On-site preparation and on-site detection type hydrogel resonance detection device and method for protein marker

Abstract

The invention discloses an on-site preparation and on-site detection type hydrogel resonance detection device and method for a protein marker, and the device comprises a step-by-step reaction module which comprises a first base, a cover plate and a plurality of flow channel units, and the flow channel units are divided into a plurality of grids through separation units; the hydrogel incubation module comprises an incubation reaction tank, a plurality of mold frames and a plurality of conversion units, and the conversion units are arranged above the mold frames and used for collecting the samples to be detected into the corresponding mold frames; and the detection module is used for forming an induction field with the split resonant ring of the corresponding mold frame for detection. The detection method comprises the following steps: sequentially loading a first reagent, a second reagent, a third reagent and a fourth reagent into the grids of the corresponding runner units, mixing different reagents when rotating different separation units, carrying out step-by-step reaction, and then detecting by the detection module. According to the present invention, the portable protein marker rapid quantitative detection of the to-be-detected sample is achieved, and the method has advantages of simple and rapid operation, high sensitivity, and simultaneous reading of the multi-target result.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a hydrogel resonance detection device and method for preparing and detecting protein markers. Background Art

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

[0003] For example, the Chinese patent with patent publication number CN220854894U proposes a PCT and CRP detection kit, which relies on the observation of the color change of the detection piece for the detection of PCT and CRP, and cannot quantitatively detect the CRP content in the sample to be tested; the Chinese patent with patent publication number CN220525831U proposes an MxA / CRP detection reagent card and kit, which requires the use of the detection reagent card with a large laboratory instrument immunoquantitative analysis instrument for quantitative detection of the sample to be tested; the Chinese patent with patent publication number CN118777610A proposes time-resolved fluorescent microspheres and their preparation method, fluorescent immunochromatography reagent plate and kit, which can detect the sample to be tested. The test requires dropping the sample to be tested on the test card and then scanning it with a blood cell analyzer for testing. The minimum detection level for CRP is 0.5 mg / mL, and the minimum detection level for SAA is 2.5 mg / mL, and the detection performance is poor. A Chinese patent with a patent publication number of CN118879941A proposes a respiratory syncytial virus detection kit, and a Chinese patent with a patent publication number of CN119101724A proposes a freeze-dried PCR reagent and a kit for respiratory virus nucleic acid detection. The detection of respiratory syncytial virus (RSV) requires the use of a fluorescent PCR detector for detection, and the detection time is long and only positive or negative results can be obtained, and quantitative detection is not possible.

[0004] In addition, the concentration of biomarkers in diluted samples is low, which challenges the practical limits of many electrochemical sensors. In recent years, more and more hydrogel sensors manufactured by different methods have been used in different fields for trace target detection. These hydrogel-based sensors have higher sensitivity, shorter response time and other characteristics, which are very suitable for the development of point-of-care (POC) devices in complex biofluid samples. However, the test kits in the prior art usually prepare the detection elements in advance, and then assemble and connect them with other elements for detection. During use, they may be affected by multiple factors, such as the limited storage time of the liquid frame stability after hydrogel preparation, and the detection activity of the antigen and antibody of the sensitive element in it is affected over time. Therefore, it is necessary to develop a rapid, highly sensitive, hydrogel-based portable sample detection solution. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned problems and to propose a ready-made and ready-tested hydrogel resonance detection device and method for protein markers, so as to realize portable rapid quantitative detection of protein markers for samples to be tested, which has the advantages of simple and rapid detection operation, high sensitivity, and simultaneous readout of multi-target results.

[0006] To achieve the above object, the technical solution adopted by the present invention is: The present invention provides a hydrogel resonance detection device for protein markers, which is prepared and tested immediately. The device comprises a step-by-step reaction module, a hydrogel incubation module and a detection module, wherein: A step-by-step reaction module comprises a first base, a cover plate and a plurality of flow channel units, wherein the first base and the cover plate 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 rotatably connected to the first base, wherein the corresponding cavities are divided into a plurality of grids for accommodating different reagents by each partition unit, and when the different partition units are rotated, the different reagents are mixed to perform step-by-step reactions; 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; 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.

[0007] 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.

[0008] 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.

[0009] Preferably, there are four partition units arranged side by side, including a lever, a rotating shaft and two spring pads, the rotating shaft is rotatably connected to the first base, the lever is connected to the rotating shaft and is obliquely arranged to penetrate 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 pads are attached to the lever and the first base, and are respectively located at two acute angles formed by the lever and the first base, the four partition units divide the corresponding cavity into four grids for accommodating different reagents, that is, a first grid is formed between the first partition unit away from the hydrogel incubation module and the first base, a second grid is formed between the first partition unit away from the hydrogel incubation module and the second partition unit, a third grid is formed between the second partition unit away from the hydrogel incubation module and the third partition unit, and a fourth grid is formed between the third partition unit away from the hydrogel incubation module and the fourth partition unit.

[0010] Preferably, the conversion unit includes a splicing frame, a conversion bracket and a button. The splicing frame includes a plurality of flip blocks flexibly connected to different side walls of the mold frame. The easy-tear film is also connected to the flip block and is provided with an easy-tear line. The conversion bracket includes a movable positioning ring and a plurality of support rods. Each support rod is cross-arranged in the movable positioning ring, and one end is arranged opposite to the button, and the other end is connected to the flip block one by one.

[0011] 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. The coil and the mold frame correspond one-to-one for induction detection and are 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.

[0012] Preferably, the detection module is also electrically connected to a network analyzer via a cable to read out the detection data.

[0013] A method for on-the-spot preparation and testing of hydrogel resonance detection for protein markers, based on any of the above-mentioned on-the-spot preparation and testing of hydrogel resonance detection devices for protein markers, comprises the following steps: S1. In the order of proximity to the hydrogel incubation module, the first reagent, the second reagent, the third reagent and the fourth reagent are sequentially loaded into the grids of the corresponding flow channel units, wherein: The first reagent is ultrapure water and acrylamide; The second reagent is a vinylation reaction product obtained by mixing 1 μg of protein marker with 0.5 μL of N-succinimidyl acrylate, which is purified by water bath and ultrafiltration, as well as 0.5 μL of N,N'-methylenebisacrylamide and 0.75 μL of N,N,N',N'-tetramethylethylenediamine; The third reagent is 0.2 μL-0.25 μL ammonium persulfate; The fourth reagent is a solution of taking 25 μL of 16 ng of protein marker antibody and adding it to 1 mL of gold nanoparticles and incubating on a shaker in the dark; Alternatively, the first reagent is ultrapure water, acrylamide, 0.5 μL N,N'-methylenebisacrylamide, and 0.75 μL N,N,N',N'-tetramethylethylenediamine; The second reagent is a vinylation reaction product obtained by mixing 1 μg of protein marker with 0.5 μL of N-succinimidyl acrylate and then sequentially purifying by water bath and ultrafiltration; The third reagent is 0.2 μL-0.25 μL ammonium persulfate; The fourth reagent is a solution of taking 25 μL of 16 ng of protein marker antibody and adding it to 1 mL of gold nanoparticles and incubating on a shaker in the dark; S2, load 1 μL-1.25 μL of ammonium persulfate into the mold holder; S3. When in use, the ready-made and tested hydrogel resonance detection device for protein markers is placed vertically, the corresponding separation unit is moved to mix the first reagent and the second reagent and shake it gently to form a first solution, and then the corresponding separation unit is moved to mix the first solution and the third reagent and shake it gently and then stand for a first preset time to form a second solution, and finally the remaining separation units are moved to mix the second solution and the fourth reagent into the mold frame and stand vertically for a second preset time, and the reagents obtained after step-by-step reaction are hydrogels, and the coupling of the hydrogel and the crack resonance ring of the corresponding mold frame is completed; S4, placing the ready-made and ready-tested hydrogel resonance detection device for protein markers horizontally, and pressing the conversion unit corresponding to the flow channel unit to tear open the tearable film; S5, using the detection module to read out the initial detection result f0; S6, dropping the sample to be tested into the hydrogel, leaving it to stand for a third preset time, and then using the detection module to read out the secondary detection result f1; S7. Calculate the content detection result of the protein marker in the sample to be tested according to the initial detection result f0 and the secondary detection result f1 in combination with the corresponding linear equation, that is, calculate (f1-f0) / f0 as the normalized resonant frequency offset of the corresponding linear equation.

[0014] Preferably, each split resonant ring is pretreated before assembly, the pretreatment comprising ultrapure water cleaning, nitrogen drying and oxygen plasma surface treatment in sequence, and the oxygen plasma surface treatment lasts for 3 minutes to 6 minutes.

[0015] Preferably, the ultrapure water is 31 μL, the acrylamide is 7.5 mg-9 mg; the concentration of N,N'-methylenebisacrylamide is 20 mg / mL, the concentration of N,N,N',N'-tetramethylethylenediamine is 0.6 mol / L-0.8 mol / L; 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.

[0016] 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 14000g, and the duration is 25 minutes-40 minutes at 2°C-6°C.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present application is an integrated device that can be quickly prepared on-site and can immediately carry out multiplexed rapid detection, including a step-by-step reaction module, a hydrogel incubation module and a detection module, to achieve multifunctional miniaturized assembly integration such as preparation of sensitive elements (such as hydrogels), sample 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 hydrogel coupling incubation in the mold frame on the crack resonant ring is 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 resonant ring in the mold frame to obtain a core detection element with high sensitivity to the detection target. The mold frame can be transformed into an incubation pool by pressing the conversion unit, and the untreated sample to be tested is dripped into the incubation pool from the sample loading port. After a period of reaction, the network analyzer can be used to connect the detection module to realize the reading of the detection result. In summary, the present application is highly operational and integrates storage, preparation, and detection that can be flexibly converted. By storing it in the form of raw materials and quickly preparing it for use when detection is needed, the immune activity of immune elements such as protein markers can be improved and stabilized over a long period of time. There are few interfering factors, and the ready-to-use design can maximize the long-term stability of the product, with a long shelf life, optimal sensitivity and specificity, and it has the advantages of being simple, accurate, and fast. The experimental results have good reproducibility and can be used for quantitative detection of protein markers, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic structural diagram of a hydrogel resonance detection device for on-the-spot production and detection of protein markers according to the present invention; Figure 2 It is a schematic diagram of the internal structure of the on-the-spot preparation and detection type hydrogel resonance detection device for protein markers of the present invention; Figure 3 For the present invention Figure 2 Exploded diagram of Figure 4 An exploded view of the hydrogel incubation module and the detection module of the present invention; Figure 5 This is a schematic diagram of the internal structure of the hydrogel incubation module of the present invention; Figure 6 For the present invention Figure 5 Exploded diagram of Figure 7 For the present invention Figure 5 Schematic diagram of the conversion state; Figure 8 It is a schematic diagram of the readout principle of the on-the-spot preparation and detection type hydrogel resonance detection device for protein markers of the present invention; Fig. 9 This is a flow chart of the on-the-spot preparation and testing type hydrogel resonance detection method for protein markers of the present invention; Fig.10Figure 1 is a result diagram of the characterization of the hydrogel preparation of the present invention, wherein Figure (a) is a scanning electron microscope image of the distribution of gold nanoparticles in the hydrogel, Figure (b) is a local magnified view of Figure (a), and Figure (c) is a line scan result diagram of energy dispersive X-ray spectroscopy; Fig.11 The diagram is a diagram showing the change of the porous structure of the hydrogel before and after the sample to be tested is added in the present invention, wherein (a) is a scanning electron microscope image of the porous structure of the hydrogel before the sample to be tested is added, and (b) is a scanning electron microscope image of the porous structure of the hydrogel after the sample to be tested is added, and the upper diagram in (c) is a diagram showing the relationship between the pore diameter and the number of the hydrogel before the sample to be tested is added, and the lower diagram in (c) is a diagram showing the relationship between the pore diameter and the number of the hydrogel after the sample to be tested is added; Fig.12 This is a response effect diagram of the first protein marker of the present invention when incubated for different time periods; Fig.13 The standard curve diagram of the present invention is a standard curve diagram after the test sample is incubated for 5 minutes at different first protein marker standard sample concentrations; Fig.14 This is a response effect diagram of the second protein marker of the present invention when incubated for different time periods; Fig.15 It is a standard curve diagram of the test sample at different second protein marker standard sample concentrations after incubation for 5 minutes.

[0019] Explanation of 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 partition column; 112. baffle plate; 113. positioning block; 121. flow channel bottom plate; 122. partition plate; 123. partition unit; 12a. first grid; 12b. second grid; 12c. third grid; 12d. fourth grid; 123a. lever; 123b. rotating shaft; 123c. spring pad; 21. incubation reaction pool; 22. mold frame ; 23. conversion unit; 24. upper cover; 25. mounting base; 26. button; 27. first gap resonance ring; 28. second gap resonance ring; 211. first flow channel inlet; 221. second flow channel inlet; 231. splicing frame; 232. easy-tear film; 233. conversion bracket; 233a. support rod; 233b. movable positioning ring; 251. sample adding port; 31. second base; 32. sealing plate; 33. coil; 34. connecting line; 35. SMA interface; 36. SMA female connector; 10. detection device; 20. network analyzer; 30. cable. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0021] It should be noted that when a component is referred to as being "connected" to another component, it may be directly connected to the other component or there may be an intermediate component. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. Example

[0022] like Figure 1-Figure 8 As shown, a hydrogel resonance detection device for protein markers that can be made and tested immediately includes a step-by-step reaction module 1, a hydrogel incubation module 2 and a detection module 3, wherein: 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 built into the cavities one by one, and the flow channel units include a plurality of partition units 123 rotatably connected to the first base 11. The corresponding cavities are divided into a plurality of grids for accommodating different reagents by each partition unit 123, and different reagents are mixed to perform step-by-step reactions when different partition units 123 are rotated; 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 rack 22 is built into the incubation reaction pool 21 and corresponds to the flow channel unit one by one. The upper wall and the lower wall of the mold rack 22 correspond to the first crack resonance ring 27 and the second crack resonance ring 28, respectively, and the opening directions are opposite. The mold rack 22 also has a receiving groove for accommodating reagents and the upper wall is sealed with an easy-tear film 232. The easy-tear film 232 is located above the first crack resonance ring 27. The incubation reaction pool 21 is opened There are a plurality of first flow channel inlets 211 corresponding to each other with the flow channel units, and a second flow channel inlet 221 corresponding to each other with the first flow channel inlet 211 is provided on the mold frame 22. Reagents after 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 and react with the reagents in the containing tank to form a hydrogel. The conversion units 23 are arranged one by one above the mold frame 22 and are used to collect the samples to be tested into the corresponding mold frame 22. The samples to be tested contain protein markers. The detection module 3 is used to form an induction field with the first split resonant ring 27 and the second split resonant ring 28 of the corresponding mold frame 22 for detection.

[0023] Among them, Figure 1 As shown, the hydrogel incubation module 2 is located directly above the detection module 3, and 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 crack resonance ring 27 and the second crack resonance ring 28 are opposite. The cover plate 14 can be an integrated structure or a split structure, such as multiple flow channel units can share one or each flow channel unit corresponds to one. The corresponding reagents can be pre-placed in advance in each corresponding cavity during the production process. When the separation unit 123 rotates, the reagents will be mixed and enter the mold frame 22 in the hydrogel incubation module 2 through the first flow channel inlet 211 and the second flow channel inlet 221 in sequence. After the reagent reaction is completed and gelled, it can be used to contact with the sample to be tested to achieve on-site detection. The detection device 10 is the abbreviation of the hydrogel resonance detection device for protein markers in this application. The sample to be tested can be an environmental liquid sample, a saliva sample, a serum sample, etc. containing protein markers, such as containing at least one of inflammatory factors and viral antigens.

[0024] 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.

[0025] 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.

[0026] 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 .

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 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 includes a movable positioning ring 233b and a plurality of support rods 233a. Each support rod 233a is cross-arranged in the movable positioning ring 233b, and one end is arranged opposite to the button 26, and the other end is connected to the flip block one by one.

[0032] Wherein, if there are three support rods 233a, and the lower ends are connected and fixed one by one with the flexibly connected flip blocks on the mold frame 22, the intersection of the three support rods 233a is flexibly positioned by the movable positioning ring 233b, and the upper ends of the support rods 233a can be movably connected with the button 26, and the button 26 is located above the support rods 233a. After the button 26 is pressed, the intersection angle of the three support rods 233a increases, the movable positioning ring 233b at the intersection moves up, and the lower ends of the support rods 233a push the flip blocks to the surroundings, completing the demoulding of the sandwich-type resonant sensor and exposing it to the incubation reaction pool 21. The sandwich hydrogel resonant sensor consists of a first crack resonant ring 27, a second crack resonant ring 28, and an immune response hydrogel coupled in the middle of the mold frame 22. Rapid demoulding and exposure can be achieved through the conversion unit 23, so that the sample to be tested can fully react.

[0033] It is easy to understand that the upper end of the support rod 233a and the button 26 can be movably connected to each other, such that the button 26 and the upper ends of the three support rods 233a are not connected or are slidably connected, so that when the button 26 is pressed, the three support rods 233a diverge (the angle increases), and at the same time, the divergence interval between the lower ends of the three support rods 233a increases, pushing down the flip block on the mold frame 22, and tearing the easy-tear film 232. Alternatively, the button 26 located above the support rod 233a can also be connected to other devices, such as being movably connected to the mounting seat 25 to realize the pressing function.

[0034] In one embodiment, the detection module 3 includes 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 one by one and are connected to the SMA interface 35 through 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.

[0035] In one embodiment, the detection module 3 is also electrically connected to the network analyzer 20 via the cable 30 to read the detection data. The network analyzer 20 is electrically connected to the SMA female connector 36 of the detection module 3 via the cable 30 to display the detection data, which helps to visually observe the detection results and improve work efficiency.

[0036] Working principle: There are several sandwich-type resonant sensors in the ready-made and tested hydrogel resonance detection device for protein markers. The sandwich-type resonant sensor is composed of a first crack resonant ring 27, a second crack resonant ring 28 and an immune response hydrogel coupled in the middle of the mold frame 22. When it contacts the free antigen in the sample to be tested, according to the competitive immune strategy, the free protein marker antigen in the sample to be tested will cause the hydrogel to swell and produce mechanical deformation, thereby increasing the distance between the two crack resonant rings. The expansion of the hydrogel directly affects the capacitance of the resonant sensor, and then affects its resonant 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, computer, etc.). Example

[0037] like Figure 9-Figure 15 As shown, a method for on-the-spot hydrogel resonance detection of protein markers, based on the on-the-spot hydrogel resonance detection device for protein markers in Example 1, comprises the following steps: 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 of the corresponding flow channel units, wherein: The first reagent is ultrapure water and acrylamide; The second reagent is a vinylation reaction product obtained by mixing 1 μg of protein marker with 0.5 μL of N-succinimidyl acrylate, which is purified by water bath and ultrafiltration, as well as 0.5 μL of N,N'-methylenebisacrylamide and 0.75 μL of N,N,N',N'-tetramethylethylenediamine; The third reagent is 0.2 μL-0.25 μL ammonium persulfate; The fourth reagent is a solution of taking 25 μL of 16 ng of protein marker antibody and adding it to 1 mL of gold nanoparticles and incubating on a shaker in the dark; Alternatively, the first reagent is ultrapure water, acrylamide, 0.5 μL N,N'-methylenebisacrylamide, and 0.75 μL N,N,N',N'-tetramethylethylenediamine; The second reagent is a vinylation reaction product obtained by mixing 1 μg of protein marker with 0.5 μL of N-succinimidyl acrylate and then sequentially purifying by water bath and ultrafiltration; The third reagent is 0.2 μL-0.25 μL ammonium persulfate; The fourth reagent is a solution of taking 25 μL of 16 ng of protein marker antibody and adding it to 1 mL of gold nanoparticles and incubating on a shaker in the dark; S2, loading 1 μL-1.25 μL of ammonium persulfate into the mold frame 22; S3. When in use, the ready-made and tested hydrogel resonance detection device for protein markers is placed vertically, and the corresponding partition unit 123 is moved to mix the first reagent and the second reagent and shake it gently to form a first solution. Then, the corresponding partition unit 123 is moved to mix the first solution and the third reagent and shake it gently, and then stand for a first preset time to form a second solution. Finally, the remaining partition units 123 are moved to collect the second solution and the fourth reagent into the mold frame 22 and stand vertically for a second preset time. The reagents obtained after step-by-step reaction are hydrogels, and the coupling of the hydrogel and the corresponding crack resonance ring of the mold frame 22 is completed. S4, placing the on-the-spot hydrogel resonance detection device for protein markers horizontally, pressing the conversion unit 23 of the corresponding flow channel unit 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, leaving it to stand for a third preset time, and then using the detection module 3 to read out the secondary detection result f1; S7. Calculate the content detection result of the protein marker in the sample to be tested according to the initial detection result f0 and the secondary detection result f1 in combination with the corresponding linear equation, that is, calculate (f1-f0) / f0 as the normalized resonant frequency offset of the corresponding linear equation.

[0038] Among them, the first preset time is preferably 20 seconds, the second preset time is preferably 2 minutes to 4 minutes, and the third preset time is preferably 5 minutes.

[0039] In one embodiment, each split resonant ring is 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.

[0040] In one embodiment, the ultrapure water is 31 μL, the acrylamide is 7.5 mg-9 mg; the concentration of N,N'-methylenebisacrylamide is 20 mg / mL, the concentration of N,N,N',N'-tetramethylethylenediamine is 0.6 mol / L-0.8 mol / L; 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.

[0041] In one embodiment, the water bath temperature is 35°C-37°C for 50 minutes-1 hour; the ultrafiltration purification is centrifugation at 14000g for 25 minutes-40 minutes at 2°C-6°C. RPM (revolutions per minute) and RCF (relative centrifugal force or g-force) are usually used to describe the centrifugal speed, that is, RCF refers to the acceleration applied to the sample to be tested, and 14000g means that the centrifugal force is 14,000 times the acceleration of the earth's gravity.

[0042] The invention has high sensitivity, high specificity, few interfering factors, and a ready-to-use design that can maximize the long-term stability of the product, a long shelf life, optimal sensitivity and specificity, and the advantages of simplicity, accuracy, and speed. The experiment is fast and accurate, the experimental results are reproducible, and it can be used for the quantitative detection of protein markers.

[0043] The instant hydrogel resonance detection device for protein markers in this embodiment includes two flow channel units arranged side by side, one of which is used to detect the content of a first protein marker, and the other is used to detect a second protein marker. The two flow channel units are symmetrically arranged, and each flow channel unit includes four side-by-side grids. The present application is verified by a specific scheme below. The first protein marker is such as an inflammatory factor, and C-reactive protein is used for experimental verification below. The second protein marker is such as a virus, and respiratory syncytial virus is used for experimental verification below, as follows: Solution 1: 1. Detection of the first protein marker content: 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 length × width 10 mm × 10 mm.

[0044] 2. 31 μL of ultrapure water and 8.5 mg of acrylamide were assembled into the first grid 12 a of the first flow channel unit 12 .

[0045] 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 .

[0046] 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 .

[0047] 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 .

[0048] 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.

[0049] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0050] 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.

[0051] 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.

[0052] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0053] 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 result f1.

[0054] 12. The detection 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, wherein 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, i.e. (f1-f0) / f0.

[0055] Experimental results, such as Figures 10 to 13 As shown, the preparation and characterization of the core sensitive element hydrogel are demonstrated. Fig.10 The distribution of gold nanoparticles in the hydrogel is shown. The presence of Au, N and S elements in the hydrogel verifies the successful polymerization of gold nanoparticles and antibodies in the hydrogel structure, and this result verifies the feasibility of the present invention. 11 clearly shows the changes in the porous structure of the hydrogel before and after the addition of the test sample. After the addition of the test sample, the pore size increased from 62.55 nm to 162.73 nm, verifying the mechanical deformation of the hydrogel after the protein in the test sample binds, and the expansion increases the distance between the two upper and lower coupled resonant rings, which in turn leads to a change in the resonant frequency of the result readout. This result verifies the mechanism from detecting the target binding signal to the frequency wireless signal readout. Fig.12The response effect of the first protein marker incubated for different times (5 minutes, 10 minutes, and 15 minutes) was demonstrated, wherein the standard sample selected a first protein marker with a concentration of 100 pg / mL (100 pg / mL CRP), and the blank control indicated that the control variable had no corresponding standard sample and the other detection solutions were consistent. This result indicated that the detection device could obtain the quantitative result of the first protein marker within 5 minutes at the fastest, indicating that it has a great detection speed advantage in portable applications. Fig.13 The standard curves of the samples to be tested at different concentrations of the first protein marker standard sample after incubation for 5 minutes are shown. 2 It is expressed as the sum of squares of the 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 detection limit (LOD) is calculated according to the formula 3σ / S, where σ represents the sensor noise and S represents the sensitivity. This result shows that a very wide linear detection range (10 -2 pg / mL to 10 5 pg / mL), and an extremely low detection limit (24 fg / mL), indicating that it has excellent detection sensitivity advantages for portable applications. The normalized resonant frequency offset is Figures 12 to 15 The normalized frequency shift in .

[0056] 2. Detection of the second protein marker content: 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.

[0057] 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 .

[0058] 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 .

[0059] 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 .

[0060] 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 .

[0061] 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.

[0062] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0063] 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.

[0064] 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.

[0065] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0066] 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 result f1.

[0067] 12. The detection result of the second protein marker content in the sample to be tested is calculated according to the linear equation y=0.44938lg(x)+1.70794, where x is the concentration of RSV (second 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, (f1-f0) / f0.

[0068] The experimental results are as follows Fig.14 , Fig.15 shown. Fig.14The response effect of the second protein marker incubated for different times (5 minutes, 10 minutes, and 15 minutes) was demonstrated, wherein the standard sample selected a second protein marker with a concentration of 100 pg / mL (100 pg / mL RSV), and the blank control indicated that the control variable had no corresponding standard sample and other detection solutions were consistent. This result indicated that the detection device could obtain the quantitative result of the second protein marker within 5 minutes at the fastest, indicating that it has a great detection speed advantage in portable applications. Fig.15 The standard curves of the samples to be tested after 5 minutes of incubation at different concentrations of the second protein marker standard sample are shown. 2 It is expressed as the sum of squares of the 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 detection limit (LOD) is calculated according to the formula 3σ / S, where σ represents the sensor noise and S represents the sensitivity. This result shows that a very wide linear detection range (10 -2 pg / mL to 10 5 pg / mL), and an extremely low detection limit (31 fg / mL), indicating that it has excellent detection sensitivity advantages for portable applications.

[0069] The above-mentioned scheme 1 has verified the feasibility and high sensitivity of this method. The experimental results presented in the following scheme have similar results to those of scheme 1, as follows.

[0070] Option 2: 1. Detection of the first protein marker content: 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 length × width 10 mm × 10 mm.

[0071] 2. 31 μL of ultrapure water and 7.5 mg of acrylamide were assembled into the first grid 12 a of the first flow channel unit 12 .

[0072] 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 .

[0073] 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 .

[0074] 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 .

[0075] 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.

[0076] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0077] 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.

[0078] 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.

[0079] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0080] 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 result f1.

[0081] 12. The detection 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, wherein 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, i.e. (f1-f0) / f0.

[0082] 2. Detection of the second protein marker content: 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.

[0083] 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 .

[0084] 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 .

[0085] 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 .

[0086] 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 .

[0087] 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.

[0088] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0089] 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.

[0090] 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.

[0091] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0092] 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 result f1.

[0093] 12. The detection result of the second protein marker content in the sample to be tested is calculated according to the linear equation y=0.44938lg(x)+1.70794, where x is the concentration of RSV (second 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, (f1-f0) / f0.

[0094] Option 3: 1. Detection of the first protein marker content: 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 length × width 10 mm × 10 mm.

[0095] 2. 31 μL of ultrapure water and 8.5 mg of acrylamide were assembled into the first grid 12 a of the first flow channel unit 12 .

[0096] 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 .

[0097] 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 .

[0098] 5. 0.2 μL of 2 mol / L ammonium persulfate was installed in the third grid 12 c of the first flow channel unit 12 .

[0099] 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.

[0100] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0101] 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.

[0102] 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.

[0103] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0104] 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 result f1.

[0105] 12. The detection 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, wherein 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, i.e. (f1-f0) / f0.

[0106] 2. Detection of the second protein marker content: 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.

[0107] 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 .

[0108] 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 .

[0109] 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 .

[0110] 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 .

[0111] 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.

[0112] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0113] 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.

[0114] 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.

[0115] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0116] 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 result f1.

[0117] 12. The detection result of the second protein marker content in the sample to be tested is calculated according to the linear equation y=0.44938lg(x)+1.70794, where x is the concentration of RSV (second 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, (f1-f0) / f0.

[0118] Option 4: 1. Detection of the first protein marker content: 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 length × width 10 mm × 10 mm.

[0119] 2. 31 μL of ultrapure water and 8.5 mg of acrylamide were assembled into the first grid 12 a of the first flow channel unit 12 .

[0120] 3. 1 μg of the first protein marker was mixed with 0.5 μL of N-succinimidyl acrylate and placed in a 37°C water bath for 50 minutes. The vinylated first protein marker was purified by ultrafiltration and assembled in the second grid 12 b of the first flow channel unit 12 .

[0121] 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 .

[0122] 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 .

[0123] 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.

[0124] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0125] 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.

[0126] 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.

[0127] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0128] 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 result f1.

[0129] 12. The detection 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, wherein 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, i.e. (f1-f0) / f0.

[0130] 2. Detection of the second protein marker content: 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.

[0131] 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 .

[0132] 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 .

[0133] 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 .

[0134] 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 .

[0135] 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.

[0136] 7. Assemble 1.25 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0137] 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.

[0138] 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.

[0139] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0140] 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 result f1.

[0141] 12. The detection result of the second protein marker content in the sample to be tested is calculated according to the linear equation y=0.44938lg(x)+1.70794, where x is the concentration of RSV (second 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, (f1-f0) / f0.

[0142] Option 5: 1. Detection of the first protein marker content: 1. After each split resonance ring is cleaned with ultrapure water and dried with nitrogen, the surface is treated with oxygen plasma for 4 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 length × width 10 mm × 10 mm.

[0143] 2. 31 μL of ultrapure water and 8.5 mg of acrylamide were assembled into the first grid 12 a of the first flow channel unit 12 .

[0144] 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 .

[0145] 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 .

[0146] 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 .

[0147] 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.

[0148] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0149] 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.

[0150] 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.

[0151] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0152] 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 result f1.

[0153] 12. The detection 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, wherein 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, i.e. (f1-f0) / f0.

[0154] 2. Detection of the second protein marker content: 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.

[0155] 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 .

[0156] 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 .

[0157] 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 .

[0158] 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 .

[0159] 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.

[0160] 7. Assemble 1 μL of 2 mol / L ammonium persulfate into the mold holder 22.

[0161] 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.

[0162] 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.

[0163] 10. Use the network analyzer connected to the detection module 3 to read out the initial detection result f0.

[0164] 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 result f1.

[0165] 12. The detection result of the second protein marker content in the sample to be tested is calculated according to the linear equation y=0.44938lg(x)+1.70794, where x is the concentration of RSV (second 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, (f1-f0) / f0.

[0166] In addition, when the first reagent is ultrapure water, acrylamide, 0.5μL N,N'-methylenebisacrylamide and 0.75μL N,N,N',N'-tetramethylethylenediamine; the second reagent is 1μg protein marker and 0.5μL acrylic acid N-succinimidyl ester, followed by water bath and ultrafiltration purification to obtain a vinylation reaction product; the third reagent is 0.2μL-0.25μL ammonium persulfate; the fourth reagent is 25μL of a solution prepared by adding 16ng protein marker antibody to 1mL gold nanoparticles and incubating on a shaker in the dark for detection operation, the specific detection method is the same as in Example 2 above, the only difference being that some reagents in the first grid 12a and the second grid 12b are exchanged during pre-configuration, but this does not affect the specific detection steps and detection results, which will not be repeated here.

[0167] It is easy to understand that, according to the different structures of the ready-made and tested hydrogel resonance detection device (hereinafter referred to as the detection device) for protein markers, the type of protein markers of the sample to be tested can be adaptively adjusted, such as each flow channel unit is also used to measure different types of protein markers or different types of protein markers are detected separately or mixed, etc. And the above parameter values ​​can also be adjusted arbitrarily within a specific limited range, which can be determined according to actual needs.

[0168] This application is only used for testing to determine whether protein markers are contained and the content of protein markers. It can be used for non-therapeutic research, such as environmental liquid sample (river water, etc.) detection of protein pollutants, food (drinking water) detection, etc. It can also be combined with other instruments in the prior art for further in-depth evaluation.

[0169] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0170] The above-described embodiments only express the more specific and detailed embodiments described in this application, but they cannot be understood as limiting the scope of the application. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of this application, which all belong to the protection scope of this application. Therefore, the protection scope of this application shall be based on the attached 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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