Multi-channel micro-fluidic chip for parallel detection of multiple pathogenic microorganisms and preparation method of multi-channel micro-fluidic chip

Abstract

The invention discloses a multi-channel micro-fluidic chip for parallel detection of multiple pathogenic microorganisms and a preparation method thereof, the multi-channel micro-fluidic chip comprises an upper layer cover plate unit and a lower layer adsorption unit, and the upper layer cover plate unit and the lower layer adsorption unit are bonded and packaged up and down; the lower-layer adsorption unit comprises a slow flow channel, an adsorption channel and a fluid flow channel; a plurality of stages of adsorption micro-chambers are arranged in the adsorption channel and are formed by cascading adsorption micro-chambers which are distributed in an array manner; the adsorption micro-chambers are in an equilateral triangle shape, every two adsorption micro-chambers are distributed in the adsorption channel in a staggered mode, the inverted-structure photonic crystal microspheres are carried in the adsorption micro-chambers, and the three vertex angles of each adsorption micro-chamber are each provided with a micro triangular pyramid to fix the corresponding inverted-structure photonic crystal microsphere. According to the invention, parallel detection of multiple pathogenic microorganisms can be realized through multiple channels, and specific recognition, efficient adsorption and sensing of different target biomolecules can be realized through the anti-structure photonic crystal microspheres.

Description

Technical Field

[0001] The invention belongs to the technical field of microfluidic chips, and in particular relates to a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms and a preparation method thereof. Background Art

[0002] During food production and distribution, it is necessary to monitor a variety of pathogens to promptly detect food safety hazards. The existing specific detection of a single pathogenic microorganism has low efficiency and large sample loss, and it is urgent to optimize it.

[0003] Parallel detection technology is an innovative technology that has developed rapidly in the field of microbial detection in recent years. This technology can detect multiple pathogenic microorganisms at the same time, significantly improving the efficiency and accuracy of detection. For example, the invention with publication number CN220214969U discloses a LAMP microfluidic chip for parallel testing of multiple samples, which can realize parallel nucleic acid detection of multiple samples. The invention with publication number CN216792246U discloses a device for detecting antigens based on electrochemical immunoassay, and the microfluidic sample collection unit is shunted to one or more parallel microfluidic detection channels. In addition, the invention with publication number CN118513094A discloses a microfluidic detection device and detection method for pathogenic microorganisms. By setting a bubble attraction component, bubbles can be attracted to the bubble puncture structure, which improves the elimination effect of bubbles in the sample liquid compared to the traditional exhaust hole exhaust method. By setting a bubble puncture structure, bubbles in the sample liquid can be actively punctured, eliminating bubble interference during the sample liquid detection process, and improving the accuracy of the detection results.

[0004] Microfluidic chip technology absorbs pathogenic microorganisms through the design of internal flow channels and the addition of adsorbent materials to enrich samples for detection. Since it can flexibly design microchannels, mix and redistribute the fluids in the chip, the amount of reagents consumed is greatly reduced, which can improve the reaction and analysis speed. For example, the invention with publication number CN216303823U discloses a microfluidic chip and its device, which is composed of a chip body composed of two layers of polydimethylsilane layers; the upper surface of the lower polydimethylsilane layer is provided with a serpentine channel, and a plurality of herringbone microstructures are provided in the serpentine channel; wherein the herringbone microstructure includes: a short arm and a long arm at a 90° angle with the short arm; one end of the short arm is connected to the inner wall of one side of the serpentine channel, the other end of the short arm is connected to one end of the long arm, and the other end of the long arm is connected to the inner wall of the other side of the serpentine channel. The microfluidic chip used in the utility model can effectively enrich and identify pathogenic bacteria, wherein the herringbone microstructure can induce chaotic mixing, strengthen the interaction between magnetic beads and bacteria, and further enhance the enrichment efficiency of magnetic beads for bacteria. The enriched magnetic bead-bacteria mixture can be directly used for MALDI-TOF MS analysis to achieve accurate identification of the enriched bacteria.

[0005] However, the existing designs still have some problems. For example, setting up an adsorption microchamber in a serpentine channel may cause the liquid flow rate to be too slow, resulting in chip clogging, the difficulty in making a herringbone adsorption microchamber, the low efficiency of single-channel detection, the low specificity of the adsorbent or the poor adsorption effect, etc. Summary of the invention

[0006] In view of the above-mentioned technical problems, the present invention discloses a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms and a preparation method thereof. The designed multi-stage adsorption microchambers and serpentine slow flow channels slow down the fluid flow rate by changing the fluid form to turbulence. Inverse-structured photonic crystal microspheres are arranged in the adsorption microchambers to increase the contact area between the sample and the adsorbent, thereby improving the adsorption efficiency of the microfluidic chip, thereby realizing parallel detection of multiple pathogenic microorganisms through multiple channels, and realizing specific recognition, efficient adsorption and sensing of different target biological molecules through the inverse-structured photonic crystal microspheres.

[0007] In a first aspect, the present invention discloses a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms, wherein the multi-channel microfluidic chip comprises an upper cover plate unit and a lower adsorption unit, wherein the upper cover plate unit and the lower adsorption unit are bonded and packaged up and down;

[0008] The lower layer adsorption unit comprises a slow flow channel, an adsorption channel and a fluid flow channel;

[0009] The adsorption channel is provided with a multi-stage adsorption microchamber, which is composed of a cascade of adsorption microchambers distributed in an array; the adsorption microchambers are in the shape of equilateral triangles, and are staggered in pairs in the adsorption channel. The adsorption microchambers are equipped with anti-structured photonic crystal microspheres, and a micro triangular pyramid is provided at each of the three vertex corners of the adsorption microchamber to fix the anti-structured photonic crystal microspheres;

[0010] The fluid flow channel includes an inlet flow channel and an outlet flow channel. The inlet flow channel is connected to the first port of the adsorption channel through a slow flow channel, and the outlet flow channel is connected to the second port of the adsorption channel for injecting and collecting fluid.

[0011] As a preferred example, the micro triangular pyramid is made of polydimethylsiloxane.

[0012] As a preferred example, the inverse structure photonic crystal microspheres are inverse opal structure microspheres covalently coupled to human IgG.

[0013] As a preferred example, the upper cover plate unit is a microfluidic chip made of polydimethylsiloxane.

[0014] As a preferred example, a liquid inlet and a liquid outlet are provided on the upper cover plate unit, and the liquid inlet and the liquid outlet are symmetrically distributed on both sides of the upper cover plate unit and are respectively connected to the liquid inlet channel and the liquid outlet channel.

[0015] As a preferred example, the lower layer adsorption unit includes N slow flow channels and N adsorption channels; the slow flow channels and the adsorption channels are connected one by one, and the adsorption channels are parallel to each other;

[0016] The liquid inlet flow channel is a branch-type liquid inlet flow channel, which includes 1 liquid inlet hole and N branch liquid outlets, and the N branch liquid outlets are connected to the N slow flow channels in a one-to-one correspondence; the liquid outlet flow channel is a confluent liquid outlet flow channel, which includes N branch liquid inlets and 1 liquid outlet hole, and the N branch liquid inlets are connected to the N adsorption channels in a one-to-one correspondence.

[0017] As a preferred example, the slow-flow channel is in a serpentine shape.

[0018] In a second aspect, the present invention discloses a method for preparing a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms, the preparation method comprising the following steps:

[0019] S1, using polydimethylsiloxane as a material and using a photolithography mask having an upper cover unit structure engraved thereon to prepare an upper cover unit;

[0020] S2. Using polydimethylsiloxane as the material, a photolithography mask having a fluid flow channel structure, a slow flow channel structure, an adsorption channel structure and a multi-stage adsorption microchamber structure is used to prepare a lower adsorption unit, and the inverse structure photonic crystal microspheres are mounted in the adsorption microchamber on the lower adsorption unit;

[0021] S3. Stack the upper cover unit and the lower adsorption unit up and down and align them, and package them together by bonding.

[0022] As a preferred example, in step S2, the method for preparing the inverse structure photonic crystal microspheres comprises:

[0023] S21: Preparation of photonic crystal sphere templates using silica nanoparticles via droplet self-assembly;

[0024] S22: activating NHS to generate NHS ester and adding it to the hydrogel prepolymer solution, infiltrating the hydrogel prepolymer solution modified with the NHS group into the photonic crystal sphere template, and forming a photonic crystal sphere-hydrogel hybrid by UV curing;

[0025] S23: removing the photonic crystal sphere template in the photonic crystal sphere-hydrogel hybrid to obtain NHS-modified inverse-structured photonic crystal microspheres;

[0026] S24: Dissolve human IgG in coupling buffer, and mix the mixture with microspheres at room temperature for 1 to 2 hours to covalently couple human IgG to the microspheres.

[0027] As a preferred example, in step S3, the bonding method of the upper cover plate unit and the lower adsorption unit is:

[0028] The upper cover plate unit and the lower adsorption unit are stacked and aligned, and the contact surfaces of the upper cover plate unit and the lower adsorption unit are plasma treated for 5 minutes and then fixed with a fixing fixture for 2 hours.

[0029] The beneficial effects of the present invention are:

[0030] First, the multi-channel microfluidic chip and its preparation method for parallel detection of multiple pathogenic microorganisms of the present invention are based on the lower adsorption channel equipped with anti-structured photonic crystal microspheres, which are bonded and packaged with the cover chip up and down, wherein the anti-structured photonic crystal microspheres covalently coupled to human IgG are used as porous adsorbents, which have excellent adsorption effects on various pathogenic microorganisms, and at the same time, the adsorption amount of other probiotics in the sample is small, and efficient and specific adsorption of target biological molecules can be achieved. At the same time, the anti-structured photonic crystal microspheres have a reflection peak of a specific wavelength. After combining with different types of target biological molecules, the displacement of its characteristic reflection peak corresponds to different types, so the sensing of the target pathogenic microorganism types can be achieved, which is efficient and labor-saving, has high sensitivity and specificity, and reduces false negative or false positive results.

[0031] Second, the multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms of the present invention and the preparation method thereof, the lower adsorption unit is provided with multiple serpentine slow-flow channels and flow channels containing multi-stage adsorption microchambers, and the inverse-structured photonic crystal microspheres are carried in the adsorption microchambers. The microfluidic chip changes the fluid state through the serpentine slow-flow channels and the staggered multi-stage adsorption microchambers to form turbulence, thereby increasing the contact time and contact area between the target pathogenic microorganisms in the fluid and the inverse-structured photonic crystal microspheres, thereby improving the adsorption rate of the microfluidic chip to the target pathogenic microorganisms.

[0032] Third, the multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms and the preparation method thereof of the present invention can simultaneously detect multiple pathogenic microorganisms in one experiment, improve detection efficiency, and significantly reduce sample consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the structure of a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms of the present invention;

[0034] Figure 2 It is a structural schematic diagram of the upper cover plate unit of the present invention;

[0035] Figure 3 It is a structural schematic diagram of the lower layer adsorption unit of the present invention;

[0036] Figure 4 It is a schematic structural diagram of the adsorption microchamber of the present invention;

[0037] The figures are marked as follows: 1 is an upper cover plate unit, 1-1 is a cover plate structure of the upper cover plate unit, 1-2 is a liquid inlet of the upper cover plate unit, and 1-3 is a liquid outlet of the upper cover plate unit; 2 is a lower adsorption unit, 2-1 is an adsorption microchamber, 2-2 is a liquid inlet channel, 2-3 is a liquid outlet channel, 2-4 is a serpentine slow flow channel, 2-5 is an adsorption channel, 2-6 is a liquid inlet hole position of the liquid inlet channel, 2-7 is a liquid outlet hole position of the liquid outlet channel, 3-1 is a micro triangular pyramid, and 3-2 is an inverse structure photonic crystal microsphere. DETAILED DESCRIPTION

[0038] The following examples will enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0039] See also Figures 1 to 4 The present invention provides a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms, including an upper cover unit 1 and a lower adsorption unit 2.

[0040] The upper cover unit 1 is a microfluidic chip made of polydimethylsiloxane (PDMS), and a liquid inlet 1-2 and a liquid outlet 1-3 are provided on the upper cover microfluidic chip 1, and the liquid inlet 1-2 and the liquid outlet 1-3 are symmetrically distributed on both sides of the cover structure 1-1.

[0041] The lower adsorption unit 2 includes a serpentine slow flow channel 2-4, an adsorption channel 2-5 and a fluid flow channel. A plurality of adsorption microchambers 2-1 are arranged in the adsorption channel. The adsorption microchamber is surrounded by three micro-triangular pyramids 3-1 at the apex of an equilateral triangle. The micro-triangular pyramid is made of polydimethylsiloxane. The inverse structure photonic crystal microspheres 3-2 are carried in the adsorption microchamber. The adsorption microchambers are staggered in pairs in the adsorption channel. The inverse structure photonic crystal microspheres are used as adsorbents to adsorb target microorganisms. The microspheres are carried in the adsorption microchambers. The surface modification group of the inverse structure photonic crystal microspheres is NHS, which can form stable peptide bonds with proteins and other molecules with primary amine groups. No EDC or glutaraldehyde activation is required. It is only necessary to dissolve human IgG in the coupling buffer and mix the mixed solution with the microspheres at room temperature for 1 to 2 hours to covalently couple the biological ligand to the microspheres to achieve specific recognition and adsorption of target microorganisms. The present invention uses the lower adsorption channel equipped with inverse-structured photonic crystal microspheres as the main body, and is bonded and packaged with the cover chip up and down, wherein the inverse-structured photonic crystal microspheres covalently coupled to human IgG are used as porous adsorbents, which have excellent adsorption effects on various pathogenic microorganisms, and at the same time, the adsorption amount of other probiotics in the sample is small, and efficient and specific adsorption of target biological molecules can be achieved. At the same time, the inverse-structured photonic crystal microspheres have a reflection peak of a specific wavelength. After combining with different types of target biological molecules, the displacement of its characteristic reflection peak corresponds to different types, so the sensing of the target pathogenic microorganism types can be achieved, which is efficient and labor-saving, has high sensitivity and specificity, and reduces false negative or false positive results.

[0042] The fluid flow channel includes an inlet channel 2-2 and an outlet channel 2-3. The inlet channel 2-2 is a split-flow inlet channel, and the outlet channel 2-3 is a confluent-flow outlet channel. The split-flow inlet channel and the confluent-flow outlet channel are arranged on both sides of the serpentine slow flow channel and the adsorption channel, and the inlet port 1-2 is directly opposite to the inlet port hole of the inlet channel 2-2, and the outlet port 1-3 is directly opposite to the outlet port hole of the outlet channel 2-3, which are used for fluid injection and collection respectively. Figure 3 As shown, the liquid inlet channel of this embodiment is a one-to-ten-flow type liquid inlet channel, and the liquid outlet channel is a ten-in-one confluent type liquid outlet channel. The liquid inlet of the upper cover plate unit is directly opposite to the liquid inlet hole of the liquid inlet channel, and the liquid outlet of the upper cover plate unit is directly opposite to the liquid outlet hole of the liquid outlet channel, which are used for fluid injection and collection respectively. The lower adsorption unit is provided with a plurality of serpentine slow-flow channels and a flow channel containing multi-stage adsorption microchambers. The inverse-structured photonic crystal microspheres are carried in the adsorption microchambers. The microfluidic chip changes the fluid state through the serpentine slow-flow channels and the staggered multi-stage adsorption microchambers to form turbulence, thereby increasing the contact time and contact area between the target pathogenic microorganisms in the fluid and the inverse-structured photonic crystal microspheres, thereby improving the adsorption rate of the microfluidic chip to the target pathogenic microorganisms.

[0043] The upper blocking unit 1 and the lower adsorption unit 2 are bonded and packaged up and down.

[0044] The present invention also provides a method for preparing the multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms, comprising the following steps:

[0045] S1. Preparation of upper cover plate unit: using polydimethylsiloxane as material and using a photolithography mask having a cover plate unit structure engraved thereon to prepare a cover plate microfluidic chip;

[0046] S2. Preparation of lower layer adsorption unit: using polydimethylsiloxane as material, using a photolithography mask plate engraved with a fluid flow channel structure, a slow flow channel structure, an adsorption channel structure and a multi-stage adsorption micro-chamber structure to prepare an adsorption base plate, and placing the inverse structure photonic crystal microspheres in the adsorption micro-chamber of the adsorption base plate;

[0047] S3, the upper cover unit and the lower adsorption unit are bonded and packaged up and down: the upper cover unit and the lower adsorption unit are stacked up and aligned, and then packaged into one by bonding.

[0048] Furthermore, in step S2, the inverse structure photonic crystal microspheres are inverse structure microspheres covalently coupled to human IgG, which are prepared by the following steps:

[0049] S21: Preparation of photonic crystal sphere templates using silica nanoparticles through droplet self-assembly method.

[0050] S22: Activate NHS to generate NHS ester and add it to the hydrogel prepolymer solution, then infiltrate the hydrogel prepolymer solution modified with NHS group into the photonic crystal sphere template, and form the photonic crystal sphere-hydrogel hybrid by UV curing. Exemplarily, the hydrogel prepolymer solution is selected from one or more of polyurethane, polyethylene glycol, polyethylene glycol diacrylate, and methacrylate gelatin. For example, the components of the hydrogel prepolymer solution are 10% v / v methacrylate gelatin, 10% v / v polyethylene glycol diacrylate, and 1% v / v photoinitiator.

[0051] S23: removing the photonic crystal sphere template in the photonic crystal sphere-hydrogel hybrid to obtain NHS-modified inverse-structured photonic crystal microspheres.

[0052] S24: Dissolve human IgG in coupling buffer, mix the mixture with microspheres at room temperature for 1 to 2 hours, and covalently couple human IgG to the microspheres. Exemplarily, the method for preparing human IgG coupling buffer is to take 200 μL of human IgG solution (50 mg / ml), dilute it with Coupling Buffer 0.1 mol / L PBS, and prepare a 2.0 mg / mL IgG solution.

[0053] Furthermore, the bonding method of the upper cover plate unit and the lower adsorption unit is: the upper cover plate unit and the lower adsorption unit are stacked and aligned, and the contact surface of the upper cover plate unit and the lower adsorption unit is plasma treated for 15 minutes and then fixed with a fixing fixture for 2 hours.

[0054] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms, characterized in that: The multi-channel microfluidic chip comprises an upper cover plate unit and a lower adsorption unit, and the upper cover plate unit and the lower adsorption unit are bonded and packaged up and down; The lower layer adsorption unit comprises a slow flow channel, an adsorption channel and a fluid flow channel; The adsorption channel is provided with a multi-stage adsorption microchamber, which is composed of a cascade of adsorption microchambers distributed in an array; the adsorption microchambers are in the shape of equilateral triangles, and are staggered in pairs in the adsorption channel. The adsorption microchambers are equipped with anti-structured photonic crystal microspheres, and a micro triangular pyramid is provided at each of the three vertex corners of the adsorption microchamber to fix the anti-structured photonic crystal microspheres; The fluid flow channel includes an inlet flow channel and an outlet flow channel. The inlet flow channel is connected to the first port of the adsorption channel through a slow flow channel, and the outlet flow channel is connected to the second port of the adsorption channel for injecting and collecting fluid.

2. The multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 1, characterized in that: The micro triangular pyramid is made of polydimethylsiloxane.

3. The multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 1, characterized in that: The inverse structure photonic crystal microspheres are inverse opal structure microspheres covalently coupled to human IgG.

4. The multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 1, characterized in that: The upper cover plate unit is a microfluidic chip made of polydimethylsiloxane.

5. The multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 1, characterized in that: The upper cover plate unit is provided with a liquid inlet and a liquid outlet, and the liquid inlet and the liquid outlet are symmetrically distributed on both sides of the upper cover plate unit and are respectively connected to the liquid inlet flow channel and the liquid outlet flow channel.

6. The multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 1, characterized in that: The lower layer adsorption unit comprises N slow flow channels and N adsorption channels; the slow flow channels and the adsorption channels are connected one by one, and the adsorption channels are parallel to each other; The liquid inlet flow channel is a branch-type liquid inlet flow channel, which includes 1 liquid inlet hole and N branch liquid outlets, and the N branch liquid outlets are connected to the N slow flow channels in a one-to-one correspondence; the liquid outlet flow channel is a confluent liquid outlet flow channel, which includes N branch liquid inlets and 1 liquid outlet hole, and the N branch liquid inlets are connected to the N adsorption channels in a one-to-one correspondence.

7. The multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 1, characterized in that: The slow-flow channel is in a serpentine shape.

8. A method for preparing a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms based on any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: S1, using polydimethylsiloxane as a material and using a photolithography mask having an upper cover unit structure engraved thereon to prepare an upper cover unit; S2. Using polydimethylsiloxane as the material, a photolithography mask having a fluid flow channel structure, a slow flow channel structure, an adsorption channel structure and a multi-stage adsorption microchamber structure is used to prepare a lower adsorption unit, and the inverse structure photonic crystal microspheres are mounted in the adsorption microchamber on the lower adsorption unit; S3. Stack the upper cover unit and the lower adsorption unit up and down and align them, and package them together by bonding.

9. The method for preparing a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 8, characterized in that: In step S2, the method for preparing the inverse structure photonic crystal microspheres comprises: S21: Preparation of photonic crystal sphere templates using silica nanoparticles via droplet self-assembly; S22: activating NHS to generate NHS ester and adding it to the hydrogel prepolymer solution, infiltrating the hydrogel prepolymer solution modified with the NHS group into the photonic crystal sphere template, and forming a photonic crystal sphere-hydrogel hybrid by UV curing; S23: removing the photonic crystal sphere template in the photonic crystal sphere-hydrogel hybrid to obtain NHS-modified inverse-structured photonic crystal microspheres; S24: Dissolve human IgG in coupling buffer, and mix the mixture with microspheres at room temperature for 1 to 2 hours to covalently couple human IgG to the microspheres.

10. The method for preparing a multi-channel microfluidic chip for parallel detection of multiple pathogenic microorganisms according to claim 8, characterized in that: In step S3, the bonding method of the upper cover plate unit and the lower adsorption unit is: The upper cover plate unit and the lower adsorption unit are stacked and aligned, and the contact surfaces of the upper cover plate unit and the lower adsorption unit are plasma treated for 5 minutes and then fixed with a fixing fixture for 2 hours.

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