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CN-121978359-A - Microfluidic mass spectrum chip with targeting separation function and preparation method and application thereof

CN121978359ACN 121978359 ACN121978359 ACN 121978359ACN-121978359-A

Abstract

The invention discloses a microfluidic mass spectrum chip with a targeting separation function and a preparation method and application thereof, and belongs to the technical field of biological chips, wherein the preparation method of the microfluidic mass spectrum chip comprises the steps of drawing a microchannel configuration, wherein the microchannel configuration comprises a first sample mixing reaction area and a second sample mixing reaction area, and a first magnetic control separation array area and a second magnetic control separation array area, and a sample inlet of the first sample mixing reaction area is used for inputting Fe 3 O 4 @PDA modified by antibodies or Fe 3 O 4 @PDA modified by Ti 4+ ; transferring the pattern onto a chromium layer glass substrate, forming a PDMS film on a convex template of the microfluidic channel, peeling the PDMS film to obtain a PDMS sheet, attaching the PDMS film to the glass sheet to form a closed microchannel, fixing a magnet under two magnetic control separation array areas, and inserting a connecting pipe into an inlet and an outlet of the chip to obtain the microfluidic mass spectrum chip. The invention has the advantages of low sample/reagent consumption and high automation degree, can rapidly complete detection with high flux, and reduces the burden of operators to the maximum extent.

Inventors

  • ZHAO YAJU

Assignees

  • 浙江工商大学

Dates

Publication Date
20260505
Application Date
20260123

Claims (10)

  1. 1. A manufacturing method of a micro-fluidic mass spectrometry chip with a targeting separation function is characterized by comprising the following steps: Drawing a microchannel configuration, and transferring a pattern corresponding to the microchannel configuration to a laser printing film, wherein the microchannel configuration comprises a first sample mixing reaction zone (1) and a second sample mixing reaction zone (2), three first sample injection ports (3) are arranged at liquid inlets of the first sample mixing reaction zone (1), one of the first sample injection ports (3) is used for inputting antibody modified Fe 3 O 4 @PDA or Ti 4+ modified Fe 3 O 4 @PDA, a liquid outlet of the first sample mixing reaction zone (1) and a liquid inlet of the second sample mixing reaction zone (2) are connected with a first magnetic control separation array zone (4), the first magnetic control separation array zone (4) is connected with a second sample injection port (5) and a first sample outlet (6), a liquid outlet of the second sample mixing reaction zone (2) is connected with a second magnetic control separation array zone (7), and the second magnetic control separation array zone (7) is connected with a second sample outlet (8); Transferring the pattern to a chromium layer glass substrate of SU-8 negative photoresist with a certain thickness by taking a laser printing film as a mask through a photoetching technology, and obtaining a micro-fluidic channel convex template; fully mixing a polymer of the PDMS prepolymer and a crosslinking agent, pouring the mixture on a glass convex template after vacuum degassing, and forming a PDMS film after heating and curing; peeling the PDMS film from the glass convex template, and then punching to obtain a PDMS sheet with a micro-flow path; bonding the PDMS sheet with the glass sheet, and fixing the PDMS sheet with a chip clamp to form a closed micro-channel; and fixing a magnet under the first magnetic control separation array region (4) and the second magnetic control separation array region (7), and inserting a connecting pipe into a sample inlet and a sample outlet of the chip to obtain the micro-fluidic mass spectrum chip.
  2. 2. The method for manufacturing the microfluidic mass spectrometry chip with the targeted separation function according to claim 1, wherein the method for preparing the antibody modified Fe 3 O 4 @PDA comprises the following steps: Dissolving ferric trichloride hexahydrate and sodium acetate in ethylene glycol, transferring the obtained mixture into a hydrothermal kettle for heating, cooling to room temperature, washing the product with ethanol for several times, and then carrying out vacuum drying to obtain Fe 3 O 4 magnetic nano particles; Taking Fe 3 O 4 magnetic nano particles in a test tube, washing the test tube for a plurality of times by using a Tris-HCl solution, adding dopamine and the Tris-HCl solution, placing the test tube into a shaking table to react under the condition of normal temperature and normal pressure, collecting a final product through magnetic separation, washing the final product by using water, dispersing the final product in deionized water, and preserving the final product to obtain Fe 3 O 4 @PDA; And (3) taking Fe 3 O 4 @PDA solution, washing with deionized water, then washing with PBS buffer solution, adding a monoclonal antibody solution of quinolone molecules, placing into a shaking table for reaction at normal temperature and normal pressure, and washing the obtained product with the PBS buffer solution.
  3. 3. The method for manufacturing the micro-fluidic mass spectrometry chip with the target separation function according to claim 1, wherein the preparation method of the Ti 4+ modified Fe 3 O 4 @PDA comprises the following steps: Adding ferric trichloride hexahydrate and trisodium citrate dihydrate into ethylene glycol, performing ultrasonic treatment, respectively adding the ultrasonic solution and anhydrous sodium acetate into a round-bottom flask, performing magnetic stirring, transferring the stirred solution into a hydrothermal kettle, heating, cooling to room temperature, washing the product with ethanol for several times, and then performing vacuum drying to obtain Fe 3 O 4 magnetic nanoparticles; Taking Fe 3 O 4 magnetic nano particles in a test tube, washing the test tube for a plurality of times by using a Tris-HCl solution, adding dopamine and the Tris-HCl solution, placing the test tube into a shaking table to react under the condition of normal temperature and normal pressure, collecting a final product through magnetic separation, washing the final product by using water, dispersing the final product in deionized water, and preserving the final product to obtain Fe 3 O 4 @PDA; Taking Fe 3 O 4 @ PDA solution, magnetically separating supernatant, adding a freshly prepared titanium sulfate solution, performing oscillation reaction at normal temperature, centrifuging to remove supernatant, washing with trifluoroacetic acid and acetonitrile solution, and removing supernatant to obtain a Ti 4+ modified Fe 3 O 4 @ PDA precipitate.
  4. 4. A microfluidic mass spectrum chip with a targeting separation function is characterized by being manufactured by the manufacturing method of the microfluidic mass spectrum chip with the targeting separation function as set forth in any one of claims 1 to 3.
  5. 5. The application of the microfluidic mass spectrometry chip with the targeting separation function according to claim 4, which is characterized by comprising the following steps of: Pushing a sample to be detected, an internal standard object Norfloxacin with known concentration and an Fe 3 O 4 @PDA solution modified by an antibody into a mass spectrum chip through a first sample injection port (3) by using a constant flow injection pump, realizing targeted enrichment of a small molecular target object in a first sample mixing reaction area (1), gradually fixing the reacted composite material in a first fixed magnetic control separation area (4), and flowing out the residual solution from a first sample outlet (6); Removing the magnet below the first fixed magnetic control separation area (4), flowing eluent from the second sample inlet (5) into the micro-fluidic chip channel for flushing, separating small molecular targets in the second sample mixing reaction area (2), and gradually fixing the eluted composite material in the second magnetic control separation array area (7) again; and directly collecting the eluted sample from the second sample outlet (8), and placing the collected liquid into a matrix-assisted laser desorption ionization time-of-flight mass spectrometer for analyzing the target.
  6. 6. The application of the micro-fluidic mass spectrometry chip with the target separation function according to claim 5, wherein the injection flow rate ratio of the sample to be detected to the internal standard Norfloxacin-d5 solution to the antibody modified Fe 3 O 4 @PDA solution is 0.8-1.2:0.8-1.2:1, and the injection duration of the constant flow injection pump is 28-32 min.
  7. 7. The application of the microfluidic mass spectrometry chip with the targeting separation function according to claim 6, wherein the injection flow rate of the antibody modified Fe 3 O 4 @PDA solution is 4.8-5.2 mu L/min, and the injection flow rate of the eluent is 4.8-5.2 mu L/min.
  8. 8. The application of the microfluidic mass spectrometry chip with the targeting separation function according to claim 4, wherein the microfluidic mass spectrometry chip is used for analyzing beta-casein digests and comprises the following steps of: Pushing a to-be-detected sample beta-casein digest, a buffer solution and a Ti 4+ modified Fe 3 O 4 @PDA solution into a mass spectrum chip through a first sample injection port (3) by using a constant flow injection pump, realizing targeting enrichment of phosphopeptide in a first sample mixing reaction zone (1), gradually fixing the reacted composite material in a first fixed magnetic control separation zone (4), and enabling the residual solution to flow out from a first sample outlet (6); Removing a magnet below the first fixed magnetic control separation area (4), flowing 10% ammonia water solution with mass fraction into a microfluidic chip channel from a second sample inlet (5) for flushing, separating a phosphopeptide target in a second sample mixing reaction area (2), and gradually fixing the eluted composite material in a second magnetic control separation array area (7); and directly collecting the eluted sample from the second sample outlet (8), and placing the collected liquid into a matrix-assisted laser desorption ionization time-of-flight mass spectrometer for analyzing the target.
  9. 9. The application of the micro-fluidic mass spectrometry chip with the target separation function according to claim 8, wherein the injection flow rate ratio of the sample to be detected to the buffer solution to the Ti 4+ modified Fe 3 O 4 @PDA is 4.8-5.2:0.8-1.2:1, and the injection duration of the constant flow injection pump is 8-12 min.
  10. 10. The application of the micro-fluidic mass spectrometry chip with the targeted separation function according to claim 9 is characterized in that the injection flow rate of Fe 3 O 4 @PDA modified by Ti 4+ is 8-12 mu L/min, the injection flow rate of 10% ammonia water solution by mass is 8-12 mu L/min, and the buffer solution comprises 50% acetonitrile, 0.1% trifluoroacetic acid and 49.9% water by mass.

Description

Microfluidic mass spectrum chip with targeting separation function and preparation method and application thereof Technical Field The invention relates to a microfluidic mass spectrum chip with a targeting separation function, and a preparation method and application thereof, belonging to the technical field of design, manufacture and application of biological chips. Background Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has been widely used as a soft ionization technique for analysis of various biomolecules such as polypeptides, proteins, oligosaccharides, nucleic acids, and antibiotics. Although this technique itself has high sensitivity, interference of the main components in the sample matrix tends to affect the detection effect of the target molecules when analyzing complex samples, and thus a pre-enrichment treatment of the target analytes is generally required. For example, in the analysis of antibiotic residues in foods, the detection accuracy and sensitivity are affected by the interference of food matrix, and in the identification of phosphorylated proteins, since phosphorylated peptide fragments are low in abundance in biological samples and are easily masked by a large amount of non-phosphorylated peptide fragment signals during mass spectrometry detection, the samples are usually pretreated, phosphorylated peptide fragments are specifically enriched from enzymatic peptide fragment mixtures, and then MALDI-TOF MS analysis is performed. In recent years, sample pre-concentration technology based on nano materials is continuously developed, and various solutions are provided for separating and enriching target substances such as antibiotics, polypeptides and the like. For example, the prior art reports the use of antibody-modified graphene oxide nanohorns for enrichment of chloramphenicol in river water and human serum, thereby enabling MALDI-TOF MS detection. The prior art also reports a core-shell Fe 3O4@PDA-Ti4+ microsphere, and by means of the magnetic separation characteristic, the Ti 4+ locus with rich surface and good water dispersibility, the efficient selective enrichment and mass spectrometry analysis of phosphopeptides in biological samples are realized. However, the above method relies on off-line extraction and purification steps, and has the advantages of limited automation degree, low detection speed, complicated operation process and large consumption of samples and reagents. Disclosure of Invention The invention aims to overcome the defects of the prior art, and provides a microfluidic mass spectrum chip with a targeting separation function, a preparation method and application thereof, which have the advantages of low sample/reagent consumption and high automation degree, can rapidly complete detection with high throughput, and furthest reduces the burden of operators. In order to solve the technical problems, the invention adopts the following technical scheme: in a first aspect, the present invention provides a method for manufacturing a microfluidic mass spectrometry chip with a targeted separation function, including the following steps: Drawing a microchannel configuration, and transferring a pattern corresponding to the microchannel configuration to a laser printing film, wherein the microchannel configuration comprises a first sample mixing reaction zone and a second sample mixing reaction zone, a liquid inlet of the first sample mixing reaction zone is provided with three first sample inlets, one of the first sample inlets is used for inputting antibody modified Fe 3O4 @PDA or Ti 4+ modified Fe 3O4 @PDA, a liquid outlet of the first sample mixing reaction zone and a liquid inlet of the second sample mixing reaction zone are connected with a first magnetic control separation array zone, the first magnetic control separation array zone is connected with a second sample inlet and a first sample outlet, a liquid outlet of the second sample mixing reaction zone is connected with a second magnetic control separation array zone, and the second magnetic control separation array zone is connected with a second sample outlet; Transferring the pattern to a chromium layer glass substrate of SU-8 negative photoresist with a certain thickness by taking a laser printing film as a mask through a photoetching technology, and obtaining a micro-fluidic channel convex template; fully mixing a polymer of the PDMS prepolymer and a crosslinking agent, pouring the mixture on a glass convex template after vacuum degassing, and forming a PDMS film after heating and curing; peeling the PDMS film from the glass convex template, and then punching to obtain a PDMS sheet with a micro-flow path; bonding the PDMS sheet with the glass sheet, and fixing the PDMS sheet with a chip clamp to form a closed micro-channel; And fixing a magnet under the first magnetic control separation array region and the second magnetic control separation array region, and inserti