Micro-fluidic mass spectrum chip with targeted separation function as well as preparation method and application of micro-fluidic mass spectrum chip

Abstract

The invention discloses a micro-fluidic mass spectrum chip with a targeted separation function and a preparation method and application thereof, and belongs to the technical field of biochips, the preparation method of the micro-fluidic mass spectrum chip comprises the following steps: drawing a micro-channel configuration, the micro-channel configuration comprises a first sample mixing reaction zone and a second sample mixing reaction zone, a first magnetic control separation array area and a second magnetic control separation array area; a sample inlet of the first sample mixing reaction area is used for inputting antibody modified Fe3O4 (at) PDA or Ti < 4 + > modified Fe3O4 (at) PDA; transferring the pattern to a chromium layer glass substrate; a PDMS film is formed on the micro-fluidic channel convex surface template; stripping the PDMS film to obtain a PDMS sheet; the micro-channel is attached to the glass sheet to form a closed micro-channel; a magnet is fixed under the two magnetic control separation array areas, and a connecting pipe is inserted into an inlet and an outlet of the chip to obtain the micro-fluidic mass spectrum chip. The method has the advantages of low sample / reagent consumption and high automation degree, can quickly complete detection in a high-throughput manner, and reduces the burden of operators to the greatest extent.

Description

Technical Field

[0001] This invention relates to a microfluidic mass spectrometry chip with targeted separation function, its preparation method and application, belonging to the field of biochip design, fabrication and application technology. Background Technology

[0002] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), as a soft ionization technique, has been widely used in the analysis of various biomolecules such as peptides, proteins, oligosaccharides, nucleic acids, and antibiotics. Although this technique itself has high sensitivity, interference from the main components of the sample matrix often affects the detection of target molecules when analyzing complex samples. Therefore, pre-enrichment of the target analytes is usually required. For example, in the analysis of antibiotic residues in food, interference from the food matrix can affect the accuracy and sensitivity of the detection. Similarly, in the identification of phosphorylated proteins, because phosphorylated peptides are relatively rare in biological samples and are easily masked by a large number of non-phosphorylated peptide signals during mass spectrometry detection, sample pretreatment is usually necessary to specifically enrich phosphorylated peptides from the enzymatically digested peptide mixture before MALDI-TOF MS analysis.

[0003] In recent years, sample preconcentration techniques based on nanomaterials have been continuously developing, providing various solutions for the separation and enrichment of target analytes such as antibiotics and peptides. For example, existing technologies have reported the use of antibody-modified graphene oxide nanohorns for the enrichment of chloramphenicol in river water and human serum, thereby enabling MALDI-TOF MS detection. Existing technologies also report a core-shell Fe3O4@PDA-Ti... 4+ Microspheres, utilizing their magnetic separation properties and abundant Ti surface 4+ Its site and good water dispersibility enable efficient and selective enrichment and mass spectrometry analysis of phosphopeptides in biological samples.

[0004] However, the above methods mostly rely on offline extraction and purification steps, have limited automation and slow detection speed, are relatively cumbersome to operate, and consume a large amount of samples and reagents. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the prior art and provide a microfluidic mass spectrometry chip with targeted separation function, its preparation method and application, which has the advantages of low sample / reagent consumption and high degree of automation, and can complete detection in high throughput and quickly, minimizing the burden on operators.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for fabricating a microfluidic mass spectrometry chip with targeted separation function, comprising the following steps:

[0008] A microchannel configuration is drawn, and the corresponding pattern is transferred to a laser printing film. The microchannel configuration includes a first sample mixing reaction zone and a second sample mixing reaction zone. The first sample mixing reaction zone has three first sample inlets, one of which is used for antibody-modified Fe3O4@PDA or Ti... 4+ The modified Fe3O4@PDA input is connected to the first sample mixing reaction zone outlet and the second sample mixing reaction zone inlet, which are connected to the first magnetic separation array zone. The first magnetic separation array zone is connected to the second sample inlet and the first sample outlet. The outlet of the second sample mixing reaction zone is connected to the second magnetic separation array zone, which is connected to the second sample outlet.

[0009] Using laser-printed film as a mask, the above pattern is transferred to a chromium-layered glass substrate with SU-8 negative photoresist of a certain thickness through photolithography etching technology, and a microfluidic channel convex template is obtained.

[0010] The polymer of PDMS prepolymer and crosslinking agent are thoroughly mixed, degassed under vacuum, and then cast onto a convex glass template. After heating and curing, a PDMS film is formed.

[0011] After peeling the PDMS film from the convex glass template, holes are punched to obtain a PDMS sheet with microflow channels.

[0012] The PDMS sheet is bonded to the glass sheet and fixed with a chip clamp to form a closed microchannel.

[0013] The magnet is fixed directly below the first and second magnetron separation array regions, and the connecting tube is inserted into the sample inlet and outlet of the chip to obtain the microfluidic mass spectrometry chip.

[0014] The preparation method of the antibody-modified Fe3O4@PDA includes:

[0015] Ferric chloride hexahydrate and sodium acetate were dissolved in ethylene glycol. The resulting mixture was then transferred to a hydrothermal reactor and heated. After cooling to room temperature, the black product was washed several times with ethanol and then vacuum dried to obtain Fe3O4 magnetic nanoparticles.

[0016] Fe3O4 magnetic nanoparticles were placed in a test tube, washed several times with Tris-HCl solution, dopamine and Tris-HCl solution were added, and the mixture was placed in a shaker to react under normal temperature and pressure conditions. The final product was collected by magnetic separation, washed with water, and dispersed in deionized water for storage, thus obtaining Fe3O4@PDA.

[0017] Take Fe3O4@PDA solution, wash with deionized water and then with PBS buffer solution, add monoclonal antibody solution of quinolone molecules, and react in a shaker at room temperature and pressure. The resulting product is then washed with PBS buffer solution.

[0018] The Ti 4+ The preparation methods of modified Fe3O4@PDA include:

[0019] Ferric chloride hexahydrate and trisodium citrate dihydrate were added to ethylene glycol and subjected to ultrasonic treatment. The ultrasonic solution and anhydrous sodium acetate were added to a round-bottom flask and magnetically stirred. The stirred solution was transferred to a hydrothermal reactor and heated. After cooling to room temperature, the product was washed several times with ethanol and then vacuum dried to obtain Fe3O4 magnetic nanoparticles.

[0020] Fe3O4 magnetic nanoparticles were placed in a test tube, washed several times with Tris-HCl solution, and then dopamine and Tris-HCl solution were added. The mixture was placed in a shaker and reacted under normal temperature and pressure conditions. The final product was collected by magnetic separation, washed with water, and dispersed in deionized water for storage, thus obtaining Fe3O4@PDA.

[0021] Take Fe3O4@PDA solution, magnetically separate to remove the supernatant, add freshly prepared titanium sulfate solution, shake at room temperature, centrifuge to remove the supernatant, wash with trifluoroacetic acid + acetonitrile solution, remove the supernatant again, and obtain Ti. 4+ Precipitate of modified Fe3O4@PDA.

[0022] Secondly, this invention discloses a microfluidic mass spectrometry chip with targeted separation function, which is fabricated by the aforementioned method for fabricating a microfluidic mass spectrometry chip with targeted separation function.

[0023] Thirdly, this invention discloses an application of a microfluidic mass spectrometry chip with targeted separation function for the analysis of norfloxacin, comprising the following steps:

[0024] The test sample, the known concentration of the internal standard Norfloxacin-d5, and the antibody-modified Fe3O4@PDA solution are injected into the mass spectrometry chip through the first sample inlet using a constant flow syringe pump. The small molecule target is enriched in the first sample mixing reaction zone. The composite material after the reaction is gradually fixed in the first fixed magnetic separation zone, and the remaining solution flows out from the first sample outlet.

[0025] Remove the magnet below the first fixed magnetic separation zone, and let the eluent flow into the microfluidic chip channel from the second sample inlet for rinsing. The small molecule target is detached in the second sample mixing reaction zone, and the eluted composite material is gradually fixed again in the second magnetic separation array zone.

[0026] The eluted sample was collected directly from the second sample outlet, and the collected liquid was placed in a MALDI-TOF MS for analysis of the target analytes.

[0027] The injection flow rate ratio of the internal standard Norfloxacin-d5 solution to the antibody-modified Fe3O4@PDA solution was 0.8~1.2:0.8~1.2:1, and the injection time of the constant flow injection pump was 28~32 min.

[0028] The injection flow rate of the antibody-modified Fe3O4@PDA solution was 4.8–5.2 μL / min, and the injection flow rate of the elution buffer was 4.8–5.2 μL / min.

[0029] Fourthly, this invention discloses an application of a microfluidic mass spectrometry chip with targeted separation function for the analysis of β-casein digests, comprising the following steps:

[0030] The β-casein digest of the sample to be tested, buffer solution and Ti 4+ The modified Fe3O4@PDA solution was injected into the mass spectrometry chip through the first sample inlet using a constant flow injection pump. Targeted enrichment of phosphopeptides was achieved in the first sample mixing reaction zone. The resulting composite material was gradually fixed in the first fixed magnetic separation zone, and the remaining solution flowed out from the first sample outlet.

[0031] Remove the magnet below the first fixed magnetic separation zone, and flush the microfluidic chip channel with a 10% mass fraction ammonia solution from the second sample inlet. The phosphopeptide target is detached in the second sample mixing reaction zone, and the eluted composite material is gradually fixed again in the second magnetic separation array zone.

[0032] The eluted sample was collected directly from the second sample outlet, and the collected liquid was placed in a MALDI-TOF MS for analysis of the target analytes.

[0033] Test sample: Buffer solution: Ti 4+ The injection flow rate ratio of the modified Fe3O4@PDA was 4.8~5.2:0.8~1.2:1, and the injection time of the constant flow injection pump was 8~12 min.

[0034] Ti 4+ The injection flow rate of the modified Fe3O4@PDA was 8~12 μL / min, and the injection flow rate of the 10% mass fraction ammonia solution was 8~12 μL / min; the buffer solution consisted of 50% acetonitrile, 0.1% trifluoroacetic acid and 49.9% water by mass fraction.

[0035] The beneficial effects of this invention: This invention provides a microfluidic mass spectrometry chip with targeted separation function, its preparation method and application, using antibodies or Ti... 4+ Modified Fe3O4@PDA, with antibody or Ti 4+ The combination of targeted separation function and magnetic separation of microfluidic chip can improve the specificity and sensitivity of detection, with the advantage of low sample / reagent consumption, which can better meet the actual detection needs. At the same time, it can realize simple sample manipulation and high degree of automation, thereby greatly improving the detection efficiency. In addition, the design of the microfluidic chip of this invention can help to realize the simultaneous manipulation of multiple samples, facilitate the simultaneous performance of multiple sets of experiments, and complete the detection with high throughput and speed, greatly improving the detection efficiency and minimizing the burden on operators. Attached Figure Description

[0036] Figure 1 This is a scanning electron microscope image of Fe3O4@PDA in Example 1 of the present invention;

[0037] Figure 2 This invention is based on antibodies or Ti. 4+ A schematic diagram of the structure of the modified Fe3O4@PDA microfluidic mass spectrometry chip;

[0038] Figure 3 This invention is based on antibodies or Ti. 4+ A physical image of the modified Fe3O4@PDA microfluidic mass spectrometry chip;

[0039] Figure 4 The image shows the analysis results of norfloxacin using the antibody-modified Fe3O4@PDA microfluidic mass spectrometry chip in Example 1 of this invention.

[0040] Figure 5 This is a scanning electron microscope image of Fe3O4@PDA in Example 4 of the present invention;

[0041] Figure 6 The image shows the MALDI-TOF MS analysis results of the β-casein dilution in Example 4 of this invention.

[0042] Figure 7 To utilize the Ti-based method in Embodiment 4 of the present invention 4+ The MALDI-TOF MS analysis results of the specific enrichment of phosphopeptides in β-casein by the microfluidic mass spectrometry chip modified Fe3O4@PDA.

[0043] The reference numerals in the figure are as follows: 1-First sample mixing reaction zone; 2-Second sample mixing reaction zone; 3-First sample inlet; 4-First magnetic separation array zone; 5-Second sample inlet; 6-First sample outlet; 7-Second magnetic separation array zone; 8-Second sample outlet. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0045] Example 1

[0046] This invention discloses a method for preparing antibody-modified Fe3O4@PDA, comprising the following steps:

[0047] Step 1: 2.15 g of ferric chloride hexahydrate and 0.49 g of sodium acetate were dissolved in 18 mL of ethylene glycol. The mixture was then transferred to a hydrothermal reactor and heated at 200 °C for 8 hours. After cooling to room temperature, the black product was washed several times with ethanol and then vacuum dried at 60 °C for 12 hours to obtain Fe3O4 magnetic nanoparticles with a particle size range of 30–180 nm.

[0048] Step 2: Take 160 mg of Fe3O4 magnetic nanoparticles into a test tube and wash three times with 10 ml of Tris-HCl solution. Add 160 mg of dopamine and 80 mL of Tris-HCl solution, place in a shaker and react for 12 hours at room temperature and pressure. The final product is collected by magnetic separation, washed with water, and dispersed in deionized water for storage to obtain Fe3O4@PDA. Figure 1 The image shows a scanning electron microscope image of Fe3O4@PDA. The obtained Fe3O4@PDA has a core-shell structure and a particle size distribution of 50 nm to 200 nm.

[0049] Step 3: Antibody modification. Take 1250 μL of 2 mg / mL Fe3O4@PDA, wash twice with deionized water, and twice with PBS buffer. Add 500 μL of a 20 μg / mL quinolone monoclonal antibody solution and react in a shaker at room temperature and pressure for 2 hours. Wash twice with PBS buffer and store in 500 μL PBS buffer at 4°C.

[0050] This invention discloses a method for fabricating a microfluidic mass spectrometry chip with targeted separation function based on antibody-modified Fe3O4@PDA, including the following steps:

[0051] Step one: Use Adobe Illustrator 10.0 software to design and draw the microchannel configuration required for the chip. For example... Figure 2 As shown, the microchannel configuration includes a first sample mixing reaction zone 1 and a second sample mixing reaction zone 2. The inlet of the first sample mixing reaction zone 1 is provided with three first sample inlets 3, one of which is used for antibody-modified Fe3O4@PDA or Ti 4+ The modified Fe3O4@PDA input is connected to the first magnetic separation array 4 via the outlet of the first sample mixing reaction zone 1 and the inlet of the second sample mixing reaction zone 2. The first magnetic separation array 4 is connected to the second sample inlet 5 and the first sample outlet 6. The outlet of the second sample mixing reaction zone 2 is connected to the second magnetic separation array 7. The second magnetic separation array 7 is connected to the second sample outlet 8.

[0052] The chip's front end features three primary sample inlets 3: a sample inlet, an internal standard inlet, and an antibody-modified Fe3O4@PDA inlet. The sample is then mixed into a channel within the primary sample mixing reaction zone 1 downstream of the inlets, where the reaction occurs. After separation, the sample is deposited in the primary magnetron separation array zone 4 downstream of the primary sample mixing reaction zone 1. A secondary sample inlet 5 is then located upstream of the secondary channel. After adding eluent, the antibody-modified Fe3O4@PDA and eluent react in the secondary sample mixing reaction zone 2, causing the analyte to detach from the antibody and flow out through the secondary sample outlet 8. The sample from the outlet is collected and analyzed by a mass spectrometer. The designed chip pattern is then transferred to a laser printing film using a high-resolution laser printer.

[0053] Step 2: The above pattern is transferred to a chromium-layered glass substrate with a certain thickness of SU-8 negative photoresist using photolithography etching technology, and a microfluidic channel convex template is obtained.

[0054] Step 3: The polymer and crosslinking agent of PDMS prepolymer are thoroughly mixed, degassed under vacuum, and then poured onto a convex glass template. After heating and curing, a PDMS film is formed.

[0055] Step 4: After peeling the PDMS film from the convex glass template, punch holes to obtain a PDMS sheet with microflow channels.

[0056] Step 5: The PDMS sheet is bonded to the glass sheet and fixed with a chip clamp to form a closed microchannel.

[0057] Step six: Fix the magnet directly below the first magnetron separation array region 4 and the second magnetron separation array region 7, and insert the connecting tube into the chip's inlet and outlet to obtain the microfluidic mass spectrometry chip. A physical image is shown below. Figure 3 As shown.

[0058] This invention also discloses the application of a microfluidic mass spectrometry chip with targeted separation function for the analysis of norfloxacin, including the following steps:

[0059] Step 1: The sample to be tested, the internal standard Norfloxacin-d5 at a known concentration, and the antibody-modified Fe3O4@PDA solution are injected through the first sample inlet 3 using a constant flow syringe pump. Figure 2 The mass spectrometry chip was used with a flow rate of 5 μL / min, where the flow rate ratio was 1:1:1 for the sample to be tested, Norfloxacin-d5 solution as internal standard, and antibody-modified Fe3O4@PDA solution. The constant flow injection time was 30 min. The targeted enrichment of small molecule targets was achieved in the first sample mixing reaction zone 1. The composite material after the reaction was gradually fixed in the first magnetic separation array zone 4, and the remaining solution flowed out from the first sample outlet 6.

[0060] Step 2: Remove the magnet below the first magnetron separation array region 4, and let the eluent flow into the microfluidic chip channel from the second sample inlet 5 at a flow rate of 5 μL / min for rinsing. The small molecule target is detached in the sample mixing reaction region 2, and the eluted composite material is gradually fixed again in the second magnetron separation array region 7.

[0061] Step 3: Collect the eluted sample directly from the second sample outlet 8, and put the collected liquid into MALDI-TOF MS for analysis of the target analytes.

[0062] Figure 4 The mass spectrum is obtained by analyzing norfloxacin in the sample using a microfluidic mass spectrometry chip based on antibody-modified Fe3O4@PDA. In positive ion mode, the mass-to-charge ratio of norfloxacin is 320.1, which is the molecular weight of [M+H]+.

[0063] Example 2

[0064] This embodiment is the same as Embodiment 1, except that in the application of the microfluidic mass spectrometry chip with targeted separation function, the flow rate of the antibody-modified Fe3O4@PDA solution in step one is 5.2 μL / min, the flow rate ratio is sample to test: internal standard Norfloxacin-d5 solution: antibody-modified Fe3O4@PDA solution = 1.2:0.8:1, the constant flow injection time is 28 min, and in step two, the eluent flows into the microfluidic chip channel from the second sample inlet 5 at a flow rate of 4.8 μL / min for rinsing.

[0065] Example 3

[0066] This embodiment is the same as Embodiment 1, except that in the application of the microfluidic mass spectrometry chip with targeted separation function, the flow rate of the antibody-modified Fe3O4@PDA solution in step one is 4.8 μL / min, the flow rate ratio is sample to test: internal standard Norfloxacin-d5 solution: antibody-modified Fe3O4@PDA solution = 0.8:1.2:1, the constant flow injection time is 32 min, and in step two, the eluent flows into the microfluidic chip channel from the second sample inlet 5 at a flow rate of 5.2 μL / min for rinsing.

[0067] Example 4

[0068] This invention discloses a Ti 4+ The preparation method of modified Fe3O4@PDA includes the following steps:

[0069] Step 1: Add 2.162 g of ferric chloride hexahydrate and 0.4 g of trisodium citrate dihydrate to 40 mL of ethylene glycol, and sonicate for 5 minutes. Add the above solution and 2.4 g of anhydrous sodium acetate to a 100 mL round-bottom flask, and stir magnetically for 30 minutes until the solution turns dark brown. Transfer the solution to a hydrothermal reactor and heat at 200 °C for 6 hours. After cooling to room temperature, wash the black product several times with ethanol, and then vacuum dry at 60 °C for 12 hours to obtain Fe3O4 magnetic nanoparticles with a particle size range of 20-80 nm.

[0070] Take 160 mg of Fe3O4 magnetic nanoparticles into a test tube and wash three times with 10 ml of Tris-HCl solution. Add 160 mg of dopamine and 80 mL of Tris-HCl solution, place in a shaker and react for 12 hours at room temperature and pressure. The final product is collected by magnetic separation, washed with water, and dispersed in deionized water for storage to obtain Fe3O4@PDA. Figure 5 This is a scanning electron microscope image of Fe3O4@PDA. The obtained Fe3O4@PDA has a core-shell structure with a particle size distribution of 40-100 nm.

[0071] Step 3, perform Ti 4+ Modification. Take 1 mL of Fe3O4@PDA solution, magnetically separate to remove the supernatant, add 1 mL of freshly prepared 100 mM titanium sulfate solution, shake at room temperature for 2 h, centrifuge to remove the supernatant, wash twice with 0.1% trifluoroacetic acid + 50% acetonitrile solution, remove the supernatant again, and obtain Ti. 4+ Precipitate of modified Fe3O4@PDA.

[0072] This invention discloses a method for fabricating a microfluidic mass spectrometry chip with targeted separation function based on antibody-modified Fe3O4@PDA. The method is the same as in Example 1, except for the three first sample inlets 3, which are the sample inlet, buffer solution inlet, and Ti inlet. 4+ Modified Fe3O4@PDA inlet.

[0073] This invention also discloses the application of a microfluidic mass spectrometry chip with targeted separation function for the analysis of β-casein digests, including the following steps:

[0074] Step 1: Enrichment analysis of phosphopeptides. 1 mg β-casein was dissolved in 1 mL of 50 mM ammonium bicarbonate solution (pH=8.3), heated at 100℃ for 5 min to denature, cooled to room temperature, and then enzymatically digested with 20 μL of 1 μg / μL trypsin at 37℃ for 16 h. Afterwards, 10 μL of trifluoroacetic acid was added to terminate the digestion. The resulting peptide mixture was stored at -20℃, which is the β-casein digest.

[0075] Step 2, Enrichment Detection of Phosphopeptides: The sample to be tested was prepared with β-casein digest, buffer solution (50% acetonitrile, 0.1% trifluoroacetic acid, and 49.9% water), and Ti... 4+ The modified Fe3O4@PDA solution was injected using a constant flow syringe pump through the first sample inlet 3. Figure 2 The mass spectrometry chip has a flow rate of 10 μL / min, where the flow rate ratio is sample:buffer solution:antibody or Ti. 4+ The modified Fe3O4@PDA solution was prepared in a ratio of 5:1:1 for 10 min. Targeted enrichment of phosphopeptides was achieved in the first sample mixing reaction zone 1. The resulting composite material was gradually fixed in the first magnetic separation array zone 4, and the remaining solution flowed out from the first sample outlet 6.

[0076] Step 3: Remove the magnet below the first magnetron separation array area 4, and flush the microfluidic chip channel with a 10% ammonia solution at a flow rate of 10 μL / min from the second sample inlet 5. The phosphopeptide target is removed in the second sample mixing reaction area 2, and the eluted composite material is gradually fixed again in the second magnetron separation array area 7.

[0077] Step 4: Collect the eluted sample directly from the second sample outlet 8, and put the collected liquid into a MALDI-TOF MS for analysis of the target analytes.

[0078] The MALDI-TOF MS analysis results of the β-casein dilution are as follows: Figure 6 As shown, the present invention is based on Ti 4+The specific enrichment of phosphopeptides in β-casein by the modified Fe3O4@PDA microfluidic mass spectrometry chip is shown in the MALDI-TOF MS analysis results. Figure 7 As shown.

[0079] Example 5

[0080] This embodiment is the same as Embodiment 4, except that in step two of the application of the microfluidic mass spectrometry chip with targeted separation function, Ti... 4+ The flow rate of the modified Fe3O4@PDA solution was 12 μL / min, with a flow rate ratio of sample:buffer solution:Ti. 4+ The modified Fe3O4@PDA solution was prepared in a ratio of 5.2:4.8:1. The injection time was 8 min using a constant flow injection pump. In step three, a 10% ammonia solution was injected into the microfluidic chip channel from the second sample inlet 5 at a flow rate of 8 μL / min for rinsing.

[0081] Example 6

[0082] This embodiment is the same as Embodiment 4, except that in step two of the application of the microfluidic mass spectrometry chip with targeted separation function, Ti... 4+ The flow rate of the modified Fe3O4@PDA solution was 8 μL / min, with a flow rate ratio of sample:buffer solution:Ti. 4+ The modified Fe3O4@PDA solution was prepared in a ratio of 4.8:5.2:1. The injection time was 12 min using a constant flow injection pump. In step three, a 10% ammonia solution was injected into the microfluidic chip channel from the second sample inlet 5 at a flow rate of 12 μL / min for rinsing.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for fabricating a microfluidic mass spectrometry chip with targeted separation function, characterized in that: Includes the following steps: The microchannel configuration is drawn, and the pattern corresponding to the microchannel configuration is transferred to a laser printing film. The microchannel configuration includes a first sample mixing reaction zone (1) and a second sample mixing reaction zone (2). The first sample mixing reaction zone (1) is equipped with three first sample inlets (3), one of which is used for antibody-modified Fe3O4@PDA or Ti 4+ The modified Fe3O4@PDA input is connected to the first magnetic separation array area (4) via the outlet of the first sample mixing reaction area (1) and the inlet of the second sample mixing reaction area (2). The first magnetic separation array area (4) is connected to the second sample inlet (5) and the first sample outlet (6). The outlet of the second sample mixing reaction area (2) is connected to the second magnetic separation array area (7). The second magnetic separation array area (7) is connected to the second sample outlet (8). Using laser-printed film as a mask, the above pattern is transferred to a chromium-layered glass substrate with SU-8 negative photoresist of a certain thickness through photolithography etching technology, and a microfluidic channel convex template is obtained. The polymer of PDMS prepolymer and crosslinking agent are thoroughly mixed, degassed under vacuum, and then cast onto a convex glass template. After heating and curing, a PDMS film is formed. After peeling the PDMS film from the convex glass template, holes are punched to obtain a PDMS sheet with microflow channels. The PDMS sheet is bonded to the glass sheet and fixed with a chip clamp to form a closed microchannel. The magnet is fixed directly below the first magnetron separation array region (4) and the second magnetron separation array region (7), and the connecting tube is inserted into the sample inlet and outlet of the chip to obtain the microfluidic mass spectrometry chip.

2. The method for fabricating a microfluidic mass spectrometry chip with targeted separation function according to claim 1, characterized in that: The preparation method of the antibody-modified Fe3O4@PDA includes: Ferric chloride hexahydrate and sodium acetate were dissolved in ethylene glycol. The resulting mixture was then transferred to a hydrothermal reactor and heated. After cooling to room temperature, the product was washed several times with ethanol and then vacuum dried to obtain Fe3O4 magnetic nanoparticles. Fe3O4 magnetic nanoparticles were placed in a test tube, washed several times with Tris-HCl solution, dopamine and Tris-HCl solution were added, and the mixture was placed in a shaker to react under normal temperature and pressure conditions. The final product was collected by magnetic separation, washed with water, and dispersed in deionized water for storage, thus obtaining Fe3O4@PDA. Take Fe3O4@PDA solution, wash with deionized water and then with PBS buffer solution, add monoclonal antibody solution of quinolone molecules, and react in a shaker at room temperature and pressure. The resulting product is then washed with PBS buffer solution.

3. The method for fabricating a microfluidic mass spectrometry chip with targeted separation function according to claim 1, characterized in that: The Ti 4+ The preparation methods of modified Fe3O4@PDA include: Ferric chloride hexahydrate and trisodium citrate dihydrate were added to ethylene glycol and subjected to ultrasonic treatment. The ultrasonic solution and anhydrous sodium acetate were added to a round-bottom flask and magnetically stirred. The stirred solution was transferred to a hydrothermal reactor and heated. After cooling to room temperature, the product was washed several times with ethanol and then vacuum dried to obtain Fe3O4 magnetic nanoparticles. Fe3O4 magnetic nanoparticles were placed in a test tube, washed several times with Tris-HCl solution, and then dopamine and Tris-HCl solution were added. The mixture was placed in a shaker and reacted under normal temperature and pressure conditions. The final product was collected by magnetic separation, washed with water, and dispersed in deionized water for storage, thus obtaining Fe3O4@PDA. Take Fe3O4@PDA solution, magnetically separate to remove the supernatant, add freshly prepared titanium sulfate solution, shake at room temperature, centrifuge to remove the supernatant, wash with trifluoroacetic acid + acetonitrile solution, remove the supernatant again, and obtain Ti. 4+ Precipitate of modified Fe3O4@PDA.

4. A microfluidic mass spectrometry chip with targeted separation function, characterized in that: It is fabricated by the microfluidic mass spectrometry chip with targeted separation function as described in any one of claims 1 to 3.

5. An application of the microfluidic mass spectrometry chip with targeted separation function as described in claim 4, characterized in that: The analysis for norfloxacin includes the following steps: The sample to be tested, the known concentration of the internal standard Norfloxacin-d5, and the antibody-modified Fe3O4@PDA solution are pushed into the mass spectrometry chip through the first sample inlet (3) using a constant flow injection pump. The small molecule target is enriched in the first sample mixing reaction zone (1). The composite material after the reaction is gradually fixed in the first fixed magnetic separation zone (4), and the remaining solution flows out from the first sample outlet (6). Remove the magnet below the first fixed magnetic separation zone (4), and let the eluent flow into the microfluidic chip channel from the second sample inlet (5) for rinsing. The small molecule target is removed in the second sample mixing reaction zone (2), and the eluted composite material is gradually fixed again in the second magnetic separation array zone (7). The eluted sample was collected directly from the second sample outlet (8), and the collected liquid was placed into a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer for analysis of the target analytes.

6. The application of the microfluidic mass spectrometry chip with targeted separation function according to claim 5, characterized in that: The injection flow rate ratio of the internal standard Norfloxacin-d5 solution to the antibody-modified Fe3O4@PDA solution was 0.8~1.2:0.8~1.2:1, and the injection time of the constant flow injection pump was 28~32 min.

7. The application of the microfluidic mass spectrometry chip with targeted separation function according to claim 6, characterized in that: The injection flow rate of the antibody-modified Fe3O4@PDA solution was 4.8–5.2 μL / min, and the injection flow rate of the elution buffer was 4.8–5.2 μL / min.

8. An application of the microfluidic mass spectrometry chip with targeted separation function as described in claim 4, characterized in that: The analysis of β-casein digests includes the following steps: The β-casein digest of the sample to be tested, buffer solution and Ti 4+ The modified Fe3O4@PDA solution was injected into the mass spectrometry chip through the first sample inlet (3) using a constant flow injection pump. The phosphopeptide was targeted and enriched in the first sample mixing reaction zone (1). The composite material after the reaction was gradually fixed in the first fixed magnetic separation zone (4). The remaining solution flowed out from the first sample outlet (6). Remove the magnet below the first fixed magnetic separation zone (4), and flush the microfluidic chip channel with a 10% mass fraction ammonia solution from the second sample inlet (5). In the second sample mixing reaction zone (2), the phosphopeptide target is removed, and the eluted composite material is gradually fixed again in the second magnetic separation array zone (7). The eluted sample was collected directly from the second sample outlet (8), and the collected liquid was placed into a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer for analysis of the target analytes.

9. The application of the microfluidic mass spectrometry chip with targeted separation function according to claim 8, characterized in that: Test sample: Buffer solution: Ti 4+ The injection flow rate ratio of the modified Fe3O4@PDA was 4.8~5.2:0.8~1.2:1, and the injection time of the constant flow injection pump was 8~12 min.

10. The application of the microfluidic mass spectrometry chip with targeted separation function according to claim 9, characterized in that: Ti 4+ The injection flow rate of the modified Fe3O4@PDA was 8~12 μL / min, and the injection flow rate of the 10% mass fraction ammonia solution was 8~12 μL / min; the buffer solution consisted of 50% acetonitrile, 0.1% trifluoroacetic acid and 49.9% water by mass fraction.

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