 20 due to the restriction of the ion selectivity of the nanoflow channel 30, as shown in Figure 4 (a). At this time, the electroosmotic flow is increased by gate voltage control, and the B particles with relatively low mobility will be flushed out of the first channel 20, as shown in Figure 4 (b). If the electroosmotic flow is continued to increase, the A particles will also be flushed out of the first channel 20, as shown in Figure 4 (c). This is a relatively simple mode of coexistence of two particles. For more complex systems, by controlling the gate voltage, we can choose particles with a certain charge-to-volume ratio to be enriched.

The gate-controlled micro-nanofluidic device 100 provided by the embodiment of the present invention can achieve efficient biochemical molecule enrichment and improve detection sensitivity and accuracy by setting the nanoflow channel 30 and then utilizing the enrichment effect of the nanoflow channel 30. At the same time, by setting the gate-controlled electrode 60 to control the electroosmotic flow speed and direction in the nanoflow channel 30, the flexible adjustment of the biochemical molecule enrichment mode on the gate-controlled micro-nanofluidic device 100 is achieved. Even if the gate-controlled micro-nanofluidic device 100 has an adjustable enrichment function, the enrichment mode can be flexibly changed according to different working conditions and requirements to meet different application requirements, and the regulating effect of the gate-controlled electrode 60 can be used to achieve the effect that cannot be achieved under the condition of no gate control, and the enrichment multiple can be increased to a greater extent, and a higher separation resolution can be achieved. Based on this, the gate-controlled micro-nanofluidic device 100 provided by the embodiment of the present invention can be applied to the fields of biosensing, clinical diagnosis, environmental monitoring, etc., and has a wide range of application prospects, and can further promote the development of micro-nanofluidic technology in the field of biochemical sensing, and provide a more flexible and efficient method for biological analysis.

In one 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.

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.

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

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.

Optionally, the characteristic size of the nanochannel 30 is less than 100 nm, wherein 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.

In one embodiment, the gate-controlled electrode 60 is a single electrode, a plurality of parallel electrodes or an interdigitated electrode, the length direction of which is perpendicular to the flow direction of the nanoflow channel 30 and is disposed above and/or below the nanoflow channel 30 .

The parallel multi-electrodes may be parallel double electrodes, parallel triple electrodes, or parallel quad electrodes. The parallel multi-electrodes do not intersect each other. Specifically, the parallel multi-electrodes may be arranged at an angle, preferably in parallel.

The interdigital electrodes include two electrodes arranged in pairs, each electrode is in the shape of connected fingers, and the fingers of the two electrodes in the pair are arranged to cross each other, wherein the length direction of the interdigital electrodes is the extending direction of the fingers.

The single electrode, the multi-electrode and the interdigital electrode are arranged above or below the nanoflow channel 30 in a manner that the length direction is parallel to the flow direction of the nanoflow channel 30, and of course, they can also be arranged in an up-down staggered manner.

Taking the designed parallel dual-electrode regulation silica nanoflow channel 30 as an example, as shown in Figures 1 and 2B, the silica nanoflow channel 30 itself is negatively charged, which will attract cations in the solution and repel anions in the solution, and has cation selective permeability. When an external bias is applied, electroosmotic flow from the positive electrode to the negative electrode will occur, as shown in Figure 3(a); when the two electrodes are given a negative potential at the same time, the surface potential of the inner surface of the nanochannel will continue to be reduced, attracting more cations in the solution, and the repulsion of anions in the solution will be stronger, which is equivalent to making the surface of the silica nanoflow channel 30 carry more negative charges, enhancing its ion selectivity, and generating positive electrode to negative electrode when a bias is applied. Electroosmotic flow in the polar direction has a higher flow rate, as shown in Figure 3(b); when the two electrodes are given a positive potential at the same time, the surface potential of the inner surface of the nanochannel will be increased. As the positive potential increases, the ion selectivity of the nanochannel 30 will first weaken, and then turn into the opposite anion selective permeability, which is equivalent to the inner surface of the nanochannel 30 being modified with a positive charge, thereby attracting anions in the solution and repelling cations in the solution. When an external bias is applied, electroosmotic flow from the negative electrode to the positive electrode will occur, as shown in Figure 3(c); when the two electrodes are given opposite potentials, the nanochannel 30 will be locked, and it will be difficult for anions and cations to pass through, and no electroosmotic flow will be generated, as shown in Figure 3(d). Normally, two electrodes can achieve the function and performance of the control device, but the control performance of the two electrodes will be defective, and better control can be achieved by three electrodes or interdigitated electrodes.

The nanochannel 30 controls the size of the electroosmotic flow through a double gate. When the double gates apply a negative potential at the same time, the charge density on the wall of the nanochannel 30 will increase, thereby increasing the electroosmotic flow rate. When the double gates apply opposite voltages, a shut-off effect will be formed, and the electroosmotic flow rate will drop to 0. Therefore, through the regulation of the double gates, an electroosmotic flow rate of 0 to a very high level can be achieved, and if necessary, a reverse electroosmotic flow can also be obtained.

In this embodiment, the upper side or the lower side of the nano-flow channel 30 is based on the paving plane of the nano-flow channel 30 .

The gate-controlled electrode 60 configured as above can be applied to nanochannels 30 of all shapes.

In one embodiment, the gate-controlled electrode 60 is tubular and is sleeved on the outside of the nanochannel.

The gate-controlled electrode 60 of this embodiment is mainly used in the nano-flow channel 30 arranged in a nano-channel array. Specifically, the gate-controlled electrode 60 is tubular and is sleeved on the outside of one or more nano-channels.

In one embodiment, the gate-controlled electrodes 60 are multiple parallel electrodes and are spaced apart between adjacent nanochannels or adjacent nano-shallow trenches.

The gate-controlled electrode 60 of this embodiment is mainly used in the nanoflow channel 30 arranged in a nanochannel array or a nano shallow groove array. Specifically, each electrode in the parallel multi-electrode is parallel to the flow direction of the nanoflow channel 30, and is arranged between adjacent nanochannels or nano shallow grooves at intervals, wherein there may be one or more nanochannels or nano shallow grooves between adjacent electrodes. In this case, the parallel multi-electrode can be arranged above or below the entire nanoflow channel 30, and can also be embedded between the nanoflow channels 30.

In one embodiment, the chip body 10 further includes an insulating layer 70, and the thickness of the insulating layer 70 is 0.5-20um. The insulating layer 70 is used to prevent the gate control electrode 60 from short-circuiting. The material of the insulating layer 70 can be silicon dioxide, hafnium oxide, etc. In order to achieve a better gate control effect, the thickness of the insulating layer 70 should be as thin as possible. If it is too thick, a high gate voltage is required, but if it is too thin, it is easy to break down. In this embodiment, the thickness of the insulating layer 70 is any value between 0.5-20um, such as 1um, 5um, 8um, 10um, 15um or 18um.

In one embodiment, the insulating layer 70 includes a first insulating layer 70 and a second insulating layer 70 which are stacked.

Specifically, the first insulating layer 70 and the second insulating layer 70 can be made of different materials, or they can be formed by two different processes of the same material. Taking silicon dioxide as an example, ALD must be used to grow a dense silicon dioxide layer, and then LPCVD, PECVD and other methods must be used to grow silicon dioxide, otherwise it is easy to break down.

In one embodiment, the first channel 20 is connected to the micro-nano flow channel 30, which is the location where the biochemical marker molecules are finally enriched. Charged particles migrate into the first channel 20 under the drive of the electric field. Due to the limitation of the micro-nano flow channel 30, they cannot pass through the micro-nano flow channel 30, but will remain in the first channel 20 and accumulate, and finally achieve high-multiple enrichment of charged particles (10 ⁴ -10 ⁷ times enrichment). At the same time, the electroosmotic flow generated by the micro-nano flow channel 30 will flow into the first channel 20. By controlling the flow flux in the first channel 20, irrelevant background impurities that migrate into the first channel 20 can be washed away to avoid protein precipitation or blockage.

In order to increase the enrichment multiple, the characteristic scale of the first channel 20 is between 1-20um, for example, the characteristic size can be 2um, 5um, 8um, 10um, 15um or 18um, and the specific size needs to be determined according to the needs. The first channel 20 has different designs according to different needs. The first channel 20 design mainly includes a straight channel, a gradient channel, a bipolar channel, a tertiary channel, a bent channel and an arbitrary waveform channel. Through mathematical modeling analysis, it is found that the shape and size of the first channel 20 have a great influence on the enrichment effect.

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

In one embodiment, the channel surface can be modified, and probes corresponding to biochemical marker molecules can be introduced into the first channel 20 to specifically identify and capture the enriched biochemical markers. For example, as shown in FIG. 5(a)-FIG. 5(b), 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 greater than that of background proteins and other impurities, thereby achieving the enrichment of only the target biochemical molecule and a small amount of molecules with a larger charge-to-body ratio into the first channel 20.

In one embodiment, the chip body 10 further includes a third channel 50 ; the first liquid inlet 51 is disposed in the third channel 50 , and the first liquid inlet 51 is connected to the first channel 20 through the third channel 50 .

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

In one embodiment, the third channel 50 is arranged in a V-shape, the first channel 20 is connected to the V-shaped tip of the third channel 50, and the first liquid inlet 51 is arranged at the two open ends of the V. When a sample is added to the third channel 50 through one first liquid inlet 51, the impurity molecules move to the other first liquid inlet 51 under the electric field force and the electroosmotic drag force.

In one embodiment, a detection area 21 is disposed on the first channel 20, or the first channel 20 is connected to a downstream analysis system.

The detection area 21 is located in the first channel 20, usually at the tip of the first channel 20. 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 adopt 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-nanofluidic technology, high-sensitivity and high-selectivity biosensing can be achieved in the detection area 21. The detection area 21 is usually designed to be disc-shaped, or the entire first channel 20 can be directly used as the detection area 21, or it can be designed as a hexagonal, square, elliptical, diamond or other structures.

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 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 modified with the probe are limited to the detection area 21 by the electric field.

In one embodiment, there are multiple nano-flow channels 30 , which are arranged at intervals along the length direction of the first channel 20 , and a gate-controlled electrode 60 is disposed on at least one nano-flow channel 30 .

In the multiple nano-flow channels 30 , each nano-flow channel 30 forms a primary enrichment structure with the corresponding first channel 20 . The multi-level enrichment structure can further improve the separation accuracy of the gate-controlled micro-nanofluidic device 100 .

Multiple nano-flow channels 30 may share the same second channel 40, and preferably each nano-flow channel 30 is provided with a second channel 40. A detection zone 21 may be provided on the first channel 20 between two adjacent nano-flow channels 30, and a detection zone 21 may be provided at the end of the first channel 20 opposite to the other end of the third channel 50, and may also be connected to a downstream analysis system.

FIG6 shows a gate-controlled micro-nanofluidic device 100 with a two-stage enrichment mechanism. As shown in FIG6, through the gate-controlled adjustment of the first-stage enrichment mechanism, the target biochemical molecules and particles with higher mobility are enriched in the first channel 20 of the first-stage enrichment mechanism, and the particles with lower mobility than the target biochemical molecules are retained in the third channel 50 by the electroosmotic drag force. In the next step, through the gate-controlled adjustment of the second-stage enrichment mechanism, the particles with high charge-to-body ratio are extracted, leaving only the target biochemical molecules in the middle of the two-stage structure, completing the separation and purification of the target biochemical molecules from the complex sample.

In one embodiment, the chip body 10 includes a substrate and a cover plate, the nanoflow channel 30 is disposed on the substrate, the first channel 20, the second channel 40 and the third channel 50 are disposed on the substrate or the cover plate, and the cover plate and the substrate are adapted for bonding and packaging.

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

The embodiment of the present application also provides a method for using the gate-controlled micro-nanofluidic device 100 .

When the gate-controlled micro-nanofluidic device 100 is used, 10uL of blocking solution is first added to the first liquid inlet 51. The blocking solution is composed of 0.1% BSA and 0.05% tween-20 added to 1mM PBS buffer, and the solution is allowed to stand for 3 minutes. After the blocking solution fills the first channel 20 and the nanofluidic channel 30, the blocking solution is added to the second liquid inlet 41, and then the solution is blocked for 10 minutes at 4°C. This process can prevent nonspecific adsorption problems in subsequent experiments.

The channel was then cleaned with a washing solution, which was 0.05% tween-20 added to 1 mM PBS buffer. The specific cleaning scheme was to slowly push PBST with a syringe to replace the original blocking solution.

Next, the sample to be enriched is added to one of the liquid inlets, and then Ag/AgCl electrodes are inserted into the first liquid inlet 51 and the second liquid inlet 41. The first liquid inlet 51 is grounded, and the second liquid inlet 41 is connected to a positive potential of 10V.

When the power is turned on, the target biochemical molecule B will be enriched in the first channel 20, and the biochemical molecules A and C will also be enriched in the first channel 20. Assume that the electrical mobility of the three molecules is A>B>C. At this time, the inner surface of the nanoflow channel 30 is SiO ₂ , which is negatively charged, and the gate electrode is suspended.

Applying a negative potential to the gate electrode, that is, increasing the negative charge density on the inner surface of the nanoflow channel 30 in disguise, the selectivity of the nanoflow channel 30 will be stronger, and the electroosmotic flow generated will also be larger. When the gate electrode is increased to negative 5V, particle C will be flushed out of the first channel 20, and only particles A and B will be enriched. At this time, we have achieved the removal of particles with a smaller mobility than the target biochemical molecules through gate control. In practical applications, the mobility of cell debris, impurity proteins, etc. is usually smaller than that of the target biochemical ions. This method can easily achieve partial purification of the target biochemical molecules, and can effectively avoid blockage of the first channel 20 and protein precipitation.

Similarly, there are many types of background proteins in actual samples, and some of them have higher electrical mobility than the target biochemical molecules. For this reason, we can add probes of the target biochemical molecules to the sample to be enriched in advance to enhance their electrical mobility. For the target protein molecules, antibodies to the target protein molecules are usually added to the sample to be enriched, and the antibodies have been modified with nucleic acids to carry more charge, which can greatly improve the electrical mobility of the complex, or adding nucleic acid aptamers can play the same role.

Still for the system of three particles A, B, and C, in order to obtain the purified target biochemical molecule B, it is necessary to separate the particle A with high electrical mobility. This can be achieved by designing a two-stage enrichment structure, as shown in Figure 6. The first-stage enrichment structure realizes the removal of particle C, and particles A and B enter the second-stage enrichment structure at the same time. Under the condition that the size parameters of the two-stage enrichment structure are the same, the internal electric field distribution and flow field distribution are roughly the same when the same bias voltage is applied. However, if we increase the gate voltage of the second-stage enrichment structure to negative 10V, the electroosmotic flow in the second-stage second channel 40 can be increased, thereby retaining particle B in the middle of the two-stage structure, thereby achieving the purification of particle B, and particles with lower mobility than particle B remain in the injection channel, and particles with higher mobility than particle B enter the outlet of the second-stage enrichment structure. The gate-controlled voltage of the two-stage enrichment structure is not a fixed value, and can be reasonably adjusted according to the actual application scenario.

When a positive potential is applied to the gate electrode, the charge on the surface of the nanoflow channel 30 is modified to become positively charged. At this time, the direction of the electroosmotic flow is reversed, and the particles in the first channel 20 are also depleted. However, when the gate voltage continues to increase and the electroosmotic flow continues to increase, for devices with some size parameters, the particles in the first channel 20 will be enriched again. The enrichment principle at this time is different from the previous one. The electroosmotic flow drags the particles to the first channel 20, and there is a strong depletion electric field in the first channel 20. The particles will accumulate and enrich at the position where the depletion electric field drops sharply.

The embodiment of the present application further provides a method for preparing a gate-controlled micro-nanofluidic device, which is used to prepare any of the gate-controlled micro-nanofluidic devices 100 described above, comprising:

S1: Execute step a, step b and step c;

S2: execute step d;

S3: Execute step e; wherein,

Step a: patterning a gate-controlled electrode on the surface of a substrate and etching it to form, sputtering a gate-controlled electrode material onto the surface of the substrate and peeling off the gate-controlled electrode material formed on the surface of the substrate to obtain a surface gate-controlled electrode;

Step b: growing an insulating layer on the surface of the substrate;

Step c: patterning the nanofluidic channel on the substrate surface and etching it into shape;

Step d: etching the first channel, the second channel, and the third channel on the substrate surface or the cover plate, and etching a gate-controlled electrode terminal on the substrate surface;

Step e: Bonding the substrate to the cover package.

Specifically, the preparation method of the gate-controlled micro-nanofluidic device is carried out in the order of S1, S2, and S3.

In one embodiment, in S1, the steps are performed in the order of step a→step b→step c, or in the order of step c→step b→step a, or in the order of step c→step a→step b.

That is, in S1,

Embodiment 1:

The gate-controlled micro-nanofluidic device 100 has a gate-controlled electrode 60 located at the bottom, the nanofluidic channel 30 is prepared into a nano-shallow groove shape, the depth of the nano-shallow groove is less than 50nm, the substrate material is quartz glass, the gate-controlled electrode 60 is located on the lower side of the nano-shallow groove array, the material of the gate-controlled electrode 60 is Cr/Au, and the structure of the gate-controlled micro-nanofluidic device 100 is shown in Figure 7.

The specific preparation process steps include:

1) Pattern the gate electrode on the surface of the quartz wafer, and then etch 120nm by IBE;

2) Sputtered metal Cr 20nm, Au 100 nm;

3) Peel off to obtain a gate electrode pattern with a flat surface and measure the step height, which must be less than 10 nm;

4) using an ALD process to grow SiO ₂ with a thickness of 50 nm, and then using CVD to continue growing SiO ₂ with a thickness of 2 μm as an insulating layer 70;

5) Use projection lithography (Stepper) to pattern a shallow groove structure array with a groove width of 500nm and a period of 2μm. IBE etches SiO2 with an etching depth of 50nm;

6) Photolithography and etching are performed to prepare the first channel 20, the first channel 20 has a width of 10 μm and a depth of 2 μm, and at the same time, etching is performed to expose the electrode to lead out the gate control electrode 60 terminal;

7) Photolithography and etching are used to prepare the second channel 40 and the third channel 50, with a channel width of 200 μm and a depth of 50 μm;

8) Bonding with PDMS cover plate for packaging.

The first channel 20, the second channel 40 and the third channel 50 can also be prepared on a PDMS cover plate, and then bonded and packaged with a quartz substrate with a patterned gate electrode and a nano-shallow groove array.

Embodiment 2:

The gate-controlled micro-nanofluidic device 100 has a gate-controlled electrode 60 located at the bottom, the nanofluidic channel 30 is prepared as an open nanochannel with an aspect ratio close to 1, and its characteristic size is less than 50nm. The substrate material is quartz glass, the gate-controlled electrode 60 is located on the lower side of the nanochannel array, and the material of the gate-controlled electrode 60 is Cr/Au. The gate-controlled micro-nanofluidic device 100 is shown in Figure 8.

The specific preparation process steps include:

1) Pattern the gate electrode on the surface of the quartz wafer, and then etch 120nm by IBE;

2) Sputtering metal Cr 20nm, Au 100nm;

3) Peel off to obtain a gate electrode pattern with a flat surface and measure the step height, which must be less than 10 nm;

4) using an ALD process to grow SiO ₂ with a thickness of 50 nm, and then using a CVD process to grow SiO ₂ with a thickness of 2 μm, as the insulating layer 70;

5) Nano-imprinting patterned nano-channel structure array, with a pattern width of 50 nm and a period of 150 nm, IBE etching SiO ₂ , and an etching depth of 50 nm;

6) Photolithography and NLD etching are used to prepare the first channel 20, the first channel 20 has a width of 10 μm and a depth of 2 μm, and at the same time, the electrode leads to the gate control electrode 60 terminal;

7) Photolithography and NLD etching are used to prepare the second channel 40 and the third channel 50, with a channel width of 200 μm and a depth of 50 μm;

8) Bonding with PDMS package.

The gate electrode control characteristics of this structure are similar to those of Example 1 and will not be described in detail. Compared with Example 1, the nanochannel 30 in Example 2 has a smaller width, which can well avoid the subsequent bonding collapse problem. The disadvantage is that the photolithography precision requirement is high, which needs to reach 50nm.

Embodiment 3:

The gate-controlled micro-nanofluidic device 100 has a gate-controlled electrode 60 located at the top, and the nanoflow channel 30 is prepared as a sealed nanopore array with a characteristic size less than 50 nm. The substrate material is quartz glass, and the gate-controlled electrode 60 is located on the upper side of the nanochannel array. The material of the gate-controlled electrode 60 is Cr/Au. The structure of the gate-controlled micro-nanofluidic device 100 is shown in Figure 9.

The specific preparation process steps include:

1) Patterning of nanopore arrays on a quartz substrate by nanoimprinting, with a channel width of 50 nm and a period of 150 nm;

2) IBE/NLD etching of nanopore arrays, with an etching depth of 100 nm;

3) using CVD process to grow SiO ₂ with a thickness of 2 μm to prepare a sealed nanopore array;

4) Patterning the gate electrode on the surface of the quartz wafer;

5) Sputtered metal Cr 20nm, Au 100 nm;

6) peeling off to obtain a surface gate electrode;

7) Photolithography and NLD etching are used to prepare the first channel 20, the first channel 20 has a width of 10 μm and a depth of 2 μm, and at the same time, the electrode leads to the gate control electrode 60 terminal;

8) Photolithography and NLD etching are used to prepare the second channel 40 and the third channel 50, with a channel width of 200 μm and a depth of 50 μm;

9) Bonding with PDMS package.

The gate electrode control characteristics of this structure are similar to those of Example 1 and will not be described in detail. Compared with Example 1, Example 3 can avoid the fluctuation of the substrate structure caused by the gate during the preparation of the nanohole array, and to a certain extent ensure the conduction characteristics of the nanohole array. Its disadvantage is that the photolithography precision requirement is high, which needs to reach 50nm.

Embodiment 4:

A gate-controlled micro-nanofluidic device 100 is provided with a gate-controlled electrode 60 arranged in a ring around a nanoflow channel 30. The nanoflow channel 30 is prepared into a sealed nanopore array with a characteristic size less than 50 nm. The substrate material is quartz glass. The gate-controlled electrode 60 adopts a ring-gate structure, surrounding the nanopores. The material of the gate-controlled electrode 60 is Cr/Au. The partial structure of the gate-controlled micro-nanofluidic device 100 is shown in FIG. 10 .

The specific preparation process steps include:

1) Patterning of nanopore arrays on a quartz substrate by nanoimprinting or stepper lithography, with a channel width of 400 nm;

2) NLD etching of nanopore arrays, with an etching depth of 400 nm;

3) Sputtering growth of metal layer, the material is Cr 20nm, Au 100nm, used as gate electrode;

4) ALD grows SiO2 with a thickness of 50 nm to prepare a gate insulating layer 70;

5) CVD growth of SiO2 with a thickness of 2 μm to prepare a sealed nanopore array;

6) Photolithography and NLD etching are used to prepare the first channel 20, the first channel 20 has a width of 10 μm and a depth of 2 μm, and at the same time, the electrode leads to the gate control electrode 60 terminal;

7) Photolithography and NLD etching are used to prepare the second channel 40 and the third channel 50, with a channel width of 200 μm and a depth of 50 μm;

8) Bonding with PDMS package.

The gate electrode control characteristics of this structure are similar to those of Example 1 and will not be described in detail. Compared with Example 1, the ring gate structure can control the nanochannel from all sides at the same time, has a better control effect, and the gate insulation layer 70 is only 50nm, which greatly reduces the gate control voltage and further improves the gate control effect. The disadvantage is that the preparation process is relatively complex, the nanochannel density is small, and the gate insulation layer 70 is thin and easy to break down.

Embodiment 5:

The gate-controlled micro-nanofluidic device 100 has a gate-controlled electrode 60 ring located on the side of the nanoflow channel 30. The nanoflow channel 30 is prepared as an open nanochannel with an aspect ratio close to 1 or greater than 1. The characteristic dimension of the nanoflow channel 30 is less than 50nm. The substrate material is quartz glass. The gate-controlled electrode 60 adopts a side gate structure and is located on both sides of the nanochannel. The material is Cr/Au. The structure of the gate-controlled micro-nanofluidic device 100 is shown in Figure 11.

The specific preparation process steps include:

1) Patterning a gate electrode array on a quartz substrate by nanoimprinting, with a pattern width of 100 nm and a period of 400 nm;

2) IBE/NLD etching, etching depth 100nm;

3) Sputtering a metal layer in the groove, the material is 20nm Cr and 30nm Au, which is used as the gate electrode;

4) Peeling;

5) Patterning a nanochannel array between the gate electrode arrays by nanoimprinting, with each two gates sandwiching a nanochannel with a channel width of 50 nm and a period of 400 nm;

6) IBE/NLD etching of nanochannel arrays, with an etching depth of 100 nm;

7) Photolithography and NLD etching are used to prepare the first channel 20, the first channel 20 has a width of 10 μm and a depth of 2 μm, and at the same time, the electrode leads to the gate control electrode 60 terminal;

8) Photolithography and NLD etching are used to prepare the second channel 40 and the third channel 50, with a channel width of 200 μm and a depth of 50 μm;

9) Bonding with PDMS package.

The control characteristics of the gate electrode of this structure are similar to those of Example 1, and will not be described in detail. Compared with Example 1, Example 5 has better control performance, but its disadvantage is that the preparation process is relatively complex and the density of nanochannels is low.

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 by 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 (9)

1. A gate-controlled micro-nanofluidic device, characterized in that it comprises a chip body, wherein the chip body is provided with: a first channel, the first channel being in communication with the first liquid inlet; a second channel, the second channel being in communication with the second liquid inlet; a nano-flow channel, the nano-flow channel having an ion selection function and connected between the first channel and the second channel; A gate-controlled electrode, the gate-controlled electrode is spaced apart from the nanoflow channel and is suitable for adjusting the ion selection function of the nanoflow channel; The chip body also includes a third channel; the first liquid inlet is provided at the end of the third channel, and the middle of the third channel is connected to the first channel; the third channel is arranged in a V shape, wherein the tip of the V shape of the third channel is connected to the first channel, and each of its open ends is connected to one of the first liquid inlets. 2. The gate-controlled micro-nanofluidic device according to claim 1 is characterized in that the nanoflow channel forms a plurality of microchannels with a characteristic size less than 100 nm by setting the following structures: a nanochannel array and a nano shallow groove array. 3. The gate-controlled micro-nanofluidic device according to claim 2, characterized in that the gate-controlled electrode is a single electrode, parallel multiple electrodes or interdigitated electrodes, the length direction of the single electrode, parallel multiple electrodes or interdigitated electrodes is perpendicular to the flow direction of the nanoflow channel, and is arranged above or below the nanoflow channel; or The gate-controlled electrode is tubular and is sleeved on the outside of the nanochannel; or The gate-controlled electrodes are parallel multi-electrodes, which are parallel to the flow direction of the nano-flow channel and are arranged at intervals between adjacent nano-channels or adjacent nano-shallow grooves. 4 . The gate-controlled micro-nanofluidic device according to claim 1 , wherein the chip body further comprises an insulating layer, and the insulating layer is arranged between the gate-controlled electrode and the nanofluidic channel. 5 . The gate-controlled micro-nanofluidic device according to claim 4 , wherein the thickness of the insulating layer is 0.5-20 um. 6 . The gate-controlled micro-nanofluidic device according to claim 1 , wherein a detection area is provided on the first channel, or the first channel is connected to a downstream analysis system. 7. The gate-controlled micro-nanofluidic device according to claim 1 is characterized in that there are multiple nanoflow channels, the multiple nanoflow channels are arranged at intervals along the length direction of the first channel, and the gate-controlled electrode is provided on at least one of the nanoflow channels. 8. A method for preparing a gate-controlled micro-nanofluidic device, characterized in that it is used to prepare the gate-controlled micro-nanofluidic device according to any one of claims 1 to 7, comprising: S1: Execute step a, step b and step c; S2: execute step d; S3: Execute step e; wherein, Step a: patterning a gate-controlled electrode on the surface of a substrate and etching it to form, sputtering a gate-controlled electrode material onto the surface of the substrate and peeling off the gate-controlled electrode material formed on the surface of the substrate to obtain a surface gate-controlled electrode; Step b: growing an insulating layer on the surface of the substrate; Step c: patterning the nanofluidic channel on the substrate surface and etching it into shape; Step d: etching the first channel, the second channel, and the third channel on the substrate surface or the cover plate, and etching a gate-controlled electrode terminal on the substrate surface; Step e: Bonding the substrate to the cover package. 9. The method for preparing a gate-controlled micro-nanofluidic device according to claim 8 is characterized in that, in S1, step a→step b→step c are performed in sequence, or step c→step b→step a are performed in sequence, or step c→step a→step b are performed in sequence.