In-situ Fabrication and Detection Type Hydrogel Resonance Detection Device and Method for Protein Markers

Abstract

The present invention discloses a ready-to-use hydrogel resonance detection device and method for protein markers. The device includes: a stepwise reaction module including a first base, a cover plate, and a plurality of flow channel units, and the flow channel units are separated into a plurality of cells by a separation unit; a hydrogel incubation module including an incubation reaction pool, a plurality of mold racks, and a plurality of conversion units, and the conversion units are arranged above the mold racks and are used to introduce the sample to be tested into the corresponding mold racks; a detection module is used to form an induction field with the crack resonance ring of the corresponding mold rack for detection. The detection method is to sequentially load the first reagent, the second reagent, the third reagent, and the fourth reagent into the cells of the corresponding flow channel unit, and when different separation units are rotated, different reagents are mixed for stepwise reaction and then detected by the detection module. The present invention realizes portable and rapid quantitative detection of protein markers in the sample to be tested, and has the advantages of simple and rapid operation, high sensitivity, and simultaneous reading of multi-target results.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a ready-to-use hydrogel resonance detection device and method for protein markers. Background Art

[0002] Currently, researchers mainly use time-consuming and complex optical detection methods to detect protein markers (such as in environmental liquid samples, saliva samples or blood samples). Recently, a concept of digital enzyme-linked immunosorbent assay has emerged for multiplex detection of protein markers with attomolar sensitivity. However, this technology either still relies on large laboratory instruments, is complex to operate, requires professionals and takes a long time, or the miniaturized portable devices usually can only achieve semi-quantitative detection, with low accuracy, lack of standardization and limited application scope.

[0003] For example, the Chinese patent with the patent publication number CN220854894U proposes a PCT and CRP detection kit, whose detection of PCT and CRP depends on observing the color change of the test piece and cannot quantitatively detect the CRP content in the test sample; the Chinese patent with the patent publication number CN220525831U proposes an MxA / CRP detection reagent card and kit, and quantitative detection of the test sample requires using the detection reagent card in combination with a large laboratory instrument, an immunoquantitative analyzer; the Chinese patent with the patent publication number CN118777610A proposes time-resolved fluorescence microspheres and their preparation method, a fluorescence immunoassay chromatography reagent plate and kit, and detection of the test sample requires dropping the test sample on the test card and then scanning it with a blood cell analyzer for detection. Among them, the lowest detection limit for CRP is 0.5 mg / mL, and the lowest detection limit for SAA is 2.5 mg / mL, with poor detection performance; the Chinese patent with the patent publication number CN118879941A proposes a respiratory syncytial virus detection kit, and the Chinese patent with the patent publication number CN119101724A proposes a freeze-dried PCR reagent and kit for respiratory virus nucleic acid detection. Detection of respiratory syncytial virus (RSV) requires using a fluorescence PCR detector for detection, with a long detection time and only positive or negative results can be obtained, and quantitative detection cannot be achieved.

[0004] In addition, the concentration of biomarkers in diluted samples is relatively low, which challenges the practical limits of many electrochemical sensors. In recent years, an increasing number of hydrogel sensors fabricated using different methods have been used in various fields for trace target detection. These hydrogel-based sensors have characteristics such as higher sensitivity and shorter response time, and are very suitable for developing point-of-care (POC) devices in complex biological fluid samples. However, in the prior art, the detection elements of the kits are usually prepared in advance and then assembled and connected with other elements for detection. During use, it may be affected by multiple factors. For example, the stability preservation time of the liquid framework after hydrogel preparation is limited, and the detection activity of the sensitive element antigen-antibody therein is affected as time goes by. Therefore, it is very necessary to develop a fast, highly sensitive, and hydrogel-based portable sample detection scheme. Summary of the Invention

[0005] The purpose of the present invention is to address the above problems and propose a hydrogel resonance detection device and method for on-site preparation and on-site detection of protein biomarkers, so as to achieve portable, rapid, and quantitative detection of protein biomarkers in a sample to be detected, and has the advantages of simple, fast, highly sensitive detection operation and simultaneous reading of multi-target results.

[0006] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0007] A hydrogel resonance detection device for on-site preparation and on-site detection of protein biomarkers proposed by the present invention includes a step-by-step reaction module, a hydrogel incubation module, and a detection module, wherein:

[0008] The step-by-step reaction module includes a first base, a cover plate, and a plurality of flow channel units. The first base and the cover plate form a plurality of cavities. The flow channel units are correspondingly placed inside the cavities one by one. Each flow channel unit includes a plurality of partition units rotatably connected to the first base. By rotating different partition units, the corresponding cavity is divided into a plurality of grids for accommodating different reagents, and different reagents are mixed to carry out step-by-step reactions.

[0009] A hydrogel incubation module comprises an incubation reaction pool, a plurality of mold racks and a plurality of conversion units, wherein the mold rack is built into the incubation reaction pool and corresponds to the flow channel unit one by one, the upper wall and the lower wall of the mold rack are respectively attached to the first crack resonance ring and the second crack resonance ring and have opposite opening directions, the mold rack also has a containing groove for accommodating reagents and the upper wall is sealed with an easy-tear film, the easy-tear film is located above the first crack resonance ring, the incubation reaction pool is provided with a plurality of first flow channel inlets corresponding to and connected to the flow channel unit one by one, the mold rack is provided with a second flow channel inlet connected to the corresponding first flow channel inlet, the reagents after step-by-step reaction enter the mold rack through the first flow channel inlet and the second flow channel inlet in turn and react with the reagents in the containing groove to form a hydrogel, the conversion units are arranged one by one above the mold rack and are used to collect the samples to be tested into the corresponding mold rack, and the samples to be tested contain protein markers;

[0010] The detection module is used to form an induction field with the first split resonant ring and the second split resonant ring of the corresponding mold frame for detection.

[0011] Preferably, the hydrogel incubation module also includes an upper cover and a mounting base which are stacked in sequence above the incubation reaction pool, the upper cover is provided with a first through hole and a plurality of second through holes, the mounting base is provided with a sample loading port and a plurality of through slots, the sample loading port corresponds to the first through hole, the through slot corresponds one-to-one with the second through hole, and the second through hole corresponds one-to-one with the mold frame, and the conversion unit is sequentially passed through the corresponding second through hole and through slot.

[0012] Preferably, the step-by-step reaction module includes two flow channel units arranged side by side, which are respectively recorded as the first flow channel unit and the second flow channel unit. A flow channel dividing column and two baffles are arranged side by side on the first base. The cover plate includes two shell plates. The flow channel dividing column is located between the two baffles for isolation. The two shell plates are correspondingly covered on the baffles and are respectively attached to the two sides of the flow channel dividing column to form two cavities. The flow channel unit also includes a flow channel bottom plate and a partition. The flow channel bottom plate is attached to the inner bottom wall of the corresponding cavity. The partition is connected to one end of the flow channel bottom plate away from the hydrogel incubation module, and is vertically attached to the first base and the shell plate to realize the sealing of the corresponding grid. A number of positioning blocks are also arranged side by side on each baffle, and the partition unit is rotatably connected with the positioning block in a one-to-one correspondence.

[0013] Preferably, there are four separating units arranged side by side, including a lever, a rotating shaft and two spring washers. The rotating shaft is rotatably connected to the first base. The lever is connected to the rotating shaft and is inclined and penetrates through the first base. One end of the lever abuts against the inner wall of the first base, and the other end extends out of the first base. The two spring washers are attached to the lever and the first base and are respectively located in two acute-angle regions formed by the lever and the first base. The four separating units divide the corresponding cavity into four grids for accommodating different reagents. That is, a first grid is formed between the first separating unit away from the hydrogel incubation module and the first base, a second grid is formed between the first separating unit away from the hydrogel incubation module and the second separating unit, a third grid is formed between the second separating unit away from the hydrogel incubation module and the third separating unit, and a fourth grid is formed between the third separating unit away from the hydrogel incubation module and the fourth separating unit.

[0014] Preferably, the conversion unit includes a splicing frame, a conversion bracket and a key. The splicing frame includes several flipping blocks flexibly connected to different side walls of the mold frame. The tearable film is also connected to the flipping blocks and is provided with tear lines. The conversion bracket includes a live-jaw positioning ring and a plurality of support rods. Each support rod is cross-penetrated through the live-jaw positioning ring, and one end is arranged opposite to the key, and the other end is connected to the flipping blocks one by one.

[0015] Preferably, the detection module includes a second base, a sealing plate, at least one coil, a connecting wire, an SMA interface and an SMA female head. The sealing plate is located between the incubation reaction pool and the second base. The coil, the connecting wire and the SMA interface are all arranged on the sealing plate, and the coil performs inductive detection corresponding to the mold frame one by one and is connected to the SMA interface through the connecting wire. The SMA female head is electrically connected to the SMA interface and is located on the second base.

[0016] Preferably, the detection module is also electrically connected to a network analyzer through a cable to realize the reading of detection data.

[0017] A method for on-site preparation and on-site detection of hydrogel resonance for protein markers, based on any of the above on-site preparation and on-site detection devices for hydrogel resonance for protein markers, includes the following steps:

[0018] S1. Load the first reagent, the second reagent, the third reagent and the fourth reagent into the corresponding grids of the corresponding flow channel unit in sequence according to the order close to the hydrogel incubation module, where:

[0019] The first reagent is ultrapure water and acrylamide;

[0020] The second reagent is a vinylated reactant obtained by mixing 1 μg of protein marker and 0.5 μL of N-succinimidyl acrylate and then successively passing through a water bath and ultrafiltration purification, as well as 0.5 μL of N,N'-methylenebisacrylamide and 0.75 μL of N,N,N',N'-tetramethylethylenediamine;

[0021] The third reagent is 0.2 μL - 0.25 μL of ammonium persulfate;

[0022] The fourth reagent is a solution obtained by adding 25 μL of a solution after incubating 16 ng of a protein biomarker antibody in 1 mL of gold nanoparticles in a shaker in the dark;

[0023] Alternatively, the first reagent is ultrapure water, acrylamide, 0.5 μL of N,N'-methylenebisacrylamide, and 0.75 μL of N,N,N’,N’-tetramethylethylenediamine;

[0024] The second reagent is a vinylated reactant obtained by mixing 1 μg of a protein biomarker with 0.5 μL of N-succinimidyl acrylate and then successively undergoing a water bath and ultrafiltration purification;

[0025] The third reagent is 0.2 μL - 0.25 μL of ammonium persulfate;

[0026] The fourth reagent is a solution obtained by adding 25 μL of a solution after incubating 16 ng of a protein biomarker antibody in 1 mL of gold nanoparticles in a shaker in the dark;

[0027] S2. Load 1 μL - 1.25 μL of ammonium persulfate into the mold holder;

[0028] S3. When in use, place the on-demand in-situ hydrogel resonance detection device for the protein biomarker vertically, turn the corresponding partition unit to mix the first reagent and the second reagent and shake gently to form a first solution, then turn the corresponding partition unit to mix the first solution and the third reagent, shake gently and let stand for a first preset time to form a second solution, and finally turn the remaining partition units to pour the second solution and the fourth reagent into the mold holder and let stand vertically for a second preset time to obtain the reagent after step-by-step reaction, which is the hydrogel, and complete the coupling of the hydrogel with the crack resonance ring of the corresponding mold holder;

[0029] S4. Place the on-demand in-situ hydrogel resonance detection device for the protein biomarker horizontally, press the conversion unit of the corresponding flow channel unit to tear the tear film;

[0030] S5. Use the detection module to read out the initial detection result f 0 ;

[0031] S6. Drop the sample to be tested into the hydrogel, let stand for a third preset time, and then use the detection module to read out the secondary detection result f 1 ;

[0032] S7. According to the initial detection result f 0 and the secondary detection result f 1 and combined with the corresponding linear equation, calculate the content detection result of the protein biomarker in the sample to be tested, that is, (f 1 - f 0 ) / f0 It is calculated as the normalized resonant frequency offset corresponding to the linear equation.

[0033] Preferably, each split resonant ring is pretreated before assembly. The pretreatment includes sequentially performing ultra-pure water cleaning, nitrogen drying, and oxygen plasma surface treatment. The duration of the oxygen plasma surface treatment is 3 min - 6 min.

[0034] Preferably, the ultra-pure water is 31 μL, and the acrylamide is 7.5 mg - 9 mg; the concentration of N,N'-methylenebisacrylamide is 20 mg / mL, and the concentration of N,N,N’,N’-tetramethylethylenediamine is 0.6 mol / L - 0.8 mol / L; the temperature for incubating in the shaker in the dark is 4°C, and the time is 8 hours - 14 hours; the concentration of ammonium persulfate is 2 mol / L.

[0035] Preferably, the temperature of the water bath is 35°C - 37°C, and the time is 50 minutes - 1 hour; the ultrafiltration purification is centrifugation at 14000 g and lasts for 25 minutes - 40 minutes under the condition of 2°C - 6°C.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] This application is an integrated device that can be rapidly prepared on-site and can immediately carry out multiplex rapid detection. It includes a step-by-step reaction module, a hydrogel incubation module, and a detection module, realizing the miniaturized assembly integration of functions such as the preparation of sensitive elements (such as hydrogels), sample addition and incubation, and result reading. Among them, the hydrogel incubation module can be transformed by pressing the conversion unit, and the influence of the deformation generated by the coupled incubation of the hydrogel in the mold rack on the split resonant ring is detected. That is, the preparation raw materials of the core sensitive element, the multi-target immune hydrogel, are assembled in the step-by-step reaction module, and after step-by-step raw material mixing, gel formation and coupling with the split resonant ring are realized in the mold rack to obtain a core detection element with high sensitivity to the detection target. By pressing the conversion unit, the mold rack can be transformed into an incubation pool. The untreated sample to be detected is dropped from the sample addition port into the incubation pool. After reacting for a period of time, a network analyzer can be used to connect to the detection module to read the detection result. In summary, this application has strong operability and integrates the flexible conversion of storage, preparation, and detection. It is stored in the form of raw materials and can be rapidly prepared and used when detection is needed. It can improve the long-term stability of the immune activity of immune elements such as protein markers, has few interfering factors, and the design of preparing and using on-site can maximize the long-term stability of the product. It has a long shelf life, the best sensitivity and specificity, and has the advantages of simplicity, accuracy, and rapidity. The experimental results have good repeatability and can be used for quantitative detection of protein markers, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic structural diagram of the in-situ preparation and in-situ detection type hydrogel resonance detection device for protein markers of the present invention;

[0039] Figure 2 Schematic diagram of the internal structure of the on-site preparation and on-site detection type hydrogel resonance detection device for protein markers of the present invention;

[0040] Figure 3 For the present invention Figure 2 exploded view;

[0041] Figure 4 Exploded view of the hydrogel incubation module and detection module of the present invention;

[0042] Figure 5 Schematic diagram of the internal structure of the hydrogel incubation module of the present invention;

[0043] Figure 6 For the present invention Figure 5 exploded view;

[0044] Figure 7 For the present invention Figure 5 schematic diagram of conversion states;

[0045] Figure 8 Schematic diagram of the readout principle of the on-site preparation and on-site detection type hydrogel resonance detection device for protein markers of the present invention;

[0046] Figure 9 Flow chart of the on-site preparation and on-site detection type hydrogel resonance detection method for protein markers of the present invention;

[0047] Figure 10 Characterization result diagram of the hydrogel preparation of the present invention. Among them, Fig. (a) is a scanning electron microscope image of the distribution of gold nanoparticles in the hydrogel, Fig. (b) is a partial enlarged view of Fig. (a), and Fig. (c) is the line scan result diagram of energy dispersive X-ray spectroscopy;

[0048] Figure 11 Change diagram of the porous structure of the hydrogel before and after adding the sample to be tested of the present invention. Among them, Fig. (a) is a scanning electron microscope image of the porous structure of the hydrogel before adding the sample to be tested, Fig. (b) is a scanning electron microscope image of the porous structure of the hydrogel after adding the sample to be tested, the upper part of Fig. (c) is a relationship diagram between the pore diameter and the number of the hydrogel before adding the sample to be tested, and the lower part of Fig. (c) is a relationship diagram between the pore diameter and the number of the hydrogel after adding the sample to be tested;

[0049] Figure 12 Response effect diagram of the first protein marker of the present invention incubated for different times;

[0050] Figure 13 Standard curve diagram of the sample to be tested incubated for 5 minutes under different concentrations of the first protein marker standard sample of the present invention;

[0051] Figure 14 The response effect diagram of incubating the second protein marker of the present invention for different times;

[0052] Figure 15 The standard curve graph of the test sample after incubating for 5 minutes under different standard sample concentrations of the second protein marker of the present invention.

[0053] Explanation of the reference numerals: 1. Step-by-step reaction module; 2. Hydrogel incubation module; 3. Detection module; 11. First base; 12. First flow channel unit; 13. Second flow channel unit; 14. Cover plate; 111. Flow channel separation column; 112. Baffle; 113. Positioning block; 121. Flow channel bottom plate; 122. Partition; 123. Separation unit; 12a. First grid; 12b. Second grid; 12c. Third grid; 12d. Fourth grid; 123a. Pushing rod; 123b. Rotating shaft; 123c. Elastic pad; 21. Incubation reaction pool; 22. Mold rack; 23. Conversion unit; 24. Upper cover; 25. Mounting seat; 26. Button; 27. First split resonant ring; 28. Second split resonant ring; 211. First flow channel inlet; 221. Second flow channel inlet; 231. Splicing rack; 232. Easy-to-tear film; 233. Conversion bracket; 233a. Support rod; 233b. Movable mouth positioning ring; 251. Sampling port; 31. Second base; 32. Sealing plate; 33. Coil; 34. Connecting wire; 35. SMA interface; 36. SMA female head; 10. Detection device; 20. Network analyzer; 30. Cable. Detailed implementation manners

[0054] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0055] It should be noted that when a component is referred to as being "connected" to another component, it can be directly connected to the other component or there may also be an intermediate component. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present application. The terms used in the description of the present application in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present application. Embodiment

[0056] As Figures 1 - 8 shown, a ready-to-use and on-site detection type hydrogel resonance detection device for protein markers includes a step-by-step reaction module 1, a hydrogel incubation module 2, and a detection module 3, wherein:

[0057] The step-by-step reaction module 1 includes a first base 11, a cover plate 14 and a plurality of flow channel units. The first base 11 and the cover plate 14 form a plurality of cavities. The flow channel units are correspondingly disposed inside the cavities one by one. Each flow channel unit includes a plurality of partition units 123 rotatably connected to the first base 11. By each partition unit 123, the corresponding cavity is divided into a plurality of grids for accommodating different reagents. When different partition units 123 are rotated, different reagents are mixed to perform step-by-step reactions;

[0058] The hydrogel incubation module 2 includes an incubation reaction pool 21, a plurality of mold racks 22 and a plurality of conversion units 23. The mold racks 22 are disposed inside the incubation reaction pool 21 and correspond to the flow channel units one by one. The upper wall and the lower wall of the mold rack 22 are respectively attached with a first split resonance ring 27 and a second split resonance ring 28 with opposite opening directions. The mold rack 22 also has a receiving groove for accommodating reagents, and the upper wall is sealed with an easily torn film 232. The easily torn film 232 is located above the first split resonance ring 27. A plurality of first flow channel inlets 211 corresponding to the flow channel units one by one are opened on the incubation reaction pool 21. A second flow channel inlet 221 communicating with the corresponding first flow channel inlet 211 is opened on the mold rack 22. The reagents after step-by-step reactions sequentially pass through the first flow channel inlet 211 and the second flow channel inlet 221 and enter the mold rack 22 to react with the reagents in the receiving groove to form a hydrogel. The conversion units 23 are correspondingly disposed above the mold racks 22 one by one and are used for introducing the test samples into the corresponding mold racks 22. The test samples contain protein markers;

[0059] The detection module 3 is used to form an induction field with the first split resonance ring 27 and the second split resonance ring 28 of the corresponding mold rack 22 for detection.

[0060] Among them, as Figure 1 shown, the hydrogel incubation module 2 is located directly above the detection module 3. The step-by-step reaction module 1 is located on the side where the first flow channel inlet 211 of the hydrogel incubation module 2 is located. The number of flow channel units of the step-by-step reaction module 1 can be adjusted according to actual needs. The opening directions of the first split resonance ring 27 and the second split resonance ring 28 are opposite. The cover plate 14 can be an integral structure or a split structure, such as one cover plate for multiple flow channel units or one cover plate for each flow channel unit. The corresponding reagents can be pre-placed in the respective corresponding cavities during the production process. When the partition unit 123 rotates, the reagents will be mixed and sequentially pass through the first flow channel inlet 211 and the second flow channel inlet 221 and enter the mold rack 22 in the hydrogel incubation module 2. After the reagents react and form a gel, they can be used to contact the test samples for on-site detection. The detection device 10 is the abbreviation of the on-site preparation and on-site detection type hydrogel resonance detection device for protein markers in this application. The test samples can be environmental liquid samples, saliva samples, serum samples, etc. containing protein markers, such as containing at least one of inflammatory factors and virus antigens.

[0061] The instant hydrogel resonance detection device for protein markers is an integrated device that can be quickly prepared on site and can immediately carry out multiplexed rapid detection. It includes a step-by-step reaction module, a hydrogel incubation module and a detection module, which realizes the preparation of sensitive elements (such as hydrogels), sample incubation, result reading and other multifunctional miniaturized assembly integration, wherein the hydrogel incubation module can be transformed by pressing the conversion unit, and the influence of the deformation generated by the hydrogel coupling incubation in the mold frame on the crack resonance ring can be detected, that is, the preparation raw materials of the core sensitive element multi-target immune hydrogel are assembled in the step-by-step reaction module, and the step-by-step raw materials are mixed to achieve gelation and coupling with the crack resonance ring in the mold frame, so as to obtain a core detection element with high sensitivity to the detection target. By pressing the conversion unit, the mold frame can transform the incubation pool, and the untreated sample to be tested can be dripped into the incubation pool from the sample addition port for a period of reaction, and the detection result can be read out by connecting the detection module with a network analyzer. Rapid on-site preparation has few interference factors, which can maximize the long-term stability of the product, long shelf life, optimal sensitivity and specificity, and has the advantages of simplicity, accuracy and speed.

[0062] In one embodiment, the hydrogel incubation module 2 also includes an upper cover 24 and a mounting base 25 which are stacked in sequence above the incubation reaction pool 21, the upper cover 24 is provided with a first through hole and a plurality of second through holes, the mounting base 25 is provided with a sample addition port 251 and a plurality of through grooves, the sample addition port 251 corresponds to the first through hole, the through groove corresponds one-to-one with the second through hole, and the second through hole corresponds one-to-one with the mold frame 22, and the conversion unit 23 is sequentially penetrated through the corresponding second through hole and through groove.

[0063] The number of the sample adding ports 251 can be adjusted according to actual needs, and can correspond to the flow channel units one by one, or can be shared by multiple flow channel units. The upper cover 24 and the mounting seat 25 are arranged to help avoid accidental contact of the conversion unit 23 .

[0064] In one embodiment, the step-by-step reaction module 1 includes two flow channel units arranged side by side, which are respectively recorded as a first flow channel unit 12 and a second flow channel unit 13. A flow channel dividing column 111 and two baffles 112 are arranged side by side on the first base 11. The cover plate 14 includes two shell plates. The flow channel dividing column 111 is located between the two baffles 112 for isolation. The two shell plates are correspondingly covered on the baffles 112 and are respectively attached to the two sides of the flow channel dividing column 111 to form two cavities. The flow channel unit also includes a flow channel bottom plate 121 and a partition plate 122. The flow channel bottom plate 121 is attached to the inner bottom wall of the corresponding cavity. The partition plate 122 is connected to one end of the flow channel bottom plate 121 away from the hydrogel incubation module 2, and is vertically attached to the first base 11 and the shell plate to realize the sealing of the corresponding grid. A plurality of positioning blocks 113 are also arranged side by side on each baffle 112, and the partition unit 123 is rotatably connected with the positioning block 113 in a one-to-one correspondence.

[0065] Among them, the partition 122 is used to ensure the unidirectional flow of the reagent liquid in the first flow channel unit 12 and the second flow channel unit 13, and does not leak from the partition 122. The flow channel bottom plate 121 is used to increase the bottom height of the first flow channel unit 12 and the second flow channel unit 13, form a good close contact with the separation unit 123, ensure the relative sealing of each grid, and the reagent does not mix when the separation unit 123 is not rotated. At the same time, the surface hydrophobic property of the flow channel bottom plate 121 makes the reagent no longer remain in the flow channel and completely enter the next chamber (such as the grid or the receiving groove of the mold frame). The separation unit 123 is rotatably connected with the positioning block 113 on the baffle 112 in a one-to-one correspondence, and the separation unit 123 on each flow channel unit is arranged side by side. When the step-by-step reaction module 1 includes two flow channel units arranged side by side, the separation units 123 of the two flow channel units can be arranged in an eight-shaped shape, and are at an acute angle with the flow channel separation column 111 to facilitate the circulation of the reagent.

[0066] In one embodiment, there are four partition units 123 arranged side by side, including a lever 123a, a rotating shaft 123b and two spring washers 123c. The rotating shaft 123b is rotatably connected to the first base 11. The lever 123a is connected to the rotating shaft 123b and is obliquely arranged to penetrate the first base 11. One end of the lever 123a abuts against the inner wall of the first base 11, and the other end extends out of the first base 11. The two spring washers 123c are attached to the lever 123a and the first base 11, and are respectively located at two acute angles formed by the lever 123a and the first base 11. The four partition units 123 are connected to the first base 11. The cavity should be divided into four grids for accommodating different reagents, that is, a first grid 12a is formed between the first partition unit 123 away from the hydrogel incubation module 2 and the first base 11, a second grid 12b is formed between the first partition unit 123 away from the hydrogel incubation module 2 and the second partition unit 123, a third grid 12c is formed between the second partition unit 123 away from the hydrogel incubation module 2 and the third partition unit 123, and a fourth grid 12d is formed between the third partition unit 123 away from the hydrogel incubation module 2 and the fourth partition unit 123.

[0067] Among them, by attaching the spring pad 123c to the lever 123a and the first base 11 and respectively located at the two acute angles formed by the lever 123a and the first base 11, the lever 123a always keeps the corresponding grid closed by elastic force when it is not moved, and only opens and releases the reagent in the corresponding grid when the lever 123a is moved. The number of the partition units 123 can also be adjusted according to actual needs.

[0068] In one embodiment, the conversion unit 23 includes a splicing frame 231, a conversion bracket 233, and a button 26. The splicing frame 231 includes a number of flipping blocks that are flexibly connected to different side walls of the mold frame 22. The easy-to-tear film 232 is also connected to the flipping blocks and is provided with an easy-to-tear line. The conversion bracket 233 includes a live-opening positioning ring 233b and a plurality of support rods 233a. Each support rod 233a is cross-passed through the live-opening positioning ring 233b, and one end is disposed opposite to the button 26, and the other end is correspondingly connected to the flipping block one by one.

[0069] Among them, if there are three support rods 233a, and the lower ends are fixedly connected to the flipping blocks flexibly connected to the mold frame 22 one by one. The intersection of the three support rods 233a is flexibly positioned through the live-opening positioning ring 233b. The upper ends of the support rods 233a can be movably connected to the button 26, and the button 26 is located above the support rods 233a. After the button 26 is pressed, the crossing angle of the three support rods 233a increases, the live-opening positioning ring 233b at the intersection moves upward, and the lower ends of the support rods 233a push the flipping blocks around, completing the demolding of the sandwich-type resonant sensor and exposing it to the incubation reaction pool 21. The sandwich hydrogel resonant sensor is composed of a first crack resonant ring 27, a second crack resonant ring 28, and an immune-responsive hydrogel coupled in the middle of the mold frame 22. Through the conversion unit 23, rapid demolding and exposure can be achieved, so that the sample to be tested can fully react.

[0070] It is easy to understand that the upper end of the support rod 233a being movably connected to the button 26 can be that the button 26 is not connected or slidably connected to the upper ends of the three support rods 233a, so that when the button 26 is pressed, the three support rods 233a diverge (the included angle increases), and at the same time, the divergence distance at the lower ends of the three support rods 233a increases, pushing down the flipping blocks on the mold frame 22 and realizing the tearing of the easy-to-tear film 232. Or, the button 26 being located above the support rods 233a can also be connected to other devices, such as being movably connected to the mounting base 25 to achieve the pressing function.

[0071] In one embodiment, the detection module 3 includes a second base 31, a sealing plate 32, at least one coil 33, a connecting wire 34, an SMA interface 35, and an SMA female head 36. The sealing plate 32 is located between the incubation reaction pool 21 and the second base 31. The coil 33, the connecting wire 34, and the SMA interface 35 are all arranged on the sealing plate 32, and the coil 33 performs induction detection corresponding to the mold frame 22 one by one and is connected to the SMA interface 35 through the connecting wire 34. The SMA female head 36 is electrically connected to the SMA interface 35 and is located on the second base 31.

[0072] In one embodiment, the detection module 3 is also electrically connected to the network analyzer 20 through a cable 30 for reading detection data. The network analyzer 20 is electrically connected to the SMA female head 36 of the detection module 3 through the cable 30 for displaying detection data, which helps to visually observe the detection results and improve work efficiency.

[0073] Working principle: There are several sandwich - type resonance sensors in the on - site preparation and on - site detection hydrogel resonance detection device for protein markers. The sandwich - type resonance sensor consists of a first split - ring resonator 27, a second split - ring resonator 28, and an immune - responsive hydrogel coupled in the middle by a mold holder 22. When contacting the free antigen in the sample to be tested, according to the competitive immunoassay strategy, the free protein marker antigen in the sample to be tested will cause the hydrogel to swell and generate mechanical deformation, thereby increasing the distance between the two split - ring resonators. The swelling of the hydrogel directly affects the capacitance of the resonance sensor, and further affects its resonance frequency. At the result output end, the network analyzer is connected to the coil 33 of the detection module 3 to realize wireless signal reading of the detection data and can transmit the detection data to a mobile terminal (such as a tablet, a computer, etc.). Example

[0074] As Figures 9 - 15 shown, an on - site preparation and on - site detection hydrogel resonance detection method for protein markers, based on the on - site preparation and on - site detection hydrogel resonance detection device for protein markers in Example 1, includes the following steps:

[0075] S1. Load the first reagent, the second reagent, the third reagent, and the fourth reagent into the corresponding grids of the flow - channel unit in sequence according to the order close to the hydrogel incubation module 2, where:

[0076] The first reagent is ultrapure water and acrylamide;

[0077] The second reagent is the vinylated reactant obtained by mixing 1 μg of protein marker and 0.5 μL of N - succinimidyl acrylate and then successively passing through water bath and ultrafiltration purification, 0.5 μL of N,N'-methylenebisacrylamide, and 0.75 μL of N,N,N’,N’-tetramethylethylenediamine;

[0078] The third reagent is 0.2 μL - 0.25 μL of ammonium persulfate;

[0079] The fourth reagent is a solution obtained by adding 25 μL of a solution after incubating 16 ng of protein marker antibody in 1 mL of gold nanoparticles in a shaker in the dark;

[0080] Or, the first reagent is ultrapure water, acrylamide, 0.5 μL of N,N'-methylenebisacrylamide, and 0.75 μL of N,N,N’,N’-tetramethylethylenediamine;

[0081] The second reagent is the vinylated reactant obtained by mixing 1 μg of protein marker and 0.5 μL of N - succinimidyl acrylate and then successively passing through water bath and ultrafiltration purification;

[0082] The third reagent is 0.2 μL - 0.25 μL of ammonium persulfate;

[0083] The fourth reagent is a solution obtained by adding 25 μL of an antibody against a protein biomarker to 1 mL of gold nanoparticles and incubating in the dark on a shaker.

[0084] S2: Load 1 μL - 1.25 μL of ammonium persulfate into the mold holder 22.

[0085] S3: When in use, place the ready-to-use and on-site-detectable hydrogel resonance detection device for protein biomarkers vertically. Toggle the corresponding partition unit 123 to mix the first reagent and the second reagent and gently shake to form a first solution. Then toggle the corresponding partition unit 123 to mix the first solution and the third reagent, gently shake and let stand for a first preset time to form a second solution. Finally, toggle the remaining partition units 123, pour the second solution and the fourth reagent into the mold holder 22 and vertically let stand for a second preset time to obtain the reagent after step-by-step reaction, which is the hydrogel, and complete the coupling of the hydrogel with the crack resonance ring of the corresponding mold holder 22.

[0086] S4: Place the ready-to-use and on-site-detectable hydrogel resonance detection device for protein biomarkers horizontally, and press the conversion unit 23 of the corresponding flow channel unit to tear the easy-to-tear film 232.

[0087] S5: Use the detection module 3 to read out the initial detection result f 0 ;

[0088] S6: Drop the sample to be tested into the hydrogel. After standing for a third preset time, use the detection module 3 to read out the secondary detection result f 1 ;

[0089] S7: According to the initial detection result f 0 and the secondary detection result f 1 and combined with the corresponding linear equation, calculate the content detection result of the protein biomarker in the sample to be tested, that is, take (f 1 -f 0 ) / f 0 as the normalized resonance frequency offset of the corresponding linear equation for calculation.

[0090] Among them, the first preset time is preferably 20 seconds, the second preset time is preferably 2 - 4 minutes, and the third preset time is preferably 5 minutes.

[0091] In one embodiment, the pre-treatment is also carried out before assembling each crack resonance ring. The pre-treatment includes sequentially performing ultra-pure water cleaning, nitrogen drying, and oxygen plasma surface treatment, and the duration of the oxygen plasma surface treatment is 3 min - 6 min.

[0092] In one embodiment, the ultrapure water is 31 μL, and the acrylamide is 7.5 mg - 9 mg; the concentration of N,N'-methylenebisacrylamide is 20 mg / mL, and the concentration of N,N,N’,N’-tetramethylethylenediamine is 0.6 mol / L - 0.8 mol / L; the temperature for incubation in the shaker in the dark is 4°C, and the time is 8 hours - 14 hours; the concentration of ammonium persulfate is 2 mol / L.

[0093] In one embodiment, the temperature of the water bath is 35°C - 37°C, and the time is 50 minutes - 1 hour; the ultrafiltration purification is centrifugation at 14000g and lasts for 25 minutes - 40 minutes under the condition of 2°C - 6°C. Among them, RPM (revolutions per minute) and RCF (relative centrifugal force or g-force) are usually used to describe the centrifugation speed, that is, RCF refers to the acceleration applied to the sample to be tested, and 14000g means that the centrifugal force is 14000 times the acceleration of gravity on the earth.

[0094] The present invention has high sensitivity, high specificity, few interfering factors. The design of preparing and using immediately can maximize the long-term stability of the product, has a long shelf life, and the best sensitivity and specificity. It has the advantages of simplicity, accuracy, and rapidity. The experiment is fast and accurate, and the experimental results have good repeatability. It can be used for the quantitative detection of protein markers.

[0095] The hydrogel resonance detection device for immediate preparation and measurement of protein markers in this embodiment includes two side-by-side flow channel units. One flow channel unit is used to detect the content of the first protein marker, and the other flow channel unit is used to detect the second protein marker. The two flow channel units are symmetrically arranged. Each flow channel unit includes four side-by-side grids. The following verifies this application through specific solutions. The first protein marker is such as an inflammatory factor. Here, C-reactive protein is taken for experimental verification. The second protein marker is such as a virus. Here, respiratory syncytial virus is taken for experimental verification. The specific details are as follows:

[0096] Solution 1:

[0097] I. Detection of the content of the first protein marker:

[0098] 1. After cleaning the crack resonance ring with ultrapure water and drying it with nitrogen, perform oxygen plasma surface treatment for 3 minutes to enhance hydrophilicity. Then assemble the crack resonance ring on the upper and lower walls of the mold rack, and the cracks of the two crack resonance rings face in opposite directions. The size of the crack resonance ring can be selected according to actual needs. For example, in this embodiment, the size of the crack resonance ring is 10 mm × 10 mm in length and width.

[0099] 2. Assemble 31 μL of ultrapure water and 8.5 mg of acrylamide in the first grid 12a of the first flow channel unit 12.

[0100] 3. 1 μg of the first protein marker was mixed with 0.5 μL of N-succinimidyl acrylate and placed in a 36°C water bath for one hour. The vinylated first protein marker was purified by ultrafiltration and assembled in the second grid 12 b of the first flow channel unit 12 .

[0101] 4. 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N',N'-tetramethylethylenediamine were assembled into the second grid 12 b of the first flow channel unit 12 .

[0102] 5. 0.25 μL of 2 mol / L ammonium persulfate was installed in the third grid 12 c of the first flow channel unit 12 .

[0103] 6. Add 16 ng of the first protein marker antibody to 1 mL of gold nanoparticles and incubate in a shaker at 4°C in the dark for 12 hours. Take 25 μL of the incubated solution and install it in the fourth grid 12d of the first flow channel unit 12.

[0104] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0105] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3 minutes, thereby completing the preparation of the core response element first protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0106] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the first flow channel unit 12, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0107] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0108] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0109] 12. The detection result of the content of the first protein biomarker in the sample to be measured is calculated according to the linear equation y = 0.48188lg(x) + 3.62886, where x is the concentration of the first protein biomarker in the sample to be measured, with the unit of pg / mL, and y is the normalized resonance frequency shift, which is the ratio of the difference between the initial detection result and the secondary detection result to the initial detection result, that is, (f 1 -f 0 ) / f 0 .

[0110] The experimental results, as Figures 10 to 13 shown, demonstrate the preparation and characterization of the core sensitive element hydrogel. Among them, Figure 10 shows the distribution of gold nanoparticles in the hydrogel. The presence of Au, N, and S elements in the hydrogel verifies the successful polymerization of gold nanoparticles and antibodies on the hydrogel structure, and this result verifies the feasibility of the present invention. 11 clearly shows the change in the porous structure of the hydrogel before and after adding the sample to be measured. After adding the sample to be measured, the pore size increases from 62.55 nm to 162.73 nm, verifying the mechanical deformation of the hydrogel caused by the binding of proteins in the sample to be measured. The swelling increases the inter-ring distance between the upper and lower coupled split-ring resonators, thereby causing a change in the resonance frequency read out as a result. This result verifies the mechanism from detecting the target binding signal to reading out the frequency wireless signal. Figure 12 shows the response effects of incubating the first protein biomarker for different times (5 minutes, 10 minutes, 15 minutes). Among them, the standard sample selects the first protein biomarker with a concentration of 100 pg / mL (100 pg / mL CRP), and the blank control represents the detection solution with the control variable having no corresponding standard sample and other conditions being the same. This result shows that the fastest this detection device can obtain the quantitative result of the first protein biomarker within 5 minutes, indicating its great advantage in detection speed for portable applications. Figure 13 shows the standard curve of the sample to be measured after incubating for 5 minutes at different concentrations of the first protein biomarker standard sample. In the figure, R 2 represents the sum of the squares of the differences between the sample points and the mean line, blank represents the response result in the blank saliva control group without the standard sample, and the limit of detection (LOD) is calculated according to the formula 3σ / S, where σ represents the sensor noise and S represents the sensitivity. This result shows that an extremely wide linear detection range (10 -2 pg / mL to 10 5 pg / mL) and an extremely low detection limit (24 fg / mL) can be obtained within 5 minutes of detection, indicating its excellent advantage in high detection sensitivity for portable applications. The normalized resonance frequency shift is the normalized frequency shift in Figures 12 to 15 .

[0111] II. Detection of the content of the second protein biomarker:

[0112] 1. After cleaning the split resonant rings with ultrapure water and drying them with nitrogen, perform oxygen plasma surface treatment for 3 minutes to enhance hydrophilicity. Then, assemble the split resonant rings on the upper and lower walls of the mold holder, with the splits of the two split resonant rings facing in opposite directions. The size of the split resonant rings can be selected according to actual needs. For example, in this embodiment, the size of the split resonant rings is 10 mm × 10 mm in length × width.

[0113] 2. Assemble 31 μL of ultrapure water and 8.5 mg of acrylamide in the first grid 12a of the second flow channel unit 13.

[0114] 3. Mix 1 μg of the second protein marker antigen with 0.5 μL of N-succinimidyl acrylate and place it in a 36°C water bath for one hour. After ultrafiltration purification, obtain the vinylated second protein marker antigen and assemble it in the second grid 12b of the second flow channel unit 13.

[0115] 4. Assemble 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N',N'-tetramethylethylenediamine in the second grid 12b of the second flow channel unit 13.

[0116] 5. Assemble 0.25 μL of 2 mol / L ammonium persulfate in the third grid 12c of the second flow channel unit 13.

[0117] 6. Add 16 ng of the second protein marker antibody to 1 mL of gold nanoparticles, incubate in the dark on a shaker at 4°C for 12 hours, and take 25 μL to assemble in the fourth grid 12d of the second flow channel unit 13.

[0118] 7. Assemble 1 μL of 2 mol / L ammonium persulfate in the mold holder 22.

[0119] 8. When starting to use, place the detection device vertically, toggle the first partition unit 123 to mix the reagents in the first grid 12a and the second grid 12b, gently shake for 5 seconds, then toggle the second partition unit 123 to mix the solution in the second grid 12b after mixing with the reagent in the third grid 12c, gently shake for 5 seconds and then let it stand for 20 seconds. Toggle the third and fourth partition units 123 to allow the solution in the third grid 12c after mixing and the reagent in the fourth grid 12d to flow into the mold holder 22 and then stand vertically for 3 minutes. Thus, the preparation of the second protein marker detection hydrogel of the core response element and its coupling with the split resonant ring are completed.

[0120] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the second flow channel unit 13, the conversion bracket 233 will drive the splicing frame 231 to fall down in all directions, the tearable film 232 will be split and separated accordingly, and the core response element (i.e., hydrogel) will complete demolding.

[0121] 10. Read out the initial detection result f using the network analyzer connected to the detection module 3 0 。

[0122] 11. Drop more than 600 μL of the sample to be tested from the sample addition port 251, after standing for 5 minutes, use the network analyzer to read out the secondary detection result f 1 。

[0123] 12. Calculate the detection result of the content of the second protein biomarker in the sample to be tested according to the linear equation y = 0.44938lg(x) + 1.70794, where x is the concentration of RSV (the second protein biomarker) in the sample to be tested, in units of pg / mL, and y is the normalized resonance frequency shift, which is the ratio of the difference between the initial detection result and the secondary detection result to the initial detection result, i.e., (f 1 -f 0 ) / f 0 。

[0124] The experimental results are as Figure 14 、 Figure 15 shown. Figure 14 It shows the response effects of the second protein biomarker incubated for different times (5 minutes, 10 minutes, 15 minutes). Among them, the standard sample selects the second protein biomarker with a concentration of 100 pg / mL (100 pg / mL RSV), and the blank control represents the detection solution with the control variable having no corresponding standard sample and other conditions being the same. This result indicates that the fastest this detection device can obtain the quantitative result of the second protein biomarker within 5 minutes, indicating its great advantage in detection speed for portable applications. Figure 15 It shows the standard curve of the sample to be tested incubated for 5 minutes under different concentrations of the second protein biomarker standard samples. In the figure, R 2 represents the sum of the squared differences between the sample points and the mean line, blank represents the response result in the blank saliva control group without standard samples, and the limit of detection (LOD) is calculated according to the formula 3σ / S, where σ represents the sensor noise and S represents the sensitivity. This result indicates that an extremely wide linear detection range (10 -2 pg / mL to 10 5 pg / mL) and an extremely low detection limit (31 fg / mL) can be obtained within 5 minutes of detection, indicating its excellent advantage in high detection sensitivity for portable applications.

[0125] The above-mentioned first solution has verified the feasibility and excellent high sensitivity of this method. The experimental results presented in the following solutions are similar to those of the first solution, which are specifically as follows.

[0126] Solution two:

[0127] I. Detection of the content of the first protein marker:

[0128] 1. After cleaning each cracked resonant ring with ultrapure water and drying it with nitrogen, perform oxygen plasma surface treatment for 3 minutes to enhance hydrophilicity. Then, assemble the cracked resonant rings on the upper and lower walls of the mold holder, and the cracks of the two cracked resonant rings face in opposite directions. The size of the cracked resonant ring can be selected according to actual needs. For example, in this embodiment, the size of the cracked resonant ring is 10 mm × 10 mm in length × width.

[0129] 2. Assemble 31 μL of ultrapure water and 7.5 mg of acrylamide in the first grid 12a of the first flow channel unit 12.

[0130] 3. Mix 1 μg of the first protein marker with 0.5 μL of N-succinimidyl acrylate and place it in a 36°C water bath for one hour. After ultrafiltration purification, obtain the vinylated first protein marker and assemble it in the second grid 12b of the first flow channel unit 12.

[0131] 4. Assemble 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N’,N’-tetramethylethylenediamine in the second grid 12b of the first flow channel unit 12.

[0132] 5. Assemble 0.25 μL of 2 mol / L ammonium persulfate in the third grid 12c of the first flow channel unit 12.

[0133] 6. Add 16 ng of the first protein marker antibody to 1 mL of gold nanoparticles and incubate in the dark on a shaker at 4°C for 12 hours. Take 25 μL of the incubated solution and assemble it in the fourth grid 12d of the first flow channel unit 12.

[0134] 7. Assemble 1 μL of 2 mol / L ammonium persulfate in the mold holder 22.

[0135] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3 minutes, thereby completing the preparation of the core response element first protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0136] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the first flow channel unit 12, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0137] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0138] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0139] 12. The test result of the first protein marker content in the sample to be tested is calculated according to the linear equation y=0.48188lg(x)+3.62886, where x is the concentration of the first protein marker in the sample to be tested, in pg / mL, and y is the normalized resonant frequency offset, which is the ratio of the difference between the initial test result and the secondary test result to the initial test result, that is, (f 1 -f 0 ) / f 0 .

[0140] 2. Detection of the second protein marker content:

[0141] 1. After each split resonance ring is cleaned with ultrapure water and dried with nitrogen, the surface is treated with oxygen plasma for 3 minutes to enhance hydrophilicity, and then the split resonance ring is assembled on the upper and lower walls of the mold frame, and the splits of the two split resonance rings are opposite. The size of the split resonance ring can be selected according to actual needs. For example, the size of the split resonance ring in this embodiment is 10 mm × 10 mm in length × width.

[0142] 2. 31 μL of ultrapure water and 9 mg of acrylamide were assembled into the first grid 12 a of the second flow channel unit 13 .

[0143] 3. 1 μg of the second protein marker antigen was mixed with 0.5 μL of N-succinimidyl acrylate and placed in a 36°C water bath for one hour. The vinylated second protein marker antigen was purified by ultrafiltration and assembled in the second grid 12 b of the second flow channel unit 13 .

[0144] 4. 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N',N'-tetramethylethylenediamine were assembled into the second grid 12 b of the second flow channel unit 13 .

[0145] 5. 0.25 μL of 2 mol / L ammonium persulfate was installed in the third grid 12 c of the second flow channel unit 13 .

[0146] 6. Add 16 ng of the second protein marker antibody to 1 mL of gold nanoparticles, incubate in a shaker at 4°C in the dark for 12 hours, and take 25 μL to assemble in the fourth grid 12d of the second flow channel unit 13.

[0147] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0148] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3 minutes, thereby completing the preparation of the core response element second protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0149] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the second flow channel unit 13, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0150] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0151] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0152] 12. Calculate the detection result of the content of the second protein marker in the sample to be measured according to the linear equation y = 0.44938lg(x) + 1.70794, where x is the concentration of RSV (the second protein marker) in the sample to be measured, with the unit of pg / mL, and y is the normalized resonance frequency shift, which is the ratio of the difference between the initial detection result and the secondary detection result to the initial detection result, i.e., (f 1 -f 0 ) / f 0 .

[0153] Scheme Three:

[0154] I. Detection of the content of the first protein marker:

[0155] 1. After cleaning the split resonator rings with ultrapure water and drying them with nitrogen, perform oxygen plasma surface treatment for 3 minutes to enhance hydrophilicity, and then assemble the split resonator rings on the upper and lower walls of the mold holder, with the splits of the two split resonator rings facing in opposite directions. The size of the split resonator rings can be selected according to actual needs. For example, in this embodiment, the size of the split resonator rings is 10 mm × 10 mm in length and width.

[0156] 2. Assemble 31 μL of ultrapure water and 8.5 mg of acrylamide in the first grid 12a of the first flow channel unit 12.

[0157] 3. Mix 1 μg of the first protein marker with 0.5 μL of N-succinimidyl acrylate and place it in a 36°C water bath for one hour. After ultrafiltration purification, obtain the vinylated first protein marker and assemble it in the second grid 12b of the first flow channel unit 12.

[0158] 4. Assemble 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N',N'-tetramethylethylenediamine in the second grid 12b of the first flow channel unit 12.

[0159] 5. Assemble 0.2 μL of 2 mol / L ammonium persulfate in the third grid 12c of the first flow channel unit 12.

[0160] 6. Add 16 ng of the first protein marker antibody to 1 mL of gold nanoparticles and incubate in the dark on a shaker at 4°C for 12 hours. Take 25 μL of the incubated solution and assemble it in the fourth grid 12d of the first flow channel unit 12.

[0161] 7. Assemble 1 μL of 2 mol / L ammonium persulfate in the mold holder 22.

[0162] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3.5 minutes, thereby completing the preparation of the core response element first protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0163] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the first flow channel unit 12, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0164] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0165] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0166] 12. The test result of the first protein marker content in the sample to be tested is calculated according to the linear equation y=0.48188lg(x)+3.62886, where x is the concentration of the first protein marker in the sample to be tested, in pg / mL, and y is the normalized resonant frequency offset, which is the ratio of the difference between the initial test result and the secondary test result to the initial test result, that is, (f 1 -f 0 ) / f 0 .

[0167] 2. Detection of the second protein marker content:

[0168] 1. After each split resonance ring is cleaned with ultrapure water and dried with nitrogen, the surface is treated with oxygen plasma for 3 minutes to enhance hydrophilicity, and then the split resonance ring is assembled on the upper and lower walls of the mold frame, and the splits of the two split resonance rings are opposite. The size of the split resonance ring can be selected according to actual needs. For example, the size of the split resonance ring in this embodiment is 10 mm × 10 mm in length × width.

[0169] 2. 31 μL of ultrapure water and 8.5 mg of acrylamide were assembled into the first grid 12 a of the second flow channel unit 13 .

[0170] 3. 1 μg of the second protein marker antigen was mixed with 0.5 μL of N-succinimidyl acrylate and placed in a 36°C water bath for one hour. The vinylated second protein marker antigen was purified by ultrafiltration and assembled in the second grid 12 b of the second flow channel unit 13 .

[0171] 4. 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N',N'-tetramethylethylenediamine were assembled into the second grid 12 b of the second flow channel unit 13 .

[0172] 5. 0.25 μL of 2 mol / L ammonium persulfate was installed in the third grid 12 c of the second flow channel unit 13 .

[0173] 6. Add 16 ng of the second protein marker antibody to 1 mL of gold nanoparticles, incubate in a shaker at 4°C in the dark for 8 hours, and take 25 μL to assemble in the fourth grid 12d of the second flow channel unit 13.

[0174] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0175] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 4 minutes, thereby completing the preparation of the core response element second protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0176] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the second flow channel unit 13, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0177] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0178] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0179] 12. Calculate the detection result of the content of the second protein marker in the sample to be measured according to the linear equation y = 0.44938lg(x) + 1.70794, where x is the concentration of RSV (the second protein marker) in the sample to be measured, with the unit of pg / mL, and y is the normalized resonance frequency shift, which is the ratio of the difference between the initial detection result and the secondary detection result to the initial detection result, that is, (f 1 -f 0 ) / f 0 .

[0180] Solution 4:

[0181] I. Detection of the content of the first protein marker:

[0182] 1. After cleaning the split resonator rings with ultrapure water and drying them with nitrogen, perform oxygen plasma surface treatment for 3 minutes to enhance hydrophilicity, and then assemble the split resonator rings on the upper and lower walls of the mold holder, with the splits of the two split resonator rings facing in opposite directions. The size of the split resonator rings can be selected according to actual needs. For example, in this embodiment, the size of the split resonator rings is 10 mm × 10 mm in length and width.

[0183] 2. Assemble 31 μL of ultrapure water and 8.5 mg of acrylamide in the first grid 12a of the first flow channel unit 12.

[0184] 3. Mix 1 μg of the first protein marker with 0.5 μL of N-succinimidyl acrylate and place it in a 37°C water bath for 50 minutes. After ultrafiltration purification, obtain the vinylated first protein marker and assemble it in the second grid 12b of the first flow channel unit 12.

[0185] 4. Assemble 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N’,N’-tetramethylethylenediamine in the second grid 12b of the first flow channel unit 12.

[0186] 5. Assemble 0.25 μL of 2 mol / L ammonium persulfate in the third grid 12c of the first flow channel unit 12.

[0187] 6. Add 16 ng of the first protein marker antibody to 1 mL of gold nanoparticles and incubate in the dark on a shaker at 4°C for 12 hours. Take 25 μL of the incubated solution and assemble it in the fourth grid 12d of the first flow channel unit 12.

[0188] 7. Assemble 1 μL of 2 mol / L ammonium persulfate in the mold holder 22.

[0189] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3 minutes, thereby completing the preparation of the core response element first protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0190] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the first flow channel unit 12, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0191] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0192] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0193] 12. The test result of the first protein marker content in the sample to be tested is calculated according to the linear equation y=0.48188lg(x)+3.62886, where x is the concentration of the first protein marker in the sample to be tested, in pg / mL, and y is the normalized resonant frequency offset, which is the ratio of the difference between the initial test result and the secondary test result to the initial test result, that is, (f 1 -f 0 ) / f 0 .

[0194] 2. Detection of the second protein marker content:

[0195] 1. After each split resonance ring is cleaned with ultrapure water and dried with nitrogen, the surface is treated with oxygen plasma for 3 minutes to enhance hydrophilicity, and then the split resonance ring is assembled on the upper and lower walls of the mold frame, and the splits of the two split resonance rings are opposite. The size of the split resonance ring can be selected according to actual needs. For example, the size of the split resonance ring in this embodiment is 10 mm × 10 mm in length × width.

[0196] 2. 31 μL of ultrapure water and 8.5 mg of acrylamide were assembled into the first grid 12 a of the second flow channel unit 13 .

[0197] 3. 1 μg of the second protein marker antigen was mixed with 0.5 μL of N-succinimidyl acrylate and placed in a 36°C water bath for one hour. The vinylated second protein marker antigen was purified by ultrafiltration and assembled in the second grid 12 b of the second flow channel unit 13 .

[0198] 4. 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N',N'-tetramethylethylenediamine were assembled into the second grid 12 b of the second flow channel unit 13 .

[0199] 5. 0.25 μL of 2 mol / L ammonium persulfate was installed in the third grid 12 c of the second flow channel unit 13 .

[0200] 6. Add 16 ng of the second protein marker antibody to 1 mL of gold nanoparticles, incubate in a shaker at 4°C in the dark for 12 hours, and take 25 μL to assemble in the fourth grid 12d of the second flow channel unit 13.

[0201] 7. Assemble 1.25 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0202] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3 minutes, thereby completing the preparation of the core response element second protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0203] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the second flow channel unit 13, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0204] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0205] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0206] 12. Calculate the detection result of the content of the second protein marker in the sample to be measured according to the linear equation y = 0.44938lg(x) + 1.70794, where x is the concentration of RSV (the second protein marker) in the sample to be measured, in pg / mL, and y is the normalized resonance frequency shift, which is the ratio of the difference between the initial detection result and the secondary detection result to the initial detection result, that is, (f 1 -f 0 ) / f 0 .

[0207] Scheme Five:

[0208] I. Detection of the content of the first protein marker:

[0209] 1. After cleaning the split resonant rings with ultrapure water and drying them with nitrogen, perform oxygen plasma surface treatment for 4 minutes to enhance hydrophilicity, and then assemble the split resonant rings on the upper and lower walls of the mold holder, with the splits of the two split resonant rings facing in opposite directions. The size of the split resonant rings can be selected according to actual needs. For example, in this embodiment, the size of the split resonant rings is 10 mm × 10 mm in length × width.

[0210] 2. Assemble 31 μL of ultrapure water and 8.5 mg of acrylamide in the first grid 12a of the first flow channel unit 12.

[0211] 3. Mix 1 μg of the first protein marker with 0.5 μL of N-succinimidyl acrylate and place it in a 36°C water bath for one hour, then ultrafilter and purify to obtain vinylated first protein marker, and assemble it in the second grid 12b of the first flow channel unit 12.

[0212] 4. Assemble 0.5 μL of 20 mg / mL N,N'-methylenebisacrylamide and 0.75 μL of 0.8 mol / L N,N,N',N'-tetramethylethylenediamine in the second grid 12b of the first flow channel unit 12.

[0213] 5. Assemble 0.25 μL of 2 mol / L ammonium persulfate in the third grid 12c of the first flow channel unit 12.

[0214] 6. Add 16 ng of the first protein marker antibody to 1 mL of gold nanoparticles, incubate in the dark on a shaker at 4°C for 12 hours. Take 25 μL of the incubated solution and assemble it in the fourth grid 12d of the first flow channel unit 12.

[0215] 7. Assemble 1 μL of 2 mol / L ammonium persulfate in the mold holder 22.

[0216] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3 minutes, thereby completing the preparation of the core response element first protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0217] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the first flow channel unit 12, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0218] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0219] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0220] 12. The test result of the first protein marker content in the sample to be tested is calculated according to the linear equation y=0.48188lg(x)+3.62886, where x is the concentration of the first protein marker in the sample to be tested, in pg / mL, and y is the normalized resonant frequency offset, which is the ratio of the difference between the initial test result and the secondary test result to the initial test result, that is, (f 1 -f 0 ) / f 0 .

[0221] 2. Detection of the second protein marker content:

[0222] 1. After each split resonance ring is cleaned with ultrapure water and dried with nitrogen, the surface is treated with oxygen plasma for 3 minutes to enhance hydrophilicity, and then the split resonance ring is assembled on the upper and lower walls of the mold frame, and the splits of the two split resonance rings are opposite. The size of the split resonance ring can be selected according to actual needs. For example, the size of the split resonance ring in this embodiment is 10 mm × 10 mm in length × width.

[0223] 2. 31 μL of ultrapure water and 8.5 mg of acrylamide were assembled into the first grid 12 a of the second flow channel unit 13 .

[0224] 3. 1 μg of the second protein marker antigen was mixed with 0.5 μL of N-succinimidyl acrylate and placed in a 36°C water bath for one hour. The vinylated second protein marker antigen was purified by ultrafiltration and assembled in the second grid 12 b of the second flow channel unit 13 .

[0225] 4. 0.5 μL of 20 mg / mL N,N′-methylenebisacrylamide and 1 μL of 0.8 mol / L N,N,N′,N′-tetramethylethylenediamine were assembled into the second grid 12 b of the second flow channel unit 13 .

[0226] 5. 0.25 μL of 2 mol / L ammonium persulfate was installed in the third grid 12 c of the second flow channel unit 13 .

[0227] 6. Add 16 ng of the second protein marker antibody to 1 mL of gold nanoparticles, incubate in a shaker at 4°C in the dark for 12 hours, and take 25 μL to assemble in the fourth grid 12d of the second flow channel unit 13.

[0228] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0229] 8. When starting to use, place the detection device vertically, move the first partition unit 123, mix the reagents in the first grid 12a and the second grid 12b, shake gently for 5 seconds, then move the second partition unit 123, mix the mixed solution in the second grid 12b and the reagent in the third grid 12c, shake gently for 5 seconds and let it stand for 20 seconds, move the third and fourth partition units 123, let the mixed solution in the third grid 12c and the reagent in the fourth grid 12d flow into the mold frame 22 and let it stand vertically for 3 minutes, thereby completing the preparation of the core response element second protein marker detection hydrogel and its coupling with the cleft resonant ring.

[0230] 9. Place the detection device horizontally, press the button 26 of the conversion unit 23 of the second flow channel unit 13, and the conversion bracket 233 will push the splicing frame 231 to the four sides, and the easy-tear film 232 will be separated and detached, and the core response element (i.e., hydrogel) will be demoulded.

[0231] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f 0 .

[0232] 11. Add more than 600 μL of the sample to be tested from the sample injection port 251, let it stand for 5 minutes, and then use the network analyzer to read the secondary test results. 1 .

[0233] 12. Calculate the detection result of the content of the second protein marker in the sample to be tested according to the linear equation y = 0.44938lg(x) + 1.70794, where x is the concentration of RSV (the second protein marker) in the sample to be tested, in pg / mL, and y is the normalized resonance frequency shift, which is the ratio of the difference between the initial detection result and the secondary detection result to the initial detection result, i.e., (f 1 -f 0 ) / f 0 .

[0234] In addition, for the first reagent being ultrapure water, acrylamide, 0.5 μL of N,N'-methylenebisacrylamide, and 0.75 μL of N,N,N’,N’-tetramethylethylenediamine; the second reagent being the vinylated reactant obtained by mixing 1 μg of the protein marker with 0.5 μL of N-succinimidyl acrylate and then successively undergoing water bath and ultrafiltration purification; the third reagent being 0.2 μL - 0.25 μL of ammonium persulfate; the fourth reagent being when taking 25 μL of the solution after incubating 16 ng of the protein marker antibody in 1 mL of gold nanoparticles in a shaker in the dark for detection operation, the specific detection method implemented is the same as that in Example 2 above, with the only difference being that some reagents in the first grid 12a and the second grid 12b are swapped during pre-configuration, but it does not affect the specific detection steps and detection results, so it will not be elaborated here.

[0235] It is easy to understand that according to the different structures of the on-site preparation and on-site detection type hydrogel resonance detection device (abbreviated as the detection device) for protein markers, the types of protein markers in the sample to be tested can be adaptively adjusted. For example, each flow channel unit can also be used to measure different types of protein markers, or different types of protein markers can be detected individually or mixedly, etc. And the above parameter values can also be arbitrarily adjusted within the specific limited range and can be determined according to actual needs.

[0236] And this application is only used for detection to determine whether there is a protein marker and the content of the protein marker, and can be used for non-therapeutic purpose research, such as the detection of environmental liquid samples (such as river water) containing protein pollutants, the detection of food (drinking water), etc. Specifically, it can also be combined with other instruments in the prior art for further in-depth evaluation.

[0237] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brief description, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combinations of these technical features do not conflict, they should all be considered as the scope recorded in this specification.

[0238] The above-described embodiments only represent relatively specific and detailed embodiments of the present application, but should not be construed as limiting the scope of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A hydrogel resonance detection device for protein markers, characterized in that: include: A step-by-step reaction module (1) comprises a first base (11), a cover plate (14) and a plurality of flow channel units, wherein the first base (11) and the cover plate (14) form a plurality of cavities, the flow channel units are built into the cavities in a one-to-one correspondence, and the flow channel units comprise a plurality of partition units (123) rotatably connected to the first base (11) so as to partition the corresponding cavities into a plurality of grids for accommodating different reagents, and when the different partition units (123) are rotated, the different reagents are mixed to perform a step-by-step reaction; A hydrogel incubation module (2) comprises an incubation reaction pool (21), a plurality of mold racks (22) and a plurality of conversion units (23), wherein the mold rack (22) is built into the incubation reaction pool (21) and corresponds to the flow channel units one by one, a first crack resonance ring (27) and a second crack resonance ring (28) are attached to the mold rack (22), the mold rack (22) also has a containing groove for containing reagents and an easy-tear film (232) is sealed on the upper wall, the reagents after step-by-step reaction enter the mold rack (22) and react with the reagents in the containing groove to form a hydrogel, and the conversion unit (23) is used to collect the sample to be tested into the corresponding mold rack (22), and the sample to be tested contains a protein marker; The detection module (3) is used to form an induction field with the two split resonant rings for detection.

2. The on-the-spot production and testing type hydrogel resonance detection device for protein markers according to claim 1, characterized in that: The hydrogel incubation module (2) further comprises an upper cover (24) and a mounting seat (25) which are sequentially stacked above the incubation reaction pool (21); the upper cover (24) is provided with a first through hole and a plurality of second through holes; the mounting seat (25) is provided with a sample injection port (251) and a plurality of through slots; the sample injection port (251) corresponds to the first through hole; the through slot corresponds to the second through hole one-to-one; and the second through hole corresponds to the mold frame (22) one-to-one; the conversion unit (23) is sequentially penetrated in the corresponding second through hole and through slot; The first split resonant ring (27) and the second split resonant ring (28) are respectively attached to the upper wall and the lower wall of the mold frame (22) and have opposite opening directions, and the easy-tear film (232) is located above the first split resonant ring (27); The incubation reaction pool (21) is provided with a plurality of first flow channel inlets (211) which are connected one-to-one with the flow channel units, and the mold frame (22) is provided with a second flow channel inlet (221) which is connected with the corresponding first flow channel inlet (211). The reagents after the step-by-step reaction enter the mold frame (22) through the first flow channel inlet (211) and the second flow channel inlet (221) in sequence.

3. The on-the-spot production and testing type hydrogel resonance detection device for protein markers according to claim 1, characterized in that: The step-by-step reaction module (1) comprises two flow channel units arranged side by side, respectively denoted as a first flow channel unit (12) and a second flow channel unit (13); a flow channel partition column (111) and two baffles (112) are arranged side by side on the first base (11); the cover plate (14) comprises two shell plates; the flow channel partition column (111) is located between the two baffles (112) for isolation; the two shell plates are correspondingly covered on the baffles (112) and are respectively attached to the two sides of the flow channel partition column (111) to form two cavities The flow channel unit also includes a flow channel bottom plate (121) and a partition plate (122), wherein the flow channel bottom plate (121) is attached to the inner bottom wall of the corresponding cavity, and the partition plate (122) is connected to the end of the flow channel bottom plate (121) away from the hydrogel incubation module (2), and is vertically attached to the first base (11) and the shell plate to achieve the sealing of the corresponding grid. A plurality of positioning blocks (113) are also arranged side by side on each baffle (112), and the partition unit (123) is rotatably connected to the positioning blocks (113) in a one-to-one correspondence.

4. The on-the-spot production and testing type hydrogel resonance detection device for protein markers according to claim 1, characterized in that: The partition units (123) are four and arranged side by side, and include a lever (123a), a rotating shaft (123b) and two spring washers (123c). The rotating shaft (123b) is rotatably connected to the first base (11). The lever (123a) is connected to the rotating shaft (123b) and is obliquely arranged to penetrate the first base (11). One end of the lever (123a) abuts against the inner wall of the first base (11), and the other end extends out of the first base (11). The two spring washers (123c) are attached to the lever (123a) and the first base (11), and are respectively located at two acute angles formed by the lever (123a) and the first base (11). The four partition units (123) correspond to the The cavity is divided into four grids for accommodating different reagents, namely, a first grid (12a) is formed between the first partition unit (123) away from the hydrogel incubation module (2) and the first base (11), a second grid (12b) is formed between the first partition unit (123) away from the hydrogel incubation module (2) and the second partition unit (123), a third grid (12c) is formed between the second partition unit (123) away from the hydrogel incubation module (2) and the third partition unit (123), and a fourth grid (12d) is formed between the third partition unit (123) away from the hydrogel incubation module (2) and the fourth partition unit (123).

5. The on-the-spot production and testing type hydrogel resonance detection device for protein markers according to claim 1, characterized in that: The conversion unit (23) is arranged one-to-one above the mold frame (22), and comprises a splicing frame (231), a conversion bracket (233) and a button (26); the splicing frame (231) comprises a plurality of flip blocks flexibly connected to different side walls of the mold frame (22); the easy-tear film (232) is also connected to the flip block and is provided with an easy-tear line; the conversion bracket (233) comprises a movable positioning ring (233b) and a plurality of support rods (233a); each of the support rods (233a) is cross-arranged in the movable positioning ring (233b), and one end of each support rod is arranged opposite to the button (26), and the other end is connected one-to-one with the flip block.

6. The on-the-spot production and testing type hydrogel resonance detection device for protein markers according to claim 1, characterized in that: The detection module (3) comprises a second base (31), a sealing plate (32), at least one coil (33), a connecting line (34), an SMA interface (35) and an SMA female connector (36); the sealing plate (32) is located between the incubation reaction pool (21) and the second base (31); the coil (33), the connecting line (34) and the SMA interface (35) are all arranged on the sealing plate (32); the coil (33) and the mold frame (22) are inductively detected in a one-to-one correspondence and are connected to the SMA interface (35) via the connecting line (34); the SMA female connector (36) is electrically connected to the SMA interface (35) and is located on the second base (31).

7. The on-the-spot production and testing type hydrogel resonance detection device for protein markers according to claim 1, characterized in that: The detection module (3) is also electrically connected to the network analyzer (20) via a cable (30) to read out detection data.

8. A method for on-the-spot preparation and testing of hydrogel resonance detection of protein markers, based on the on-the-spot preparation and testing of hydrogel resonance detection device for protein markers according to any one of claims 1 to 7, characterized in that: The steps include: S1. In the order of proximity to the hydrogel incubation module (2), the first reagent, the second reagent, the third reagent and the fourth reagent are sequentially loaded into the grids corresponding to the flow channel unit, wherein: The first reagent is ultrapure water and acrylamide; The second reagent is a vinylation reaction product obtained by mixing a protein marker with N-succinimidyl acrylate and then purifying it in a water bath and ultrafiltration, as well as N,N'-methylenebisacrylamide and N,N,N',N'-tetramethylethylenediamine; Alternatively, the first reagent is ultrapure water, acrylamide, N,N'-methylenebisacrylamide and N,N,N',N'-tetramethylethylenediamine; The second reagent is a vinylation reaction product obtained by mixing a protein marker with N-succinimidyl acrylate and then sequentially purifying by water bathing and ultrafiltration; The third reagent is a first preset amount of ammonium persulfate; The fourth reagent is a solution obtained by adding a protein marker antibody to gold nanoparticles and incubating on a shaker in the dark; S2, loading a second preset amount of ammonium persulfate into the mold frame (22); S3, when in use, the ready-made and ready-tested hydrogel resonance detection device for protein markers is placed vertically, and the corresponding separation units (123) are sequentially moved to obtain the reagents after step-by-step reactions, namely the hydrogel, and the coupling of the hydrogel with the corresponding split resonance ring of the mold frame (22) is completed; S4, pressing the corresponding conversion unit (23) to tear open the easy-tear film (232); S5, using the detection module (3) to read out the initial detection result f0; S6, dropping the sample to be tested into the hydrogel and letting it stand, and then using the detection module (3) to read out the secondary detection result f1; S7. Calculate the content of protein markers in the sample to be tested according to the initial test result f0 and the secondary test result f1 in combination with the corresponding linear equation, that is, use (f1-f0) / f0 as the normalized resonant frequency offset of the corresponding linear equation.

9. The method for preparing and testing protein markers on-the-spot hydrogel resonance detection according to claim 8, characterized in that: Each of the split resonant rings is also pre-treated before assembly. The pre-treatment includes ultrapure water cleaning, nitrogen drying and oxygen plasma surface treatment in sequence. The oxygen plasma surface treatment lasts for 3 minutes to 6 minutes.

10. The on-the-spot preparation and detection type hydrogel resonance detection method for protein markers according to claim 8, characterized in that: The ultrapure water is 31 μL, the acrylamide is 7.5 mg-9 mg; the protein marker is 1 μg, the N-succinimidyl acrylate is 0.5 μL, the N,N'-methylenebisacrylamide is 0.5 μL and the concentration is 20 mg / mL, the N,N,N',N'-tetramethylethylenediamine is 0.75 μL and the concentration is 0.6 mol / L-0.8 mol / L; the solution after the protein marker antibody is added to the gold nanoparticles and incubated on a shaker in the dark is 25 μL, the protein marker antibody is 16 ng, the gold nanoparticles are 1 mL, the temperature of the shaker incubation in the dark is 4°C, and the time is 8 hours-14 hours; the concentration of ammonium persulfate is 2 mol / L, the first preset amount is 0.2 μL-0.25 μL, and the second preset amount is 1 μL-1.25 μL.

11. The method for on-site preparation and on-site detection of protein markers according to claim 8, characterized in that: The temperature of the water bath is 35°C-37°C, and the time is 50 minutes-1 hour; the ultrafiltration purification is centrifuged at 14000g, and lasts for 25 minutes-40 minutes at 2°C-6°C.

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