C-reactive protein rapid detection method

Abstract

The invention discloses a C-reactive protein rapid detection method, and belongs to the technical field of C-reactive protein detection.The C-reactive protein rapid detection device comprises a disc provided with a first liquid storage bin, a second liquid storage bin and a reaction bin, and the first liquid storage bin and the second liquid storage bin are communicated with the reaction bin through corresponding microfluidic channels; a mixed solution of polystyrene microspheres, Tween20, sodium azide, a phosphate buffer and a C reactive protein polyclonal antibody is added into the first liquid storage bin; a mixed solution of a phosphate buffer solution with the pH value of 7.4, Tween 20, polyethylene glycol, sodium chloride and sodium azide is added into the second liquid storage bin; adding a to-be-detected sample into the second liquid storage bin; the disc is driven to rotate; the flow (such as starting, stopping and path switching) of the solution in the micro-channel can be accurately controlled by driving the disc to rotate without externally connecting a pump, a valve and other complex mechanical devices and adjusting the rotating speed of the disc, uniform mixing of a sample and a solution reagent is promoted, the mixing efficiency is remarkably improved compared with passive diffusion, and the reaction is sufficient.

Description

Technical Field

[0001] This invention relates to the field of C-reactive protein detection technology, and in particular to a rapid method for detecting C-reactive protein. Background Technology

[0002] C-reactive protein (CRP), as a key inflammatory marker, has irreplaceable and multifaceted value in clinical diagnosis and treatment, and its detection and application span multiple disciplines in disease management. For example, in the field of infectious diseases, dynamic changes in CRP levels are an important basis for distinguishing between bacterial and viral infections. Furthermore, by monitoring the decline in CRP levels during treatment, the effectiveness of anti-infective therapy can be quickly assessed, guiding adjustments to the treatment course and shortening the average hospital stay by 1-2 days. In addition, CRP plays a crucial role in connective tissue diseases (such as monitoring active rheumatoid arthritis), metabolic syndrome (positively correlated with insulin resistance), cancer (an auxiliary indicator of tumor progression and metastasis), neurological diseases (assessment of inflammatory responses after cerebral hemorrhage), and in obstetrics and gynecology, pediatrics, and surgery (early warning of postoperative infection).

[0003] Therefore, CRP testing is crucial. Traditional CRP testing relies on large biochemical analyzers, which takes a long time from sample collection to report generation and requires professional laboratory support, making it difficult to meet the immediate needs of emergency departments, primary healthcare institutions, and POCT scenarios.

[0004] In contrast, the latest microfluidic detection, by constructing channels and reaction chambers in a (10-100) μm microscale space, shortens the diffusion distance of biomolecules to 1 / 100 of that in traditional systems. The probability of antigen-antibody collision increases exponentially, the kinetic rate is greatly improved, and the immune reaction that originally took several hours can be completed in just a few minutes.

[0005] Microfluidic detection devices for C-reactive protein detection have been developed. The core of these devices is to drop various solution reagents onto a reaction plate for reaction. However, they often require external pumps or modules to control the flow of the solution and accelerate the immune reaction. This process is cumbersome and has low reaction efficiency. There is still much room for improvement in terms of sample processing convenience and the versatility of detection and analysis. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problems of existing technologies that require external pumps or modules to control solution flow and accelerate immune reactions, resulting in cumbersome operation and low reaction efficiency. Therefore, this invention proposes a rapid detection method for C-reactive protein.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A rapid detection method for C-reactive protein includes a disk with a first reservoir, a second reservoir, and a reaction chamber, wherein the disk has multiple sets of microfluidic channels; the first and second reservoirs are respectively connected to the reaction chamber through corresponding microfluidic channels; a mixed solution of polystyrene microspheres, Tween 20, sodium azide, phosphate buffer, and C-reactive protein polyclonal antibody is added to the first reservoir; a mixed solution of phosphate buffer (pH 7.4), Tween 20, polyethylene glycol, sodium chloride, and sodium azide is added to the second reservoir; the sample to be tested is added to the second reservoir; and the disk is driven to rotate.

[0008] Preferably, the disk rotates at 200 rpm for 15 seconds; then the rotation speed is increased to 1000 rpm for 15 seconds, and the solution enters the reaction chamber.

[0009] Preferably, the mixed solution in the first storage tank is 125 μL.

[0010] Preferably, the mixed solution in the second storage tank is 500 μL.

[0011] Preferably, the sample to be tested is 2 μL.

[0012] Preferably, the reaction solution in the reaction chamber is subjected to spectrophotometric detection, and the absorbance value is recorded.

[0013] Preferably, the disk is further provided with a third liquid storage chamber, which is also connected to the reaction chamber through a corresponding microfluidic channel; 3% (wt / vol%) C18 microsphere methanol solution is added to the third liquid storage chamber.

[0014] Preferably, the disk is rotated to 1500 rpm and then rotated for 15 seconds to allow the C18 microsphere methanol solution to fully react with the solution in the reaction chamber, thereby removing proteins and peptides from the sample.

[0015] Preferably, the disc is further provided with a centrifuge chamber; the centrifuge chamber is also connected to the reaction chamber through a corresponding microfluidic channel; the disc rotates and the sample enters the centrifuge chamber.

[0016] Preferably, the second liquid storage tank is heated.

[0017] Compared with existing technologies, this invention provides a rapid detection method for C-reactive protein, which has the following advantages: 1. This rapid C-reactive protein (CRP) detection method utilizes a rotating disk to achieve a rapid immune response to CRP in biological samples. The operation is simple and convenient, significantly reducing reaction time. It can be used to improve the efficiency of collecting baseline CRP data from large sample sizes of healthy individuals, enhancing the application value of CRP detection in precision medicine. 2. This rapid detection method for C-reactive protein, by driving the disk to rotate, eliminates the need for complex mechanical devices such as external pumps and valves. By adjusting the disk rotation speed, the flow of the solution in the microchannel can be precisely controlled (such as starting, stopping, and switching paths), promoting uniform mixing of the sample and solution reagents. The mixing efficiency is significantly improved compared to passive diffusion, and the reaction is more complete. 3. In this rapid detection method for C-reactive protein, a highly sensitive and specific C-reactive protein antibody is directionally coupled to the surface of latex microspheres in the immune reaction. After the C-reactive protein in the sample to be tested binds to the antibody, an antigen-antibody complex is formed, and the latex microspheres aggregate, resulting in a change in the actual turbidity. The reaction solution in the reaction chamber is taken for spectrophotometric detection, the absorbance value is recorded, and a standard working curve of absorbance versus C-reactive protein concentration is plotted. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the structure of a disk used in a rapid detection method for C-reactive protein proposed in this invention. Figure 2 This invention proposes a rapid detection method for C-reactive protein. Figure 1 Enlarged view of section A; Figure 3 This is a front view of a disk representing a rapid detection method for C-reactive protein proposed in this invention. Figure 4 This is the standard working curve for C-reactive protein.

[0019] In the diagram: 1. Disc; 2. First liquid storage chamber; 3. Second liquid storage chamber; 4. Third liquid storage chamber; 5. Reaction chamber; 6. Centrifuge chamber; 7. Microfluidic channel; 8. Chamfer; 9. Heating chamber. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0021] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0022] Example: like Figures 1-3A rapid detection method for C-reactive protein includes a circular disk 1, which serves as a carrier for the reaction disk. The center of the disk is fixedly connected to the output end of a motor.

[0023] The disc 1 is equipped with a first liquid storage chamber 2, a second liquid storage chamber 3, and a reaction chamber 5.

[0024] The first storage chamber 2 has a volume of 135 μL. During the test, a mixed solution of polystyrene microspheres, Tween 20, sodium azide, phosphate buffer, and C-reactive protein polyclonal antibody was added, totaling 125 μL.

[0025] The second storage chamber 3 has a volume of 530 μL. During testing, a mixed solution of phosphate buffer, Tween 20, polyethylene glycol, sodium chloride, and sodium azide is added, totaling 500 μL.

[0026] The pH of the phosphate buffer is 7.4.

[0027] The volume of reaction chamber 5 is 640 μL.

[0028] The disk 1 is provided with multiple sets of microfluidic channels 7, the width of which is 0.6-1mm, preferably 0.8mm.

[0029] The first liquid storage tank 2 and the second liquid storage tank 3 are respectively connected to the reaction tank 5 through corresponding microfluidic channels 7; for example Figure 1 and Figure 3 The first liquid storage chamber 2 and the second liquid storage chamber 3 are each connected to the reaction chamber 5 through a microfluidic channel 7.

[0030] like Figure 1 During the test, 2 μL of the sample to be tested is added to the second storage chamber 3. Then, the motor drives the disk 1 to rotate at a centrifugal speed of 200 rpm. After mixing for 15 seconds, the centrifugal speed is increased to 1000 rpm and rotated for 15 seconds. Through centrifugal force, the solutions in the first storage chamber 2 and the second storage chamber 3 are transferred to the reaction chamber 5 through the microfluidic channel 7 for mixing and immune reaction.

[0031] In this immune reaction, highly sensitive and specific C-reactive protein antibodies are directionally coupled to the surface of latex microspheres. After the C-reactive protein in the sample binds to the antibody, an antigen-antibody complex is formed, causing the latex microspheres to aggregate and resulting in a change in actual turbidity. The reaction solution in reaction chamber 5 is taken for spectrophotometric detection, and the absorbance value is recorded. A standard working curve of absorbance versus C-reactive protein concentration is plotted, as shown below. Figure 4 As shown.

[0032] The linear fitting equation for the standard working curve is y = 0.063x + 1.28, R0 2 =0.998.

[0033] The accuracy and repeatability of this testing method were verified using nationally certified reference materials.

[0034] Accuracy: Using the nationally certified standard reference material GBW09228, C-reactive protein solution purity standard, 2 μL of C-reactive protein standard material of known concentration was mixed and reacted through disk 1. After the reaction, the sample was detected by a spectrophotometer. The spectra were observed, the absorbance was recorded, and the measured value of C-reactive protein was calculated using a working curve, rounded to one decimal place. Δc was calculated according to the following formula as the evaluation result of the accuracy of this method.

[0035] Δc=| - | In the formula: : Measured value, unit mg / L; Theoretical value, unit: mg / L.

[0036] The C-reactive protein concentration was 54.9 mg / L, with an expanded uncertainty of 2.8 mg / L and k=2. After repeated testing three times using the method described in the project, the inter-batch measured values ​​were obtained using the standard curve: 55.6, 52.7, and 53.1 mg / L, respectively. The average value was calculated to be 53.8 mg / L, with Δc=1.1 mg / L.

[0037] Repeatability: The above detection was performed by adding a C-reactive protein standard solution of known concentration, and the measured values ​​were obtained. The relative standard deviation (RSD) was calculated after six consecutive tests as the result of repeatability of the method.

[0038] In the formula: xi: The measured value of the i-th measurement; The arithmetic mean of 6 measurements; i: Measurement sequence number.

[0039] Through six experiments, the measured values ​​of C-reactive protein were 55.6, 52.7, 53.1, 53.7, 52.1, and 55.9 mg / L, respectively. The RSD was calculated to be 2.86% using the above formula.

[0040] This method can balance the efficiency, portability and low cost of detecting complex biological samples, and has important advantages in areas such as point-of-care testing and high-throughput screening, providing a technical direction for the clinical application of microfluidic technology.

[0041] By driving the disk 1 to rotate, without the need for complex mechanical devices such as external pumps and valves, the flow of the solution in the microchannel can be precisely controlled by adjusting the rotation speed of the disk 1, such as starting, stopping, and switching paths, which promotes the uniform mixing of the sample and the solution reagent. The mixing efficiency is significantly improved compared with passive diffusion, and the reaction is more complete.

[0042] In addition, centrifugal force can suppress the residue and adsorption of solution in microfluidic channel 7. During the rotation of disk 1, the solution in microfluidic channel 7 is rapidly transferred by centrifugal force which is greater than capillary force and surface tension of solution, thereby improving reaction efficiency.

[0043] It should be noted that in actual testing, the disc 1 is located inside the housing of the testing equipment, and there is a cover on top. During the inspection, that is, when the disc 1 rotates, the cover is closed and the cover is in contact with the upper surface of the disc 1 to prevent various solutions from splashing out during the test.

[0044] like Figure 1 and Figure 3 The disk 1 is also equipped with a third liquid storage chamber 4; the third liquid storage chamber 4 is also connected to the reaction chamber 5 through a corresponding microfluidic channel 7. The third liquid storage chamber 4 is also connected to the reaction chamber 5 through a separate microfluidic channel 7.

[0045] Add 3% wt / vol% C18 microsphere methanol solution to the third storage tank 4.

[0046] Increase the centrifugation speed to 1000 rpm in disc 1 and rotate for 15 seconds. Then, continue to adjust the speed to 1500 rpm and rotate for another 15 seconds. The C18 microspheres in the third storage chamber 4 enter the reaction chamber 5 for mixing and immune reaction to remove proteins and peptides from the sample.

[0047] like Figure 1 and Figure 3 The disc 1 is also equipped with a centrifuge chamber 6; the centrifuge chamber 6 is also connected to the reaction chamber 5 through a corresponding microfluidic channel 7; the centrifuge chamber 6 is also connected to the reaction chamber 5 through a separate microfluidic channel 7.

[0048] In other words, there are four microfluidic channels 7: the first liquid storage chamber 2, the second liquid storage chamber 3, the third liquid storage chamber 4, and the centrifuge chamber 6, all of which are connected to the reaction chamber 5 through a microfluidic channel 7.

[0049] like Figure 1 The first liquid storage chamber 2, the second liquid storage chamber 3, and the third liquid storage chamber 4 are located on the side of the reaction chamber 5 closest to the circular side of the disk 1; Centrifuge chamber 6 is located on the side of reaction chamber 5 away from the circular shape of disk 1.

[0050] This ensures that the solution enters the reaction chamber 5 and the centrifuge chamber 6 sequentially through centrifugal force.

[0051] The reacted sample is separated into centrifuge chamber 6 by centrifugation, and can then be analyzed by large instruments to obtain a cleaner detection background and better detection signal.

[0052] like Figures 1-3 The connections between the microfluidic channel 7 and the first liquid storage tank 2, the second liquid storage tank 3, and the third liquid storage tank 4 are all designed with chamfers 8.

[0053] The connection between the microfluidic channel 7 and the centrifuge chamber 6 is also designed with a chamfer 8.

[0054] The chamfered 8 design facilitates the flow of solution in and out.

[0055] like Figures 1-3 The second liquid storage tank 3 is heated to 37°C, specifically as follows: The disc 1 is also equipped with a heating chamber 9; the second liquid storage chamber 3 is located inside the heating chamber 9, and the heating chamber 9 and the second liquid storage chamber 3 are separated by a partition. The partition is thermally conductive and heats the solution in the second liquid storage chamber 3.

[0056] A heating component, such as a heating wire, is installed inside the heating chamber 9 to heat the solution in the second storage chamber 3 to 37°C, thereby accelerating the subsequent reaction.

[0057] A rapid C-reactive protein (CRP) detection method utilizes a rotating disk (1) to achieve a rapid immune response to CRP in biological samples. The method is simple and convenient, significantly reducing reaction time. It can be used to improve the efficiency of collecting baseline CRP data from large sample sizes of healthy individuals, enhancing the application value of CRP detection in precision medicine.

[0058] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A rapid detection method for C-reactive protein, comprising a disk (1) having a first reservoir (2), a second reservoir (3), and a reaction chamber (5), characterized in that, The disk (1) is provided with multiple sets of microfluidic channels (7); The first liquid storage tank (2) and the second liquid storage tank (3) are respectively connected to the reaction tank (5) through corresponding microfluidic channels (7); Add a mixed solution of polystyrene microspheres, Tween 20, sodium azide, phosphate buffer and C-reactive protein polyclonal antibody to the first storage tank (2); Add a mixed solution of phosphate buffer, Tween 20, polyethylene glycol, sodium chloride, and sodium azide to the second storage tank (3); Add the sample to be tested to the second liquid storage tank (3); Drive the disk (1) to rotate.

2. The rapid detection method for C-reactive protein according to claim 1, characterized in that, The disk (1) rotates at 200 rpm for 15 seconds; Then the rotation speed is increased to 1000 rpm, and after 15 seconds of rotation, the solution enters the reaction chamber (5).

3. The rapid detection method for C-reactive protein according to claim 1, characterized in that, The mixed solution in the first storage tank (2) is 125 μL.

4. The rapid detection method for C-reactive protein according to claim 1, characterized in that, The mixed solution in the second storage tank (3) is 500 μL.

5. The rapid detection method for C-reactive protein according to claim 1, characterized in that, The sample to be tested is 2 μL.

6. The rapid detection method for C-reactive protein according to claim 1, characterized in that, Take the reaction solution in the reaction chamber (5) for spectrophotometric detection and record the absorbance value.

7. The rapid detection method for C-reactive protein according to claim 2, characterized in that, The disk (1) is also provided with a third liquid storage chamber (4), which is also connected to the reaction chamber (5) through a corresponding microfluidic channel (7); Add 3% (wt / vol%) C18 microsphere methanol solution to the third storage tank (4).

8. The rapid detection method for C-reactive protein according to claim 7, characterized in that, The disk (1) is rotated to 1500 rpm and continues to rotate for 15 seconds; The C18 microsphere methanol solution is allowed to react fully with the solution in the reaction chamber (5) to remove proteins and peptides from the sample.

9. A rapid detection method for C-reactive protein according to any one of claims 1-8, characterized in that, The disc (1) is also provided with a centrifuge chamber (6); The centrifuge chamber (6) is also connected to the reaction chamber (5) through the corresponding microfluidic channel (7).

10. The rapid detection method for C-reactive protein according to claim 1, characterized in that, The second liquid storage tank (3) is heated.

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