CN115184415B - A kind of microfluidic chip and its preparation method and application - Google Patents

Abstract

The invention discloses a microfluidic chip and a preparation method and application thereof. The micro-fluidic chip comprises a chip body and an acoustic micro-flow initiating mechanism, wherein the chip body sequentially comprises an electrode layer, a micro-flow channel layer and a top layer, a micro-flow channel is arranged in the micro-flow channel layer, the electrode layer and the top layer are respectively communicated with the micro-flow channel, and the electrode layer comprises a micro-column array electrode; during detection, under the triggering action of the acoustic micro-flow triggering mechanism, acoustic micro-flow is integrated in the micro-flow channel, and the acoustic micro-flow increases the contact between a sample to be detected in the micro-flow channel and the micro-column array electrode, so that a large response signal is realized. The microfluidic chip provided by the invention is applied to electrochemical detection and has the characteristics of high sensitivity and low temperature rise.

Description

A kind of microfluidic chip and its preparation method and application

Technical field

The invention belongs to the field of microfluidic technology, and specifically relates to a microfluidic chip and its preparation method and application.

Background technique

Electrochemical detection is an analytical method that uses the electrochemical properties of substances to characterize and measure. The use of electrochemical analysis methods can not only automatically record analysis results, but also facilitate the detection of trace substances, including glucose, sarcosine and urea, etc., and are widely used in industry, agriculture, food safety and other aspects. Microfluidic technology is a scientific technology whose main feature is the manipulation of fluids in micron-scale space, and can realize multi-step biochemical reactions on a chip. Microfluidic technology has special advantages in electrochemical detection research, including small sample volume, low cost, rapid sample analysis, and improved reaction reliability and reproducibility. However, at present, the sensitivity of electrochemical detection using traditional electrochemical detectors is low.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the application of a microfluidic chip with an acoustic microflow initiating mechanism and a micropillar array electrode in electrochemical detection, which has the characteristics of high detection sensitivity.

The invention also proposes a microfluidic chip.

The invention also proposes a method for preparing a microfluidic chip.

The invention also proposes a microfluidic platform.

The invention also proposes an electrochemical detector.

The invention also proposes an electrochemical detection platform.

The invention also proposes the application of the above-mentioned microfluidic chip, microfluidic platform, electrochemical detector or electrochemical detection platform.

A first aspect of the present invention proposes the application of a microfluidic chip having an acoustic microflow initiating mechanism and a micropillar array electrode in electrochemical detection.

The application of the microfluidic chip with the acoustic microflow initiating mechanism and the micropillar array electrode in electrochemical detection according to the embodiment of the present invention has at least the following beneficial effects:

The present invention is based on acoustic microfluidic technology. The microfluidic chip is used in electrochemical detection, which can effectively increase the diffusion near the electrode surface and has a small temperature rise of the detection sample. Acoustic microfluidics can increase the mass transfer efficiency of analytes from electrodes without significant temperature rise. At the same time, in the present invention, compared with planar electrodes, the micropillar array electrode has a larger surface area and better electrical signal, which can significantly improve the sensitivity of the microfluidic chip. Therefore, when the microfluidic chip is used in electrochemical detection, it has the characteristics of high sensitivity and low temperature rise, and is very suitable for the detection of biomarkers.

In a second aspect of the present invention, a microfluidic chip is proposed, which includes a chip body and an acoustic microflow initiating mechanism. The chip body sequentially includes an electrode layer, a microfluidic channel layer and a top layer. The microfluidic channel layer is provided with There is a microfluidic channel, the electrode layer and the top layer are respectively connected with the microfluidic channel, and the electrode layer includes a micropillar array electrode;

During detection, the acoustic microflow is integrated into the microfluidic channel under the initiating action of the acoustic microfluidic initiating mechanism. The acoustic microfluidic increases the contact between the sample to be tested in the microfluidic channel and the micropillar array electrode.

The microfluidic chip according to the embodiment of the present invention has at least the following beneficial effects: The present invention is based on microfluidic technology and combines acoustic microfluidics with electrochemical detection, which can increase the temperature near the electrode surface without significant temperature rise. Mass transfer efficiency. At the same time, the present invention uses micro-pillar array electrodes. Since the micro-pillar array electrode has a large surface area and a micro-pillar structure, under the action of acoustic microfluidics, the micropillar array electrodes and the acoustic microfluidics interact synergistically, thereby achieving super sensitivity. electrochemical detection. When the microfluidic chip is used in electrochemical detection, it has the characteristics of high sensitivity and low temperature rise, and the temperature rise is within 3°C. In embodiments of the present invention, during the working process with acoustic microfluidics, the temperature change of the liquid in the microfluidic chip is less than 1°C. Usually in biomarker detection applications, the temperature rise of the sample when detecting biomarkers needs to be as small as possible, for example, within a few degrees Celsius. Therefore, the microfluidic chip of the present invention has significant advantages in electrochemical biomarker detection.

In some embodiments of the present invention, the acoustic microflow initiating mechanism includes an acoustic wave initiating component, and an array of micropits connected to the microfluidic channel is provided in the top layer;

During detection, the micropit array forms a bubble array, the acoustic wave inducing component induces a sound field, and the bubble array forms an acoustic microflow under the action of the sound field.

Through the above embodiment, after the solution of the sample to be tested is injected into the microfluidic chip, since the micro-pit array is provided on the top layer, the micro-pits can spontaneously form micro bubbles to form a bubble array, and a certain frequency of acoustic wave excitation is applied through the acoustic wave initiating component. Under the action of the bubble array, the acoustic microflow is formed in the microfluidic channel, thereby integrating the acoustic microflow in the microfluidic chip (including the generation of acoustic microflow near the surface of the micropillar array electrode). The acoustic microflow technology based on bubble excitation can effectively stir The liquid volume can be in the range of microliters, thereby effectively stirring the solution in the detection area, enhancing the contact between the electrode and the analyte, thereby improving the detection sensitivity and achieving ultra-high-sensitivity electrochemical analysis.

This invention proposes for the first time that bubble-based acoustic microfluidic technology and micro-pillar array electrode technology can be used to collaboratively assist in enhancing electrochemical sensing sensitivity, and the micro-pillar array electrode and bubble-induced acoustic microfluidic technology can be jointly integrated for high-sensitivity biomarkers. Microfluidic detection chip. The invention discloses that the combination of microarray electrode technology and acoustic microflow technology in the electrochemical detection process can greatly improve the sensitivity of electrochemical detection and obtain ultra-high electrochemical detection sensitivity. At the same time, the detection platform using microfluidic chips has the advantages of low consumption and small temperature rise during the working process. It can be used for low-abundance biological detection and has great potential in biosensing.

In some preferred embodiments of the present invention, the number of micro pits in the micro pit array is 1-1000.

In some more preferred embodiments of the present invention, the number of micro pits in the micro pit array is 10-1000.

In some preferred embodiments of the present invention, each micro pit in the micro pit array is arranged in a circular array, a sector array, an annular array, an elliptical array, a triangular array, a quadrilateral array, a pentagonal array or a hexagonal array. At least one of the array.

In some more preferred embodiments of the present invention, each micro pit in the micro pit array is arranged in an equidistant hexagonal array.

In some preferred embodiments of the present invention, the micro pits in the micro pit array are cylindrical, cone-shaped, truncated cone-shaped or micro-conical pits.

In some more preferred embodiments of the present invention, the micro pits in the micro pit array are micro-tapered pits.

In some preferred embodiments of the present invention, the depth of the micro pits in the micro pit array is 10 μm-10 mm.

In some more preferred embodiments of the present invention, the depth of the micro pits in the micro pit array is 10 μm-1 mm.

In the present invention, the intensity of the induced acoustic microflow is related to the depth of the prepared micropit array. The increase in sensitivity is related to the depth of the micro-pit array.

In some more preferred embodiments of the present invention, the depth of the micro pits in the micro pit array is 10-1000 μm.

In some more preferred embodiments of the present invention, the depth of the micro-pits in the micro-pit array is about 500 μm.

In some preferred embodiments of the present invention, the spacing of micro pits in the micro pit array is 10-500 μm.

In some preferred embodiments of the present invention, the diameter of the end of the micro-pit array close to the micro-channel layer is 10-300 μm, and the diameter of the end of the micro-pit away from the micro-channel layer is 10 μm. -300μm.

In some preferred embodiments of the present invention, the preparation method of the top layer includes: the top layer is made by reverse molding of a 3D printed micro-pillar array model, or by reverse molding of a micro-pillar array model prepared by a photolithography process. or by machining different substrates with micropit arrays. The substrate can be made of different materials.

In some preferred embodiments of the present invention, a micro-pillar array model produced by 3D printing is used for inversion molding to obtain the top layer provided with a micro-pit array.

In some more preferred embodiments of the present invention, in the micro-pillar array model, the height of the micro-pillars is 10 μm-10 mm, the spacing of the micro-pillars is 10-500 μm, and the diameter of the top circle of the micro-pillars is 10-300 μm. , the diameter of the bottom circle of the micro-column is 10-300μm.

In some more preferred embodiments of the present invention, in the micro-pillar array model, the height of the micro-pillars is 10-1000 μm.

In some preferred embodiments of the present invention, the micro pit array is disposed on a side of the top layer close to the microfluidic channel, and the acoustic wave initiating component is disposed on a side of the top layer away from the microfluidic channel, so The top layer is located above the microfluidic layer.

In some preferred embodiments of the invention, the acoustic wave inducing assembly includes a piezoelectric transducer.

In some more preferred embodiments of the present invention, the piezoelectric transducer is a piezoelectric sheet.

In some more preferred embodiments of the present invention, the excitation electrical signal waveform of the piezoelectric sheet is a sine wave, the peak-to-peak voltage of the waveform is 1-50V, and the frequency of the waveform is 1-15kHz.

In some embodiments of the present invention, the electrode layer includes a substrate and a working electrode, a reference electrode and a counter electrode all disposed on the substrate. The working electrode, the reference electrode and the counter electrode can all be connected to the The samples to be measured in the microfluidic channel are in contact with each other, and the working electrode is a micropillar array electrode.

In the present invention, the sensitivity of the microfluidic chip can be improved by synergistically using micropillar array electrodes and acoustic microfluidics.

In the present invention, a 3D printing model can be used as a positive mold, and a micro-pillar array can be prepared through two molding processes, and then the micro-pillar array electrode can be prepared through magnetron sputtering or other methods.

In some preferred embodiments of the present invention, the reference electrode is a planar electrode.

In some preferred embodiments of the present invention, the counter electrode is a planar electrode.

In some preferred embodiments of the present invention, the materials of the reference electrode and the counter electrode are independently selected from at least one of conductive metals, metal salts, metal oxides or conductive polymers.

In some more preferred embodiments of the present invention, the materials of the reference electrode and the counter electrode are independently selected from at least one of gold, silver, platinum, silver chloride or ITO.

In some more preferred embodiments of the invention, both the reference electrode and the counter electrode include gold films.

In some more preferred embodiments of the present invention, the thickness of the gold thin film is 50-300 nm.

In some preferred embodiments of the present invention, the reference electrode includes a gold film and Ag/AgCl located at an end of the gold film.

In some preferred embodiments of the present invention, the projected size of the reference electrode is (0.5-3) mm × (0.5-3) mm; the projected size of the counter electrode is (0.5-3) mm × ( 0.5-3)mm.

The projection size refers to the projection size of the reference electrode or counter electrode along the vertical direction on the horizontal plane.

In some preferred embodiments of the present invention, the projected size of the working electrode is (0.5-3) mm×(0.5-3) mm.

The projected size refers to the projected size of the working electrode on the horizontal plane along the vertical direction.

In some preferred embodiments of the present invention, the substrate includes at least one of a glass substrate, a PDMS substrate or a silicon wafer substrate.

In some embodiments of the present invention, the micropillar array electrode includes a micropillar array substrate and a conductive layer disposed on the micropillar array substrate.

In some preferred embodiments of the present invention, the micropillar array substrate includes a high molecular polymer. The high molecular polymers include but are not limited to plastics.

In some more preferred embodiments of the invention, the micropillar array substrate includes polydimethylsiloxane, polyimide, organic glass, polyethylene terephthalate or epoxy resin. At least one.

Polydimethylsiloxane, referred to as: PDMS; polyethylene terephthalate, referred to as: PET.

In some preferred embodiments of the present invention, the conductive layer includes at least one of conductive metal, metal salt, metal oxide or conductive polymer.

In some more preferred embodiments of the present invention, the conductive layer includes at least one of gold, silver, iron, platinum, copper, stainless steel, nickel, chromium, silver chloride or ITO.

In some more preferred embodiments of the invention, the conductive layer includes a gold film.

In some more preferred embodiments of the present invention, the thickness of the gold thin film is 10-500 nm.

In some preferred embodiments of the present invention, the surface of the conductive layer is modified with nanoparticles.

In some more preferred embodiments of the present invention, the nanoparticles include at least one of carbon nanotubes, graphene, metal oxides, carbon-based nanosheets, metal elements or composite metals.

In some more preferred embodiments of the invention, the nanoparticles are platinum nanoparticles and palladium nanoparticles.

Modification of platinum and palladium nanoparticles improves the sensing performance of micropillar array electrodes.

In some embodiments of the present invention, the micro-pillar array electrode has micron-level or sub-micron-level precision.

In some embodiments of the present invention, the height of the micro-pillars in the micro-pillar array electrode is 1 μm-10 mm.

In some preferred embodiments of the present invention, the height of the micro-pillars in the micro-pillar array electrode is 50 μm-1 mm.

In some preferred embodiments of the present invention, the height of the micropillars in the micropillar array electrode is 10-1000 μm.

Through the above embodiments, the higher the height of the micropillars in the micropillar array electrode, the better the electrochemical testing effect of the microfluidic chip will be.

In some embodiments of the present invention, the spacing of micro-pillars in the micro-pillar array electrode is 10-500 μm.

In some preferred embodiments of the present invention, the spacing between micropillars in the micropillar array electrode is 50-500 μm.

In some embodiments of the present invention, the diameter of the top circle of micro-pillars in the micro-pillar array electrode is 1-500 μm.

In some preferred embodiments of the present invention, the diameter of the top circle of micro-pillars in the micro-pillar array electrode is 10-500 μm.

In some embodiments of the present invention, the diameter of the bottom circle of the micro-pillars in the micro-pillar array electrode is 1-500 μm.

In some preferred embodiments of the present invention, the diameter of the bottom circle of the micro-pillars in the micro-pillar array electrode is 10-500 μm.

In some embodiments of the present invention, the micro-pillars in the micro-pillar array electrode are cylindrical.

In some embodiments of the present invention, the number of micropillars in the micropillar array electrode is 10-10,000.

In some preferred embodiments of the present invention, the number of micropillars in the micropillar array electrode is 10-50.

In some embodiments of the present invention, the micro-pillars in the micro-pillar array electrode are arranged equidistantly.

In some embodiments of the present invention, each microcolumn in the microcolumn array electrode is arranged in a circular array, a sector array, an annular array, an elliptical array, a triangular array, a quadrilateral array, a pentagonal array or a hexagonal array. at least one of them.

In some preferred embodiments of the present invention, each micro-pillar in the micro-pillar array electrode is arranged in an equidistant hexagonal array.

In some embodiments of the present invention, the micro-pillar array electrode includes a micro-pillar array substrate and a conductive layer disposed on the micro-pillar array substrate, and the material of the micro-pillar array in the micro-pillar array substrate includes polymer polymer. things. The high molecular polymers include but are not limited to plastics.

In some preferred embodiments of the present invention, the material of the micropillar array in the micropillar array substrate includes polydimethylsiloxane, polyimide, organic glass, polyethylene terephthalate or At least one type of epoxy resin.

In some embodiments of the present invention, the micropillar array in the micropillar array electrode is a PDMS micropillar array.

The preparation method of the micro-pillar array in the micro-pillar array electrode includes: the micro-pillar array is obtained by using the micro-pillar array template prepared by 3D printing as a positive mold through two consecutive PDMS inversion molds, or is made by a photolithography process, Or it can be prepared by machining.

In some embodiments of the present invention, the electrode layer, the microfluidic channel layer and the top layer are arranged in a stacked manner, and the microfluidic channels are arranged parallel to the electrode layer and the top layer.

In some embodiments of the present invention, the material of the top layer includes a high molecular polymer. The high molecular polymers include but are not limited to plastics.

In some embodiments of the present invention, the material of the top layer includes polydimethylsiloxane, polyethylene terephthalate, acrylonitrile-butadiene-styrene copolymer or polymethyl methacrylate. at least one of the esters.

Acrylonitrile-butadiene-styrene copolymer, abbreviation: ABS; polymethylmethacrylate, abbreviation: PMMA.

In some preferred embodiments of the invention, the top layer is a PDMS top layer.

In some embodiments of the present invention, the material of the microfluidic channel layer includes high molecular polymer. The high molecular polymers include but are not limited to plastics.

In some embodiments of the present invention, the material of the microfluidic layer includes at least one of polydimethylsiloxane, polyethylene terephthalate or acrylonitrile-butadiene-styrene copolymer. A sort of.

In some embodiments of the present invention, the material of the microfluidic layer includes a double-sided sticker, and the microfluidic layer is a double-sided sticker patterned by laser cutting.

In some embodiments of the present invention, the size of the microfluidic channel is on the order of microns.

In some embodiments of the present invention, the width of the microfluidic channel is 1 μm-10 mm.

In some embodiments of the present invention, the depth of the microfluidic channel is 1 μm-2 mm.

In some preferred embodiments of the present invention, the depth of the microfluidic channel is 1-500 μm.

In some embodiments of the invention, the length of the microfluidic channel is 10-30 mm.

In some preferred embodiments of the present invention, the microfluidic channel has a width of 2 mm, a depth of 650 μm, and a length of 20 mm.

In some embodiments of the present invention, the microfluidic chip is used for electrochemical detection, and the analysis method of the electrochemical detection includes at least one of electrochemiluminescence, electrophoresis, electrochemical voltammetry or impedance method.

In some embodiments of the present invention, the flow rate of the solution of the sample to be tested injected into the microfluidic chip is 0.01-10 μL/min.

In a third aspect of the present invention, a method for preparing a microfluidic chip is proposed, which includes the following steps:

S1, micropillar array electrodes are prepared through photolithography or soft photolithography technology;

S2, assemble the electrode layer including the micropillar array electrode, the microfluidic channel layer, the top layer and the acoustic microfluidic initiating mechanism to obtain the microfluidic chip.

In some embodiments of the present invention, step S1 also includes the following step: using reverse mold technology to prepare a top layer with a micro-pit array.

In some preferred embodiments of the present invention, in step S1, first use 3D printing or micro-machining technology to prepare a positive mold template with a micro-pillar array, and inject PDMS mixture I into the positive mold template to obtain the desired Describe the top level.

In some more preferred embodiments of the present invention, in step S1, first use 3D printing or micro-machining technology to prepare a positive mold template with a micro-pillar array, and inject PDMS mixture I into the positive mold template. Soak, bake, and separate the cured product of PDMS mixture I and the positive mold template to obtain a PDMS top layer with a micro-pit array.

In some more preferred embodiments of the present invention, the PDMS mixture I includes a matrix material I and a curing agent I.

In some more preferred embodiments of the present invention, the mass ratio of the matrix material I and the curing agent I is (5-11):1.

In some embodiments of the present invention, the micro-pillar array electrode is manufactured by a photolithography process or a soft photolithography process.

In some embodiments of the present invention, the preparation method of the micro-pillar array electrode includes the following steps: using soft photolithography reverse mold technology and sputtering metal technology to prepare the micro-pillar array electrode.

In some embodiments of the present invention, the preparation method of the micropillar array electrode includes the following steps:

S1-1, use 3D printing or micro-machining technology to prepare a micro-pillar array template, use the micro-pillar array template as the positive mold I, inject the PDMS mixture II into it, and solidify to obtain a template layer with a micro-pit array;

S1-2, use the template layer with the micro-pit array as the positive mold II, inject the PDMS mixture III into it, and solidify to prepare the PDMS layer with the micro-pillar array, and obtain the micro-pillar array substrate;

S1-3, prepare a conductive layer on the micro-pillar array substrate to obtain the micro-pillar array electrode.

In some preferred embodiments of the present invention, in step S1-1, the PDMS mixture II is injected into the micropillar array template, defoamed, and baked to obtain a template layer with a micropit array.

In some preferred embodiments of the present invention, in step S1-2, the PDMS mixture III is injected into the template layer, defoamed, and baked to obtain a PDMS layer with a micropillar array.

In some more preferred embodiments of the present invention, in step S1-1, the PDMS mixed liquid II includes a matrix material II and a curing agent II; in step S1-2, the PDMS mixed liquid III includes a matrix material III and a curing agent II. Curing agent III.

In some more preferred embodiments of the present invention, the mass ratio of the matrix material II and the curing agent II is (5-11):1; the mass ratio of the matrix material III and the curing agent III is (5-11 ):1.

In some more preferred embodiments of the present invention, in step S1-3, a conductive layer is prepared on the micro-pillar array substrate, and then nanoparticles are modified on the conductive layer to obtain the micro-pillar array electrode.

In some more preferred embodiments of the present invention, in step S1-3, the nanoparticles include platinum nanoparticles and palladium nanoparticles.

In some more preferred embodiments of the present invention, the method of modifying platinum nanoparticles and palladium nanoparticles includes an electroplating deposition process, a drop casting process or an immersion physical adsorption process.

In some more preferred embodiments of the present invention, a micropillar array substrate with a conductive layer is electroplated and deposited in an electroplating solution, and platinum nanoparticles and palladium nanoparticles are modified on the conductive layer to obtain the micropillar array electrode. .

In some more preferred embodiments of the present invention, in the electroplating deposition process, the applied potential is -0.2V and the deposition time is 100s.

In some embodiments of the present invention, the surface of the micropillar array electrode is modified with platinum nanoparticles and palladium nanoparticles.

In some embodiments of the present invention, the electrode layer includes a substrate and a working electrode, a reference electrode and a counter electrode all disposed on the substrate. The working electrode, the reference electrode and the counter electrode can all be connected to the The samples to be measured in the microfluidic channel are in contact with each other. The reference electrode and the counter electrode are planar electrodes, which are made through a coating process.

In some preferred embodiments of the present invention, the reference electrode and counter electrode are made by sputtering or printing processes.

In some embodiments of the present invention, the preparation step of the microfluidic layer includes: using double-sided tape as a raw material and using laser cutting technology to obtain a microfluidic layer provided with microfluidic channels.

In some preferred embodiments of the present invention, the microfluidic channel layer of the microfluidic chip is patterned on the double-sided tape based on laser cutting.

In some embodiments of the present invention, the preparation step of the electrode in the electrode layer includes: using glass as a substrate, and using a coating process to plate a gold film on the surface of the substrate.

In some preferred embodiments of the present invention, the glass is first ultrasonically cleaned with acetone and deionized water, a stainless steel mask is affixed to the surface of the glass using high-temperature tape, and then the gold film is plated.

A fourth aspect of the present invention provides a microfluidic platform, which includes the above-mentioned microfluidic chip.

A fifth aspect of the present invention provides an electrochemical detector, which includes the above-mentioned microfluidic chip.

In some embodiments of the invention, the electrochemical detector is an electrochemical biosensor. Can be used for immediate diagnosis.

A sixth aspect of the present invention provides an electrochemical detection platform, which includes the above-mentioned microfluidic chip or the above-mentioned electrochemical detector.

In some embodiments of the invention, the electrochemical detection platform also includes a syringe, a controller and an electrochemical workstation, wherein the solution of the sample to be tested is injected into the microfluidic chip through the syringe, and the controller is used to control the acoustic wave The acoustic wave of the initiating component is initiated, and the electrochemical detection results in the microfluidic chip are output and analyzed through the electrochemical workstation.

The seventh aspect of the present invention proposes the application of the above-mentioned microfluidic chip, microfluidic platform, electrochemical detector or electrochemical detection platform in electrochemical detection.

In some embodiments of the present invention, the above-mentioned microfluidic chip, microfluidic platform, electrochemical detector or electrochemical detection platform is used for body fluid metabolic component analysis, body fluid analysis, protein separation, in vitro detection, cancer marker analysis or Cell analysis.

In some embodiments of the present invention, the above-mentioned microfluidic chip, microfluidic platform, electrochemical detector or electrochemical detection platform is used for the detection of electrochemical biomarkers.

Description of the drawings

The present invention will be further described below in conjunction with the accompanying drawings and examples, wherein:

Figure 1 is a schematic structural diagram of the electrochemical detection platform in Embodiment 1 of the present invention;

Figure 2 is a cross-sectional view of the micropit array of the microfluidic chip in Embodiment 1 of the present invention;

Figure 3 is a scanning electron microscope image of the micropillar array electrode in the microfluidic chip in Embodiment 1 of the present invention;

Figure 4 is a schematic flow chart of the soft photolithography preparation method of micro-pit array and micro-pillar array electrodes in Embodiment 1 of the present invention;

Figure 5 is a temperature change diagram of the solution of the sample to be tested in the microfluidic chip in Example 1 of the present invention;

Figure 6 is a signal comparison diagram of the microfluidic chip in the acoustic microfluidic on/off state in Example 1 and Comparative Example 1 of the present invention;

Figure 7 is a signal comparison diagram of the microfluidic chip in the acoustic microfluidic on/off state in Examples 1-3 and Comparative Example 2 of the present invention.

Detailed ways

The concept of the present invention and the technical effects produced will be clearly and completely described below with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without exerting creative efforts are all protection scope of the present invention.

Example 1

This embodiment discloses an electrochemical detection platform, the structural schematic diagram of which is shown in Figure 1, wherein Figure 1A is a schematic diagram of the electrochemical detection platform; Figure 1B is an exploded view of a microfluidic chip; Figure 1C is a microfluidic The working principle diagram of the control chip: Figure 1C (1) is a schematic diagram of the bubble array formation, Figure 1C (2) is a schematic diagram of the piezoelectric driven acoustic microflow formation, and Figure 1C (3) is a real-time current enhancement effect diagram of increasing the acoustic microflow .

Specifically, the electrochemical detection platform includes a syringe, a microfluidic chip, a controller and an electrochemical workstation, wherein the solution of the sample to be tested is injected into the microfluidic chip through the syringe, and the controller is used to control the acoustic wave initiation of the acoustic wave initiation component, The electrochemical detection results in the microfluidic chip are output and analyzed through the electrochemical workstation.

Among them, the microfluidic chip consists of three layers: a microfluidic layer, a PDMS top layer with a micro-pit array, and an electrode layer. The microfluidic layer is the middle layer, connecting the top layer of PDMS and the electrode layer. There are microfluidic channels in the microfluidic channel layer. The top layer of PDMS and the electrode layer are connected to the microfluidic channels respectively. The micro-pit array on the top layer of PDMS is connected to the microfluidic channels. For the micro-pit array, the number of micro-pits is 78, and the arrangement is an equidistant hexagon. The spacing of the micro-pits is 300 μm; the bottom diameter of a single micro-pit is 200 μm (the micro-pit is close to the end of the microfluidic layer). The diameter of the top circle is 100 μm (the end of the micro pit away from the microfluidic layer) and the depth is 500 μm. The cross-sectional view of the micropit array observed through an electron microscope is shown in Figure 2. By measurement, the depth of the prepared micropits was 507.2±6.7μm.

Below the micropit array is the reaction zone of the microfluidic layer. The material of the microfluidic layer is a double-sided waterproof sticker patterned by laser cutting, which is used to bond the top layer of PDMS to the electrode layer. The width of the microfluidic channels in the microfluidic layer is 2mm, the depth is 650μm, and the total length is 20mm.

The electrode layer includes a glass substrate and three electrodes provided on the substrate, namely a reference electrode (RE), a counter electrode (CE) and a working electrode (WE). All three electrodes can interact with the sample to be measured in the microfluidic channel. solutions are in contact. The counter electrode and reference electrode are planar electrodes, both of which are gold films on glass substrates, and the tips of RE are coated with Ag/AgCl. The thickness of the gold film is about 250nm. The reference electrode (RE) and counter electrode (CE) are both micron and sub-nanometer precision, with projected areas of 1.5mm×1mm and 1.5mm×2.5mm respectively. The working electrode is a micro-pillar array electrode, including a PDMS micro-pillar array and a gold film with a thickness of 250 nm sputtered on the PDMS micro-pillar array as a base. The projected size of the working electrode is 1 mm × 1 mm. The number of micro-pillars is 16, and they are arranged in an equidistant rectangular arrangement. The spacing between micro-pillars is 200 μm. A single micro-pillar is cylindrical, and its specific dimensions are a base diameter of 100 μm and a height of 500 μm. The scanning electron microscope image of the micropillar array electrode is shown in Figure 3.

A piezoelectric sheet is provided on the side of the top layer of PDMS away from the microfluidic channel (above the electrode reaction area). When the solution of the sample to be tested is injected into the empty microfluidic chip, the micropits will spontaneously form a bubble array. The pit array forms a certain volume of bubbles. A voltage controller drives the piezoelectric sheet at the desired frequency to induce the formation of acoustic microflow near the bubble interface (the bubble array is near the electrode, ≤3mm from the electrode surface, forming acoustic microflow). When the electrode begins to perform electrochemical detection, the acoustic microflow will promote the diffusion of analytes to the electrode surface, thereby improving the electrochemical performance of the electrode, and the current signal will be improved, thereby realizing the integration of acoustic microflow on the microfluidic chip and achieving ultrasonic Highly sensitive electrochemical analysis.

The preparation process of the microfluidic chip includes:

(1) The flow channel layer of the microfluidic chip is patterned by double-sided tape based on laser cutting.

(2) The PDMS top layer of the microfluidic chip uses inversion molding technology to realize the micro pit array. The details include: first, use 3D printing to process the micro-pillar array template I. The height of the micro-pillars in the micro-pillar array template I is 500 μm, the number of micro-pillars is 78, the spacing of the micro-pillars is 300 μm, the diameter of the top circle of the micro-pillars is 100 μm, and The diameter of the column bottom circle is 200 μm. Then use the micro-column array template I as the positive mold, inject the PDMS mixture (matrix: curing agent = 10:1) into it, and after defoaming and baking, tear off the tape above the positive mold. PDMS top layer with micro-pit array.

(3) The electrode layer of the microfluidic chip is prepared by using two consecutive soft photolithography reverse molding techniques and a gold sputtering process to achieve the preparation of micro-pillar array electrodes. Specifically, it includes: first, use 3D printing to process the micro-pillar array template II (the number of micro-pillars is 16, the arrangement is an equidistant rectangular arrangement, the spacing of the micro-pillars is 200 μm, a single micro-pillar is cylindrical, and its specific size The bottom radius of the micro-column is 50 μm and the height is 500 μm), and then the micro-column array template Ⅱ is used as the positive mold Ⅰ, and the PDMS mixture (matrix: curing agent = 10:1) is injected into it. After defoaming and baking, Tear off the PDMS layer with the micro-pit array on top of the positive mold I; then, use the PDMS layer with the micro-pit array as the positive mold II for the second soft photolithography, and inject the PDMS mixture into it (matrix: curing agent = 10:1), after defoaming and baking, peel off the PDMS layer with the micro-pillar array on top of the male mold II; finally, after cleaning the PDMS layer with the micro-pillar array, pass the pattern of the stainless steel mask change. The PDMS layer with the micropillar array is first ultrasonically cleaned with acetone and deionized water, and then a stainless steel mask is attached to the surface of the PDMS layer with the micropillar array using high-temperature tape. A gold film with a thickness of about 250 nm is prepared on a glass substrate using a conventional coating process to form three patterned electrodes (including an electrode with a micropillar array, a planar reference electrode and a flat electrode), where the reference electrode It is necessary to apply silver/silver chloride ink on the electrode surface and wait for the ink to dry to obtain a reference electrode with silver/silver chloride at the end and obtain the electrode layer of the microfluidic chip. The preparation parameters of the gold thin film are: the sputtering power is 300W, the helium gas introduction rate is 50-80sccm, the vacuum degree is 0.2-0.5Pa, and the time is 600s.

(4) Modify the micro-pillar array electrode (working electrode). The micro-pillar array electrode is immersed in the electroplating solution, applying a potential of -0.2V, and the deposition time is 100s to obtain a working electrode modified with platinum nanoparticles and palladium nanoparticles. Among them, the components of the electroplating solution: the concentration of chloroplatinic acid is 10mmol/L; the concentration of sodium chloropalladate is 10mmol/L; the concentration of HCl is 0.5mmol/L; the solvent is deionized water.

(5) Use the microfluidic layer as the middle layer to assemble the PDMS top layer and electrode layer.

(6) Clean the chip and complete the preparation of the entire chip.

Among them, the PDMS mixture is: PDMS matrix and curing agent are products of Dow Corning, SYLGARDTM184 Silicone Elastomer Kit, Batch No.: H052L7L156.

Among them, the flow chart of the soft photolithography preparation method of micro-pit array and micro-pillar array electrodes is shown in Figure 4. The preparation method of the PDMS micropit array is steps 1-2 in Figure 4. The preparation method of the micropillar array electrode is steps 1-5 in Figure 4.

The electrochemical detection platform in this embodiment is used to conduct electrochemical testing, and the test results are shown in Figure 5:

Figure 5 shows the solution temperature inside the chip when a sine wave signal is excited to the piezoelectric chip. The sampling time is from 0s to 200s, and the increment of the working time is 40s. Under the conditions of acoustic microflow, the temperature of the solution in the chip does not increase significantly. Among them, when the working time is 200s, the solution temperature of the chip only increases by 0.2℃, that is, after working for 200s, the temperature rises by 0.2℃. Therefore, the acoustic microfluidic technology used in the present invention can effectively stir the solution without increasing the temperature, proving its biocompatibility and application advantages for biological samples. The chip facilitates high-sensitivity electrochemical detection of low-sample, low-abundance biological analytes.

Example 2

This embodiment discloses an electrochemical detection platform, which differs from Embodiment 1 in that in the microfluidic chip, the depth of the micropits in the micropit array on the top layer of PDMS is 100 μm.

Example 3

This embodiment discloses an electrochemical detection platform, which differs from Embodiment 1 in that in the microfluidic chip, the depth of the micropits in the micropit array on the top layer of PDMS is 300 μm.

In Example 2-3, the temperature rise result of the solution of the sample to be tested in the microfluidic chip is equivalent to Example 1 (the test method is the same as Example 1).

Comparative example 1

This comparative example discloses an electrochemical detection platform. The difference from Example 1 is that in the microfluidic chip, the working electrode is a planar electrode, specifically a gold film with a thickness of about 250 nm.

Comparative example 2

This comparative example discloses an electrochemical detection platform, which differs from Example 1 in that in the microfluidic chip, there is no micropit array on the top layer of PDMS.

Electrochemical testing was performed on the electrochemical detection platforms of Example 1 and Comparative Example 1, and the test results are shown in Figure 6:

Figure 6 shows the current density change of the potassium ferricyanide solution detected by the micropillar array electrode when a sine wave electrical signal with a frequency of the bubble excitation frequency is used to excite the piezoelectric sheet. At t=0s, the analyte is pumped into the microfluidic chip. At t=100s, the sine wave signal is used to excite the piezoelectric film, and at t=250s, the electrical signal input is turned off. The current reaches a plateau between t=200-250s, and this value is recorded as the final equilibrium current. The results show that chips integrating acoustic microfluidics can significantly improve detection sensitivity. The detection current of the detection chip under the condition of flat electrode and the presence of acoustic microflow is about 6.7 times that of the flat electrode and without acoustic microflow. The detection current of the detection chip in the presence of micropillar array electrodes and acoustic microflow is approximately 6.6 times that of micropillar array electrodes and without acoustic microflow. Most importantly, the detection current of a detection chip integrating micropillar array electrodes and acoustic microfluidics technology is approximately 26.6 times that of a detection chip integrating traditional planar electrodes (without acoustic microfluidics). Moreover, the chip design is simple and does not require additional circuit design (for example, the design of interdigital electrodes, etc.). The results show that microfluidic chips using acoustic microfluidics and micropillar array electrode technology can significantly improve detection sensitivity. Moreover, the chip is simple in design, does not require additional circuit design (for example, the design of interdigital electrodes, etc.) and consumes less.

Aiming at the micropit array depth of the acoustic microfluidic technology, electrochemical testing was performed using the electrochemical detection platform of Examples 1-3 and Comparative Example 2. The test results are shown in Figure 7:

Figure 7 shows the current density change of the potassium ferricyanide solution detected by the planar electrode when a sine wave electrical signal with a frequency of the bubble excitation frequency is used to excite the piezoelectric sheet. At t=0s, the analyte is pumped into the microfluidic chip. At t=100s, the sine wave signal is used to excite the piezoelectric film, and at t=250s, the electrical signal input is turned off. The current reaches a plateau between t=200-250s, and this value is recorded as the final equilibrium current. The results show that integrating chips with different micro-pit depths can significantly improve detection sensitivity, and the detection chip with a micro-pit depth of 500 μm has the largest signal increase. The detection current of the detection chip in the presence of acoustic microflow is approximately 6.6 times that in the absence of acoustic microflow.

As can be seen from Figures 5-7, the electrochemical detection chip integrated with acoustic wave microfluidic technology in the present invention has the characteristics of high sensitivity and low temperature rise. At the same time, the chip design is simple and does not require additional circuit design.

In the above test experiments of Examples 1-3 and Comparative Examples 1-2, the flow rate of the solution of the sample to be tested injected into the microfluidic chip was 5 μL/min.

In the above test experiments of Example 1-3 and Comparative Example 1-2, the waveform of the excitation electrical signal of the piezoelectric sheet is a sine wave, the peak-to-peak voltage of the waveform is 10V, and the frequency of the waveform is 12kHz.

In the above test experiments of Examples 1-3 and Comparative Examples 1-2, the potassium ferricyanide solution was a 5 mmol/L potassium ferricyanide aqueous solution.

In summary, the present invention discloses a high-sensitivity electrochemical detection microfluidic platform that integrates sonic microfluidics and micropillar array electrode technology, specifically involving the integration of sonic microfluidic technology and micropillar array technology on a microfluidic chip to achieve ultra-high sensitivity. A highly sensitive electrochemical detection method uses an ultra-deep micro-pit array integrated in a microfluidic chip to spontaneously form a larger bubble array when loading a sample. During the detection process, the bubble array near the micro-pillar array electrode will be Excite strong acoustic microflow, thereby realizing a microfluidic chip that can be used for ultra-highly sensitive electrochemical analysis. Specifically, after injecting the solution to be tested, the platform can spontaneously form a large number of microbubble arrays. These bubbles can be excited by specific frequency sound waves, and acoustic microflows are generated near the surface of the micropillar array electrode. When there are acoustic microflows, the bubbles are located far away. The analyte at the location will increase the contact with the electrode due to the action of acoustic microflow. Under the combined action of convection and diffusion, the mass transfer efficiency of the micropillar array electrode to the analyte is improved, and finally the biomarker detection sensitivity is improved; At the same time, during the working process of acoustic microfluidics, the temperature of the liquid in the chip changes less than 1°C, which demonstrates the superiority of this technology in electrochemical biomarker detection. In addition, the present invention uses bubble-induced acoustic microfluidic technology, which does not require electrodes and piezoelectric substrates, and the microfluidic chip and electrochemical detection platform have simple structures.

It should be noted that the “approximately” used in numerical values in this article means an error of ±2%.

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various modifications can be made without departing from the purpose of the present invention. Variety. In addition, the embodiments of the present invention and the features in the embodiments may be combined with each other without conflict.

Claims (15)

1. A microfluidic chip, characterized in that it includes a chip body and an acoustic microflow initiating mechanism. The chip body includes an electrode layer, a microfluidic channel layer and a top layer in sequence. A microfluidic flow is provided in the microfluidic channel layer. channel, the electrode layer and the top layer are respectively connected with the microfluidic channel, and the electrode layer includes a micro-pillar array electrode; the acoustic microflow initiating mechanism includes an acoustic wave initiating component, and the top layer is provided with the An array of micropits connected by the microfluidic channels; During detection, the micropit array forms a bubble array, the acoustic wave initiating component triggers a sound field, and the bubble array forms an acoustic microflow under the action of the sound field; the acoustic microflow is integrated in the microfluidic channel, and the acoustic microflow increases. The contact between the sample to be measured in the microfluidic channel and the micropillar array electrode; The spacing of micro-pits in the micro-pit array is 10-500 μm; the diameter of one end of the micro-pit array close to the micro-channel layer is 10-300 μm, and the end of the micro-pit away from the micro-channel layer is 10-300 μm. The diameter is 10-300μm; The height of the micro-pillars in the micro-pillar array electrode is 1 μm-10 mm, the spacing of the micro-pillars in the micro-pillar array electrode is 10-500 μm, and the diameter of the top circle of the micro-pillars in the micro-pillar array electrode is 1-500 μm, The diameter of the bottom circle of the micro-pillars in the micro-pillar array electrode is 1-500 μm. 2. A microfluidic chip according to claim 1, characterized in that the number of micro pits in the micro pit array is 1-1000. 3. A microfluidic chip according to claim 1, characterized in that the depth of the micro pits in the micro pit array is 10 μm-10 mm. 4. The microfluidic chip according to claim 1, wherein the size of the microfluidic channel is in the micron level. 5. A microfluidic chip according to claim 1, characterized in that the acoustic wave inducing component includes a piezoelectric transducer. 6. A microfluidic chip according to claim 5, characterized in that the piezoelectric transducer is a piezoelectric sheet. 7. A microfluidic chip according to claim 1, characterized in that the electrode layer includes a substrate and a working electrode, a reference electrode and a counter electrode all disposed on the substrate, and the working electrode, Both the reference electrode and the counter electrode can be in contact with the sample to be measured in the microfluidic channel, and the working electrode is a micropillar array electrode. 8. The microfluidic chip according to claim 1, wherein the micro-pillar array electrode includes a micro-pillar array base and a conductive layer provided on the micro-pillar array base. 9. A microfluidic chip according to claim 8, characterized in that the surface of the conductive layer is modified with nanoparticles. 10. A microfluidic chip according to claim 1, characterized in that the number of micro-pillars in the micro-pillar array electrode is 10-10,000. 11. A method for preparing a microfluidic chip as claimed in claim 1, characterized in that it includes the following steps: S1, micropillar array electrodes are prepared through photolithography or soft photolithography technology; S2, assemble the electrode layer including the micropillar array electrode, the microfluidic channel layer, the top layer and the acoustic microfluidic initiating mechanism to obtain the microfluidic chip. 12. A microfluidic platform, characterized in that the microfluidic platform includes the microfluidic chip according to any one of claims 1 to 10 or the microfluidic chip produced by the method according to claim 11. control chip. 13. An electrochemical detector, characterized in that the electrochemical detector includes the microfluidic chip according to any one of claims 1 to 10 or the microfluidic chip produced by the method according to claim 11. control chip. 14. An electrochemical detection platform, characterized in that the electrochemical detection platform includes the microfluidic chip according to any one of claims 1 to 10 or the microfluidic chip prepared by the method according to claim 11. control chip or the electrochemical detector according to claim 13. 15. The microfluidic chip according to any one of claims 1 to 10 or the microfluidic chip produced by the method according to claim 11 or the microfluidic platform according to claim 12 or the microfluidic platform according to claim 15. Application of the electrochemical detector according to claim 13 or the electrochemical detection platform according to claim 14 in electrochemical detection.