A device for detecting three inflammatory factors and application thereof

Abstract

The application discloses a device for triple detection of inflammatory factors and application, and particularly relates to the technical field of biological medical materials. The device is sequentially provided with a plasma separation layer, a sample detection layer, a support layer, an intermediate connecting layer and a washing layer from top to bottom. The sample detection layer is divided into a hydrophilic region and a hydrophobic region. Antibodies for detecting inflammatory factors are respectively added into detection zones of the hydrophilic region. The washing layer is rotationally connected to the lower side of the support layer through the intermediate connecting layer. The device ingeniously combines microfabrication and microfluids, integrates the detection of multiple immune reaction indexes and detection factors by taking advantage of the capillary flow function of paper itself and the excellent biological compatibility, and can quickly and accurately realize the joint detection of inflammatory factors CRP, PCT and SAA in blood. The device does not need to rely on traditional expensive professional detection instruments, and has a great application prospect in special environments or medical environments with limited conditions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a device for triple detection of inflammatory factors and application. BACKGROUND

[0002] During the invasion of pathogenic bacteria into blood, the body will produce immune response and various cytokines, and under the action of cytokines, the liver will produce inflammatory proteins, including C-reactive protein (CRP), procalcitonin (PCT), serum amyloid A (SAA), interleukin-6 (IL-6), etc. Among them, the content of CRP begins to rise 6-8h after infection, reaches a peak 24-48h after infection, the content of PCT generally rises rapidly within 2-4h after infection, and rises to a peak 6-8h after infection, and the content of SAA generally also rises rapidly by about 1000 times after infection. Therefore, the three inflammatory factors are sensitive indicators reflecting the infection condition and the treatment effect of inflammation of the body, and can be used to diagnose whether there is pathogenic infection in blood and further identify whether the infection is bacterial infection or viral infection. Specifically, the content changes of CRP and PCT are specific to bacterial infection, and the content change of SAA is closely related to viral infection and can be used as a differential diagnosis index for viral infection.

[0003] In actual medical diagnosis and treatment, the detection of inflammatory factors needs to be combined with a complex sample pretreatment program, and can only be completed by relying on professional and expensive detection equipment, which greatly limits the application of the detection device under non-laboratory conditions, and generally uses the above single inflammatory factor index for detection. However, single index detection takes a long time and is generally not very accurate. For example, in some heart diseases and inflammatory bowel diseases, CRP is usually used as a common index for diagnosing inflammatory response, so it cannot be simply identified as blood bacterial infection by the increase of a single CRP index. In addition, the prior art also uses the combination of CRP and PCT indexes to diagnose whether there is blood infection, but these two indexes are mainly aimed at bacterial infection and cannot identify viral infection.

[0004] Therefore, in view of the detection needs of multiple inflammatory factors, it is of great significance to develop a rapid detection device capable of simultaneously detecting the content of the three indexes of CRP, PCT and SAA. SUMMARY

[0005] Based on this, one of the purposes of the present application is to provide a device for triple detection of inflammatory factors, which realizes the combined detection of inflammatory factors CRP, CRP and SAA during early blood infection, does not need to rely on traditional expensive professional detection instruments, and has a greater application prospect in special environments, poor areas, community hospitals and medical environments with limited conditions.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The device for triple detection of inflammatory factors comprises, from top to bottom, a plasma separation layer, a sample detection layer, a support layer, an intermediate connecting layer and a washing layer, the plasma separation layer is composed of PES material and is fixed on the sample area of the sample detection layer, the sample detection layer is divided into a hydrophilic area and a hydrophobic area, the hydrophilic area comprises a sample area in the center, three detection areas uniformly distributed around the sample area as the center, and three reaction channels for connecting the sample area and the detection areas respectively, the detection areas are respectively added with capture antibodies for detecting inflammatory factors, the support layer is composed of a hard hydrophobic material, is closely attached below the sample detection layer and covers the sample area and the reaction channels, the washing layer is rotationally connected below the support layer through the intermediate connecting layer, a through hole is arranged in the center of the washing layer, three absorption pools are uniformly arranged around the through hole, the three absorption pools are respectively arranged along the extension direction of the reaction channels and correspond to the three detection areas one by one, and are used for absorbing excess liquid from the detection areas, and the width of the reaction channel is 3 mm.

[0008] Preferably, the support layer and the intermediate connecting layer are made of polyvinyl chloride, polyethylene or polypropylene.

[0009] Preferably, the intermediate connecting layer comprises a first fixed layer and a second fixed layer fixedly arranged below the support layer and the washing layer respectively, and a rotating shaft connected between the first fixed layer and the second fixed layer, the rotating shaft passes through the through hole on the washing layer and is connected with the second fixed layer.

[0010] Preferably, the absorption pool comprises, in sequence from the center outward, a water absorption area, a hydrophilic channel and a water storage area which are communicated with each other, and the water absorption area corresponds to the directly below of the detection area.

[0011] Preferably, the inflammatory factors are SAA, CRP and PCT respectively.

[0012] Preferably, the shape of the support layer is any one of a cylindrical shape, a cubic shape or a hexagonal prism shape, and the preferred shape is a hexagonal prism shape.

[0013] Preferably, the plasma separation layer is circular and has a diameter of 10-16 mm, and the preferred diameter is 12.5 mm.

[0014] Preferably, the centers of the plasma separation layer, the sample detection layer, the support layer, the intermediate connecting layer and the washing layer are located on the same straight line.

[0015] The second object of the present application is to provide a preparation method of the device for triple detection of inflammatory factors, and the technical scheme is as follows:

[0016] obtaining a plasma separation layer,

[0017] The detection layer is printed by the method of inkjet printing, and the plasma separation layer is fixed on the sample area of the detection layer;

[0018] The washing layer is printed by the method of inkjet printing, and a through hole is engraved in the center of the washing layer by a laser engraving machine;

[0019] The support layer and the intermediate connecting layer are cut on the PVC base plate by the laser engraving method;

[0020] The support layer is fixed and attached to the lower side of the detection layer, and the intermediate connecting layer is used to connect the support layer and the washing layer.

[0021] Preferably, the hydrophobic area of the detection layer is prepared by inkjet printing of an alkyl ketene dimer-heptane-wax dye mixed solution, the mass-volume ratio of the alkyl ketene dimer and heptane is 5%, and the mass percentage of the wax dye in the mixed solution is 1%.

[0022] Preferably, the printing frequency is 8-15 times.

[0023] Preferably, chitosan and glutaraldehyde are added dropwise in the detection area for modification.

[0024] The third object of the present application is to provide a use method of the inflammation factor triple detection device, and the technical scheme is:

[0025] The sample to be detected is added to the sample area along the plasma separation layer, the sample flows into the reaction channel, the gold nano-labeled antibody is added to the channel, the gold nano-labeled antibody is mixed with the sample and flows into the three detection areas, and the three detection areas are added with corresponding capture antibodies of CRP, PCT and SAA, respectively;

[0026] The washing layer is rotated so that the three washing pools correspond to the three detection areas of the sample detection layer one by one, the washing liquid is added dropwise to the detection areas to remove the unbound gold nano-labeled antibody, and the liquid is absorbed through the washing pool;

[0027] The color development results of the three detection areas of the sample to be detected are respectively collected and processed, the standard fitting curve established by the image processing software is used to obtain the contents of CRP, PCT and SAA in the sample to be detected.

[0028] The above description is only a summary of the technical scheme of the present application, in order to more clearly understand the technical means of the present application, the content of the specification can be implemented, and in order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described as follows BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a structural schematic diagram of the device of the present application.

[0030] Figure 2 Detection flow chart of the device of the present application.

[0031] Figure 3 Color development effect chart of the device of the present application for detecting different standard concentrations of CRP, PCT and SAA proteins.

[0032] Figure 4 Standard curve of the device of the present application for detecting CRP.

[0033] Figure 5 Standard curve of the device of the present application for detecting PCT.

[0034] Figure 6 Standard curve of the device of the present application for detecting SAA.

[0035] Figure 7 Fitting curve of the device of the present application for comparing the results of detecting CRP with those of a clinical instrument.

[0036] In the figure: 1-plasma separation layer, 2-sample detection layer, 3-support layer, 4-first fixation layer, 5-rotating shaft, 6-second fixation layer, 7-intermediate connecting layer, 8-through hole, 9-washing layer, 10-sample area, 11-detection area, 12-reaction channel, 13-water absorption area, 14-hydrophilic channel, 15-water storage area. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0038] The experimental methods carried out in the embodiments of the present application are all conventional methods unless otherwise specified, and the reagents and drugs used are all commercially purchased, and the preparation parameters and use parameters are all the best parameters optimized by the applicant through creative exploration.

[0039] As shown in Figure 1 A device for triple detection of inflammatory factors, the device sequentially comprises, from top to bottom, a plasma separation layer 1, a sample detection layer 2, a support layer 3, an intermediate connecting layer 7 and a washing layer 9, and the centers of the plasma separation layer 1, the sample detection layer 2, the support layer 3, the intermediate connecting layer 7 and the washing layer 9 are located on the same straight line.

[0040] The plasma separation layer 1 is composed of PES material and is fixed on the sample area 10 of the sample detection layer 2, and is used for vertically filtering whole blood and separating blood cells.

[0041] The shape of the plasma separation layer 1 is circular, which is adapted to the shape of the sample area 10, and the diameter is 10 mm, 12.5 mm, 14 mm or 16 mm.

[0042] In another alternative embodiment, when the sample is a serum sample, the plasma separation layer 1 can be omitted, and the serum sample can be directly added to the sample area 10 of the sample detection layer 2.

[0043] The sample detection layer 2 is circular in shape and includes a hydrophilic area and a hydrophobic area, and the hydrophilic area includes a sample area 10 in the center, three detection areas 11 uniformly distributed around the sample area, and three reaction channels 12 for connecting the sample area 10 and the detection areas 11.

[0044] The shape of the sample area 10 is circular, and the diameter is 10 mm, 12.5 mm, 14 mm or 16 mm.

[0045] The shape of the three detection areas 11 is circular, and the diameter is 7 mm, and antibodies for specific binding with the inflammatory factors SAA, CRP and PCT to be detected are added respectively.

[0046] The width of the three reaction channels 12 is 3 mm, and in another alternative embodiment, the width can also be set to 2 mm or 4 mm or 5 mm.

[0047] In another alternative embodiment, the number of detection areas and reaction channels can also be set according to actual detection needs, such as two, four, and multiple detection areas and reaction channels are uniformly arranged around the sample area.

[0048] The support layer 3 is composed of PVC and closely adheres to the lower part of the sample detection layer 2 and extends to cover the sample area 10 and the reaction channels 12. Since the filter paper above the support layer 3 has a pore size, the liquid sample may flow vertically to the lower layer, and at this time the PVC of the lower layer is a hydrophobic material and has a certain hardness, which can well support the horizontal flow of fluid and has no concern about vertical leakage, thereby improving the accuracy of measurement.

[0049] In another alternative embodiment, the support layer can also be selected from other hard hydrophobic materials, including polyethylene or polypropylene, etc.

[0050] The shape of the support layer 3 is hexagonal prism, and in another alternative embodiment, it can also be cylindrical or cubic.

[0051] The intermediate connecting layer 7 comprises a first fixed layer 4, a second fixed layer 6 and a rotating shaft 5 fixed between the first fixed layer 4 and the second fixed layer 6, the first fixed layer 4 is located below the support layer 3, the second fixed layer 6 is located below the washing layer, and the rotating shaft 5 penetrates through the through hole 8 on the washing layer and is connected with the second fixed layer 6.

[0052] The washing layer 9 is rotationally connected below the support layer 3 through the intermediate connecting layer 7, the washing layer is circular in shape and has an area larger than that of the sample detection layer 2.

[0053] The center of the washing layer 9 is provided with a through hole 8, and three absorption pools are uniformly arranged around the through hole 8, the three absorption pools are arranged along the extension line direction of the reaction channel respectively, and correspond to the three detection areas 11 on the sample detection layer 2 one by one, and are used for absorbing excess liquid from the detection areas 11.

[0054] The absorption pool sequentially comprises a water absorption area 13, a hydrophilic channel 14 and a water storage area 15 which are connected with each other in a center outward direction, and the water absorption area corresponds to the directly below of the detection area.

[0055] The embodiment provides Figure 1 The preparation method of the inflammation factor triple detection device:

[0056] Step 1: Obtain the plasma separation layer.

[0057] Specifically, the plasma separation layer is commercially purchased and is cut into a circular pattern with a diameter of 12.5 mm.

[0058] Step 2: Print the detection layer by using the inkjet printing method, and fix the plasma separation layer on the sample area of the detection layer.

[0059] Specifically, the paper substrate is Whatman 1001-70 filter paper, alkyl ketene dimer is used as the hydrophobic material, the alkyl ketene dimer is dissolved in heptane, 1% blue wax dye is added, an alkyl ketene dimer-heptane-blue wax dye solution is prepared, the mass / volume ratio of the alkyl ketene dimer and the heptane is 5%, and the alkyl ketene dimer-heptane-blue wax dye solution is replaced into the ink cartridge of the inkjet printer Hewlett Packard HP 1112.

[0060] Through the optimization experiment, the optimal reaction channel width is 3 mm, if the width is too large, the required sample amount will be increased and the reaction time will be slowed down, and if the width is too small, it is not conducive to the full reaction of the detection antibody and the target. In addition, the optimal proportion of the hydrophobic material is 5% w / v, which is less than 5% and cannot form a solid hydrophobic wall, and is higher than 5% and is easy to cause the ink cartridge to be blocked. Under the condition of 5% (w / v) alkyl ketene dimer, the printing is performed for 8-15 times, the optimal printing times are 10 times, and a solid hydrophilic and hydrophobic structure can be formed on the paper substrate.

[0061] In another alternative embodiment, the hydrophobic material can also be paraffin dissolved in heptane, and the mass volume ratio is also 5%.

[0062] The printed microfluidic pattern is printed on the filter paper by using a printer to obtain the sample detection layer, which contains a 12.5 mm diameter middle sample area, three 7 mm diameter circular detection areas, and three 3 mm width reaction channels.

[0063] The printed filter paper sample is heated in an oven at 80°C for 10 minutes to solidify the alkyl ketene dimer onto the paper fibers. The paper chip after heat solidification treatment is taken out of the oven and cooled to room temperature to obtain the detection layer containing the hydrophilic area and the hydrophobic area.

[0064] In the above detection area, 2 μL of 1.5 mg / mL CRP capture antibody, 50 μg / mL PCT capture antibody, and 1 mg / mL SAA capture antibody are added respectively, and after 30 min of reaction, 2 μL of 1% (w / v) BSA solution is added for blocking.

[0065] The plasma separation layer is fixed on the sample area of the detection layer.

[0066] In another alternative embodiment, if the sample to be tested is serum, the plasma separation layer structure is not required, and the plasma separation layer is also not required.

[0067] In another alternative embodiment, the detection area is also modified by adding chitosan and glutaraldehyde, which specifically includes: first adding 3 μL of 0.05% (w / v) chitosan solution to the detection area, and after 1 h of reaction, adding 3 μL of 0.25% (v / v) glutaraldehyde solution to the detection area, and after 3 h of reaction, washing the detection area with a washing solution to make the detection area carry aldehyde groups, which can be covalently cross-linked with the amino groups of the subsequent antibodies.

[0068] Step 3: The washing layer is printed by using the inkjet printing method, and a through hole is engraved in the center of the washing layer by using a laser engraving machine.

[0069] Specifically, the paper substrate is Whatman 1001-110 filter paper, an alkyl ketene dimer-heptane-blue wax dye solution (5%, w / v) is prepared, the alkyl ketene dimer-heptane-blue wax dye solution is replaced into the ink in the ink cartridge of the HP 1112 printer, and the printed microfluidic pattern is printed on the filter paper by using a printer, which contains three 7 mm water absorption areas, a 12 mm*12 mm water storage area, and a hydrophilic channel connecting the hydrophilic area and the water storage area.

[0070] A through hole is engraved in the center of the washing layer by using a laser engraving machine, and the through hole is a 11 mm*15 mm square through hole.

[0071] Step 4: cutting the support layer and the intermediate connecting layer on the PVC base plate by laser engraving.

[0072] Specifically, after setting the support layer with a predetermined shape on the laser engraving machine, the support layer is obtained by cutting the PVC base plate.

[0073] The first fixed layer, the rotating shaft and the second fixed layer with a predetermined shape are set on the laser engraving machine, the intermediate connecting layer is obtained by cutting the PVC base plate, and the first fixed layer is connected with the rotating shaft by using an adhesive.

[0074] Step 5: fixing and adhering the support layer to the lower side of the detection layer, and connecting the support layer and the washing layer by using the intermediate connecting layer.

[0075] The support layer is fixed and adhered to the lower side of the detection layer by using an adhesive, the first fixed layer is fixed and adhered to the lower side of the support layer, and the other end of the rotating shaft passes through the through hole of the washing layer and is fixedly connected with the second fixed layer.

[0076] As shown in Figure 2 , the embodiment provides a use method of the inflammation factor triple detection device:

[0077] Step 1: adding the sample to be detected along the plasma separation layer into the sample area, flowing the sample into the reaction channel, adding gold nano-labeled antibodies into the channel, mixing the gold nano-labeled antibodies with the sample and flowing into three detection areas, reacting and developing color in the areas, the three detection areas are respectively added with capture antibodies corresponding to CRP, PCT and SAA, the concentration OD values of the gold nano-labeled CRP antibodies, the gold nano-labeled PCT antibodies and the gold nano-labeled SAA antibodies are respectively 1.4, 1.8 and 1.8, and the added amount is 16 μL, when the sample to be detected is whole blood, the added amount is 100 μL, and when the sample to be detected is serum, the added amount is 50 μL, and the reaction time is about 10 minutes;

[0078] Step 2: rotating the washing layer so that the three washing pools correspond one by one to the three detection areas of the sample detection layer, respectively adding washing liquid to the detection areas to remove the unbound gold nano-labeled antibodies, and absorbing the liquid through the washing pool, the washing liquid is a PBS solution containing 3% Tween;

[0079] Step 3: image acquisition and data processing are respectively performed on the color development results of the three detection areas of the sample to be detected, and the CRP, PCT and SAA contents in the sample to be detected are obtained through the standard fitting curve established by Image J.

[0080] Specifically, under the same conditions, use a camera or smartphone camera to face the test area and take a picture of the color development results of the test area. Take screenshots of the color-developed test areas in the obtained pictures and export them as corresponding JPG format images. Then, import these JPG format screenshots reflecting the overall color development of each test area into Image J image processing software one by one, convert them into RGB format, and further calculate their RGB intensity.

[0081] Substitute the obtained RGB intensity into the standard curve to obtain the corresponding content.

[0082] Based on the concentration of each inflammatory marker, the inflammatory index is divided into low, medium (within the critical range) and high risk. Based on the signal intensity of each inflammatory marker, the risk level is made into a reference chart. Figure 3 The graph shows the results of protein testing at different concentrations. Generally speaking, when CRP is less than 10 μg / mL, the likelihood of infection is low, while when CRP is greater than 10 μg / mL, it indicates infection. In normal individuals, PCT is less than 0.05 ng / mL. When PCT is greater than 0.5 μg / mL, it indicates mild infection. When PCT is greater than 20 μg / mL, it indicates severe infection. When SAA is greater than 10 μg / mL, it indicates infection. Furthermore, when SAA is greater than 10 μg / mL and CRP is normal or slightly elevated, it indicates a mild viral infection.

[0083] Among them, the method for constructing a standard fitting curve includes: testing samples with different standard concentrations, obtaining the RGB color intensity corresponding to different standard concentrations through image acquisition and data processing of the color development results of the detection area, using the color development signal intensity RGB of the detection area as the vertical coordinate and the different infection indicator concentrations as the horizontal coordinate, drawing a fitting curve between the color development signal intensity and the concentration of the infection indicator to be tested, and obtaining the detection limit.

[0084] Figures 4-6 are the standard fitting curves of CRP, PCT and SAA, respectively. Among them, the standard curve of CRP is Y = -25.92055x + 168.4731, the detection limit is 5 μg / mL-100 μg / mL, and the correlation coefficient is 0.99571; the standard curve of PCT is Y = -9.85161x + 139.143.12, the detection limit is 0.05 ng / mL-100 ng / mL, and the correlation coefficient is 0.99232; the standard curve of SAA is Y = -21.66573x + 159.83026, the detection limit is 2 μg / mL-100 μg / mL, and the correlation coefficient is 0.99385. It can be seen that the content of each inflammatory factor indicator and the corresponding RGB color intensity are linearly correlated, and can be effectively used for the quantitative analysis of CRP, PCT and SAA.

[0085] In this embodiment, in order to further verify the difference between the content of the inflammation factor detected by the detection device described in the application and the content detected by the conventional detection instrument in clinic, the collected blood samples of multiple clinical patients are detected by the detection device and the clinical instrument respectively, each sample is detected three times, the average value is taken as the final detection concentration, and the obtained detection results are recorded and summarized as follows:

[0086] Table 1 Comparison of detection results of CRP, PCT and SAA

[0087]

[0088] In this embodiment, the results of the linear fitting curve of the detection results of the two detection methods are obtained as shown in the following table. Figure 7 The correlation coefficient R 2 = 0.9902, and the coefficient of variation (CV) is less than 10%; the linear fitting curve of the detection results of PCT by the two detection methods is Y = 1.00283X-3.27417, the correlation coefficient R 2 = 0.98538, and the coefficient of variation (CV) is less than 10%; the linear fitting curve of the detection results of SAA by the two detection methods is Y = 1.00513X+0.98513, the correlation coefficient R 2 = 0.98513, and the coefficient of variation (CV) is less than 10%, which indicates that the three detection results have good linear correlation with the detection results of the conventional instrument, and can replace the conventional instrument for multiple detection of inflammation factors, and can be used for early diagnosis of bacterial or viral infection, and has the advantages of portability, simple detection process, rapidness and accuracy.

[0089] In summary, compared with the prior art, the application has the following beneficial effects:

[0090] (1) The inflammation factor triple detection device provided by the application integrates the detection of multiple immune reaction indicators into one device, and combines with an intelligent photographing device, so that the combined detection of inflammation factors CRP, PCT and SAA during early blood infection can be realized, and the device does not need to rely on traditional expensive professional detection instruments, and has a great application prospect in special environments, poor areas, community hospitals and medical environments with limited conditions.

[0091] (2) The preparation method of the inflammation factor triple detection device provided by the application ingeniously combines microfabrication and microfluidics, utilizes the capillary flow function of paper itself and the excellent biocompatibility, optimizes the parameters of hydrophobic material, reaction channel size, sample addition, prints the pattern on filter paper by using an office inkjet printer combined with microfluidic technology, and forms a microfluidic detection channel through heat curing treatment, so that the preparation method is simple, the cost is low, and batch production can be realized.

[0092] (3) The use method of the inflammation factor triple detection device provided by the application can realize the simultaneous detection of multiple inflammation factors by only adding the sample to the device and taking a simple photo, the use operation is simple, strict professional technical operation is not required, the application range is wider, and the detection time from sample addition to final coloration is generally within 15-20 minutes, the detection is rapid and accurate.

[0093] Finally, it should be noted that although the preferred embodiments of the application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the application.

[0094] Obviously, those skilled in the art can make various modifications and variations to the application without departing from the spirit and scope of the application. Thus, if these modifications and variations of the application fall within the scope of the claims of the application and their equivalents, the application also intends to include these modifications and variations.

Claims

1. A device for triplex detection of inflammatory factors, characterized by: The device is sequentially provided from top to bottom with a plasma separation layer, a sample detection layer, a support layer, an intermediate connecting layer and a washing layer, the plasma separation layer is composed of PES material and is fixed on a sample area of the sample detection layer, the sample detection layer is divided into a hydrophilic area and a hydrophobic area, the hydrophilic area includes a sample area in the center, three detection areas uniformly distributed around the sample area as the center and three reaction channels respectively used for connecting the sample area and the detection areas, antibodies for detecting inflammatory factors are respectively added in the detection areas, the support layer is composed of hard hydrophobic PVC material, is closely attached below the sample detection layer and completely covers the sample area and the reaction channels for supporting fluid horizontal flow, the washing layer is rotationally connected to below the support layer through the intermediate connecting layer, a through hole is arranged in the center of the washing layer, three absorption pools are uniformly arranged around the through hole, the three absorption pools are respectively arranged along the extension direction of the reaction channels and correspond to the three detection areas one by one for absorbing excess liquid from the detection areas, the absorption pools sequentially include a water absorption area, a hydrophilic channel and a water storage area which are connected to each other along the outward direction from the center, the water absorption area corresponds to directly below the detection area, the width of the reaction channel is 3 mm, the inflammatory factors are respectively SAA, CRP and PCT, and the sample is a whole blood sample. The intermediate connecting layer includes a first fixed layer and a second fixed layer which are respectively fixedly arranged below the support layer and the washing layer and a rotating shaft which is connected between the first fixed layer and the second fixed layer, the rotating shaft passes through the through hole on the washing layer and is connected with the second fixed layer. The washing layer is rotationally connected below the support layer through the intermediate connecting layer, the washing layer is in a circular shape and has an area greater than that of the sample detection layer.

2. The device for detecting three inflammatory factors according to claim 1, characterized in that: The support layer and the intermediate connecting layer are made of polyvinyl chloride, polyethylene or polypropylene.

3. A preparation method of the device for detecting three inflammatory factors according to any one of claims 1-2, characterized in that: a plasma separation layer is obtained; a detection layer is prepared by an inkjet printing method, and the plasma separation layer is fixed on a sample area of the detection layer; a washing layer is prepared by an inkjet printing method, and a through hole is engraved in the center of the washing layer by using an engraving machine; a support layer and an intermediate connecting layer are obtained by cutting a PVC base plate by using an engraving method; the support layer is fixedly attached below the detection layer, and the support layer and the washing layer are connected by using the intermediate connecting layer; the hydrophobic area of the detection layer is prepared by inkjet printing of an alkyl ketene dimer-heptane-wax dye mixed solution, the mass-volume ratio of the alkyl ketene dimer and heptane is 5%, and the wax dye accounts for 1% of the mass percentage of the mixed solution; chitosan and glutaraldehyde are respectively added dropwise in the detection areas for modification.

4. The method of claim 3, wherein: The printing times are 8-15 times.

5. A use method of the device for detecting three inflammatory factors according to any one of claims 1-2, characterized in that: The sample to be detected is added to the sample area along the plasma separation layer, the sample flows into the reaction channel, gold nano-labeled antibodies are added to the channel, the gold nano-labeled antibodies are mixed with the sample and flow into three detection areas, the sample reacts and develops color in the areas, and the three detection areas are respectively added with capture antibodies corresponding to CRP, PCT and SAA; The rotating washing layer is matched with the three detection areas of the sample detection layer one by one, and the washing liquid is added dropwise to the detection areas to remove the unbound gold nano-labeled antibodies, and the liquid is absorbed through the washing pool; The color development results of the three detection areas of the sample to be detected are respectively collected and data processed, the standard fitting curve established by the image processing software is used to obtain the contents of CRP, PCT and SAA in the sample to be detected; The sample is a whole blood sample.

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