Microfluidic chip for sdma optical chemiluminescence detection and method of use

Abstract

The application provides a microfluidic chip for SDMA optical chemiluminescence detection and a use method, relates to the field of biomedical detection, and the microfluidic chip comprises a fan-shaped substrate, a sample introduction unit and a diluent unit are arranged on the fan-shaped substrate, a mixing groove is arranged on the side of the diluent unit away from the center of the fan-shaped substrate, and the mixing groove is connected with the sample introduction unit and the diluent unit; a plurality of detection units are arranged on the side of the mixing groove away from the center in a circumferential direction, each detection unit comprises a first reaction groove and a second reaction groove arranged in a radial direction of the fan-shaped substrate and connected with each other, and the first reaction groove is connected with the mixing groove; a first compound and a second compound are arranged in the first reaction groove, and a third compound is arranged in the second reaction groove; by arranging the two reaction grooves, the competition reaction is ensured not to be disturbed by a subsequent signal amplification step in space, so that hook effect is effectively avoided, liquid crosstalk to adjacent detection units is avoided, and the independence of each detection unit and the accuracy of results are ensured.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, and in particular to a microfluidic chip for SDMA photo-induced chemiluminescence detection and its usage method. Background Technology

[0002] SDMA (symmetric dimethylarginine) is a small molecule metabolite produced by the hydrolysis of proteins after methylation modification in vivo. In recent years, SDMA has evolved from a common metabolite into a novel biomarker with high specificity and sensitivity, widely recognized in clinical practice for assessing early renal function impairment. Compared with traditional indicators such as serum creatinine, SDMA is almost entirely excreted through the kidneys and is not affected by non-renal factors such as age, sex, or muscle mass. Therefore, it can show a significant increase in the early stages of renal function decline, providing a crucial window for early warning of acute kidney injury and chronic kidney disease. However, current mainstream detection methods for SDMA (such as high-performance liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay) usually rely on large central laboratory equipment, which are complex and time-consuming, making it difficult to meet the urgent needs of clinical departments, emergency departments, or primary healthcare settings for rapid, bedside, and immediate testing.

[0003] Photo-induced chemiluminescence (PET) is an advanced chemiluminescent immunoassay technique based on a homogeneous, wash-free reaction principle. Its core mechanism relies on a pair of functionalized microspheres: donor microspheres are coated with a photosensitizer, generating transiently diffusing singlet oxygen under 680 nm laser excitation; acceptor microspheres are coated with a chemiluminescent agent and can be drawn closer to the effective diffusion distance of the singlet oxygen generated by the donor microspheres through biomolecular interactions (such as antigen-antibody reactions), thereby being excited to generate a fluorescence signal. This technique eliminates the cumbersome solid-phase carrier separation and multiple washing steps required in traditional heterogeneous chemiluminescence detection, simplifying the entire reaction system into a single liquid phase environment, greatly enhancing the automation potential and ease of operation. Furthermore, since an effective signal is only generated when the target molecule causes the two microspheres to bind close together, this method has significant advantages such as low background noise, high sensitivity, and a wide kinetic range. However, this technique also faces unique challenges when applied to point-of-care testing: in the presence of high concentrations of analyte, the microspheres are prone to saturation, leading to a "hook effect" that affects detection accuracy.

[0004] Therefore, there is an urgent need to develop a highly integrated microfluidic chip that is deeply adapted to the characteristics of photochemiluminescence reaction and the requirements of the entire SDMA detection process. Summary of the Invention

[0005] The purpose of this application is to address the above problems by providing a microfluidic chip for SDMA photo-induced chemiluminescence detection and its usage method.

[0006] In a first aspect, this application provides a microfluidic chip for SDMA photochemiluminescence detection, comprising: A fan-shaped substrate, wherein a sample introduction unit and a diluent unit are provided on the fan-shaped substrate; A mixing tank is located on the side of the diluent unit away from the center of the fan-shaped substrate and is connected to the sample introduction unit and the diluent unit respectively. The mixing tank is used to mix the sample to be tested and the diluent. The detection unit comprises multiple detection units located on the side of the mixing tank away from the center of the fan-shaped substrate and distributed circumferentially along the fan-shaped substrate. Each detection unit includes a first reaction tank and a second reaction tank arranged radially along the fan-shaped substrate and connected thereto. Multiple first reaction tanks are connected to the mixing tank. The first reaction tank contains a first complex containing an SDMA antibody and a second complex containing a biotin-labeled SDMA antigen. The second reaction tank contains a third complex containing streptavidin.

[0007] According to the technical solutions provided in certain embodiments of this application, a fluid shut-off valve is provided between the first reaction tank and the second reaction tank. The fluid shut-off valve is configured such that when the pressure on the fluid shut-off valve reaches a threshold, the fluid shut-off valve opens, and the first reaction tank connects to the second reaction tank.

[0008] According to the technical solutions provided in certain embodiments of this application, the sample introduction unit includes a sample loading groove, a sample quantitative groove, and a sample flow channel. The sample loading groove and the sample quantitative groove are arranged radially along the fan-shaped substrate and are connected. The sample quantitative groove is connected to the mixing groove through the sample flow channel.

[0009] According to the technical solutions provided in certain embodiments of this application, the diluent unit includes a diluent storage tank, a diluent metering tank, and a diluent flow channel. The diluent storage tank is provided with a diluent bladder, and the diluent storage tank, the diluent metering tank, the diluent flow channel, and the mixing tank are connected in sequence.

[0010] According to the technical solutions provided in some embodiments of this application, an annular groove is further provided between the plurality of detection units and the mixing tank, the mixing tank is connected to the annular groove through a mixing channel, and the annular groove is respectively connected to each of the first reaction tanks.

[0011] According to the technical solutions provided in certain embodiments of this application, a separation groove is provided on the side of the sample quantification groove away from the center of the fan-shaped substrate, and the separation groove is connected to the sample quantification groove.

[0012] According to the technical solutions provided in certain embodiments of this application, the inner surfaces of the sample channel, the diluent channel, and the mixing channel are treated with hydrophilicity so that the liquid fills the channel by relying on surface tension.

[0013] Secondly, this application provides a method for using the microfluidic chip for SDMA photochemiluminescence detection as described above, comprising: The sample to be tested is added to the sample loading slot, and the microfluidic chip is fixed on the testing platform; Start the drive motor to make the microfluidic chip rotate at a first set speed for a first set time, so that the sample to be tested enters the sample quantitative tank and the diluent enters the diluent quantitative tank; The microfluidic chip is rotated at a first set speed for a first set time so that the sample to be tested and the diluent are mixed in the mixing tank to form the test mixture. The microfluidic chip is rotated at a second set speed for a second set time so that the mixture to be tested enters the first reaction tank. The mixture to be tested reacts with the first complex and the second complex to form the first reactant. The microfluidic chip is rotated at a third set speed for a third set time to open the fluid shut-off valve, allowing the first reactant to enter the second reaction tank. The first reactant reacts with the third complex to form the second reactant. The third set speed is greater than the second set speed. The second reactant is irradiated with a laser, and the chemiluminescence signal generated by the excited second reactant is detected.

[0014] According to the technical solutions provided in certain embodiments of this application, the range of the first set rotational speed is 2000rpm-6000rpm, and the range of the first set time is 10s-300s; the range of the second set rotational speed is 200rpm-2000rpm, and the range of the second set time is 30s-300s; the range of the third set rotational speed is 3000rpm-6000rpm, and the range of the third set time is 20s-200s.

[0015] Compared with the prior art, the beneficial effects of this application are as follows: This application provides a microfluidic chip and a method of using it for SDMA photoluminescence detection. The microfluidic chip includes a fan-shaped substrate, on which a sample introduction unit and a diluent unit are provided. A mixing tank is provided on the side of the diluent unit away from the center of the fan-shaped substrate. The mixing tank is connected to the sample introduction unit and the diluent unit respectively, for mixing the sample to be detected and the diluent. Multiple detection units are provided on the side of the mixing tank away from the center of the fan-shaped substrate. The multiple detection units are distributed along the circumference of the fan-shaped substrate. Each detection unit includes a first reaction tank and a second reaction tank arranged radially along the fan-shaped substrate and connected to it. Multiple first reaction tanks are connected to the mixing tank. A first complex containing an SDMA antibody and a second complex containing a biotin-labeled SDMA antigen are provided in the first reaction tank. A third complex containing streptavidin is provided in the second reaction tank. By integrating the sample introduction unit, dilution unit, mixing tank, and multiple detection units onto a single sector substrate, the SDMA detection process is integrated and miniaturized, significantly reducing reliance on large external equipment and complex operations, making it suitable for point-of-care clinical testing. The competitive detection principle is adopted. A first complex containing SDMA antibody and a second complex containing biotin-labeled SDMA antigen are simultaneously placed in the first reaction cell. This allows SDMA in the sample to compete with the labeled antigen for binding to the antibody, thereby maintaining a linear response even at high sample concentrations, thus improving the accuracy and reliability of the detection. By setting up a first reaction chamber and a second reaction chamber, only the competitive reaction occurs in the first reaction chamber, where SDMA in the sample fully competes with the biotin-labeled SDMA antigen, completely eliminating the possibility of false signals due to non-specific effects or premature binding. The signal generation reaction occurs in the second reaction chamber, ensuring that only the results of specific competition can be converted into optical signals, greatly improving the signal-to-noise ratio. Compared with the traditional one-step homogeneous reaction, spatial separation prevents the competitive reaction from being interfered with by subsequent signal amplification steps, thus effectively avoiding the hook effect and ensuring the accuracy and wide linear range of high-concentration sample detection. Each detection unit includes an independent first reaction tank and a second reaction tank connected in radial series. During the process of centrifugal force driving the liquid, the reaction liquid can be effectively constrained within the flow channel and tank of its respective unit, preventing liquid crosstalk to adjacent detection units. This ensures the independence of each detection unit and the accuracy of the results when multiple items or multiple samples are detected in parallel on the same chip, thereby improving the detection reliability of the chip.

[0016] It should be understood that the descriptions of technical features, technical solutions, beneficial effects, or similar language in this application do not imply that all features and advantages can be achieved in any single embodiment. Rather, it is understood that the description of a feature or beneficial effect means that a specific technical feature, technical solution, or beneficial effect is included in at least one embodiment. Therefore, the descriptions of technical features, technical solutions, or beneficial effects in this specification do not necessarily refer to the same embodiment. Furthermore, the technical features, technical solutions, and beneficial effects described in this embodiment can be combined in any suitable manner. Those skilled in the art will understand that embodiments can be implemented without one or more specific technical features, technical solutions, or beneficial effects of a particular embodiment. In other embodiments, additional technical features and beneficial effects may be identified in specific embodiments that do not embody all embodiments. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the structure of a microfluidic chip for SDMA photochemiluminescence detection provided in Embodiment 1 of this application; Figure 2 This is a flowchart illustrating a method of using a microfluidic chip for SDMA photoluminescence detection, as provided in Embodiment 2 of this application.

[0019] The text labels in the image represent: 1. Fan-shaped substrate; 2. Mixing tank; 3. First reaction tank; 4. Second reaction tank; 5. Fluid shut-off valve; 6. Sample loading tank; 7. Sample quantitative tank; 8. Sample flow channel; 9. Diluent storage tank; 10. Diluent quantitative tank; 11. Diluent flow channel; 12. Mixing flow channel; 13. Annular tank; 14. Separation tank; 15. Vent. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. The descriptions in this section are merely illustrative and explanatory, and should not be construed as limiting the scope of protection of this application. Specifically, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort should fall within the scope of protection of this application.

[0021] It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such process, method, product, or apparatus.

[0022] Example 1 As mentioned in the background section, to address the problems existing in the prior art, this embodiment provides a microfluidic chip for SDMA photochemiluminescence detection, comprising: A sector-shaped substrate 1 is provided with a sample introduction unit and a diluent unit; Mixing tank 2 is located on the side of the diluent unit away from the center of the fan-shaped substrate 1, and is connected to the sample introduction unit and the diluent unit respectively. Mixing tank 2 is used to mix the sample to be tested and the diluent. The detection unit comprises multiple detection units located on the side of the mixing tank 2 away from the center of the fan-shaped substrate 1 and distributed circumferentially along the fan-shaped substrate 1. Each detection unit includes a first reaction tank 3 and a second reaction tank 4 arranged radially along the fan-shaped substrate 1 and connected to it. Multiple first reaction tanks 3 are connected to the mixing tank 2. The first reaction tank 3 contains a first complex containing SDMA antibody and a second complex containing biotin-labeled SDMA antigen. The second reaction tank 4 contains a third complex containing streptavidin.

[0023] like Figure 1As shown, the sample introduction unit and the diluent unit are distributed along the circumference of the fan-shaped substrate 1. The sample introduction unit is used to receive and introduce the sample to be tested, and the diluent unit is used to store and release the diluent. The mixing tank 2 is approximately annular in structure, with the diluent unit located on the side away from the center of the fan-shaped substrate 1. The mixing tank 2 is connected to both the sample introduction unit and the diluent unit, and is used to mix the sample to be tested and the diluent. Multiple detection units are distributed along the circumference of the fan-shaped substrate 1 on the side of the mixing tank 2 away from the center of the fan-shaped substrate 1. The first reaction tank 3 and the second reaction tank 4 are arranged radially along the fan-shaped substrate 1 and are interconnected. The first complex... The main components of the first complex include Tris (tris(hydroxymethyl)aminomethane) buffer, sodium chloride, surfactants, SDMA antibody-crosslinked receptor microspheres, excipients, and shaping agents. The main components of the second complex include biotin-labeled SDMA antigen, bovine serum albumin, sucrose, excipients, and shaping agents. The main components of the third complex include streptavidin-crosslinked donor microspheres, excipients, and shaping agents. Excipients and shaping agents include, but are not limited to, sugars, amino acids, proteins, polyols, surfactants, and polymers. During detection, the centrifugal force generated by the rotation of the fan-shaped substrate 1 introduces the sample into the unit for detection. The sample and the diluent from the diluent unit are diluted and mixed in mixing tank 2. After mixing, the mixture enters each of the first reaction tanks 3 under centrifugal force. The SDMA in the mixture and the biotin-labeled SDMA antigen in the second complex compete for binding with the receptor microspheres cross-linked with the SDMA antibody in the first complex. After the biotin-labeled SDMA antigen binds to the receptor microspheres cross-linked with the SDMA antibody, a biotin-labeled "signal complex" is generated. After the competitive reaction is completed, the "reactants" in each of the first reaction tanks 3 enter the corresponding second reaction tank 4 under centrifugal force. Because streptavidin has a strong affinity for biotin... With extremely high affinity, the donor microspheres capture biotin in the "reactants" formed during the competitive reaction via streptavidin. If the "signal complex" is captured, a bridging structure is formed between the donor and recipient microspheres, bringing them closer together. When the donor microspheres are irradiated by laser, they generate singlet oxygen, which in turn excites the recipient microspheres to produce a fluorescent signal. That is, the higher the SDMA concentration in the sample, the fewer "signal complexes" are generated after the competitive reaction, and the weaker the fluorescent signal after laser irradiation. Conversely, the lower the SDMA concentration in the sample, the more "signal complexes" are generated after the competitive reaction, and the stronger the fluorescent signal after laser irradiation.

[0024] Because the microfluidic chip has a fan-shaped structure, several fan-shaped substrates 1 can be freely combined on the detection platform during a single detection process, thereby enabling simultaneous detection of multiple samples and multiple detection items in a single test. By integrating the sample introduction unit, dilution unit, mixing tank 2, and multiple detection units onto a single sector substrate 1, the SDMA detection process is integrated and miniaturized, significantly reducing reliance on large external equipment and complex operations, making it suitable for point-of-care clinical testing. The competitive detection principle is adopted. A first complex containing SDMA antibody and a second complex containing biotin-labeled SDMA antigen are simultaneously placed in the first reaction cell 3. This allows SDMA in the sample to compete with the labeled antigen for binding to the antibody, thereby maintaining a linear response even at high sample concentrations, thus improving the accuracy and reliability of the detection. By setting up a first reaction chamber 3 and a second reaction chamber 4, only the competitive reaction takes place in the first reaction chamber 3, where SDMA in the sample competes fully with the biotin-labeled SDMA antigen, completely eliminating the possibility of false signals due to non-specific effects or premature binding. The signal generation reaction takes place in the second reaction chamber 4, ensuring that only the results of specific competition can be converted into light signals, greatly improving the signal-to-noise ratio. Compared with the traditional one-step homogeneous reaction, spatial separation prevents the competitive reaction from being interfered with by subsequent signal amplification steps, thereby effectively avoiding the hook effect and ensuring the accuracy and wide linear range of high-concentration sample detection. Each detection unit includes an independent first reaction tank 3 and a second reaction tank 4 connected in a radial series. During the process of centrifugal force driving the liquid, the reaction liquid can be effectively constrained within the flow channel and tank of each unit, preventing liquid crosstalk to adjacent detection units. This ensures the independence of each detection unit and the accuracy of the results when multiple items or multiple samples are detected in parallel on the same chip, thus improving the detection reliability of the chip.

[0025] Furthermore, a fluid shut-off valve 5 is provided between the first reaction tank 3 and the second reaction tank 4. The fluid shut-off valve 5 is configured such that when the pressure on the fluid shut-off valve 5 reaches a threshold, the fluid shut-off valve 5 opens, and the first reaction tank 3 connects to the second reaction tank 4. For details, please refer to Figure 1A fluid shut-off valve 5 is installed between the first reaction tank 3 and the second reaction tank 4. When the microfluidic chip rotates at a low speed, the fluid shut-off valve 5 cuts off the first reaction tank 3 and the second reaction tank 4, allowing the mixture to remain in the first reaction tank 3 and compete with the first and second complexes for a reaction. After the competitive reaction is completed, by increasing the rotation speed of the microfluidic chip, the reactants after the competitive reaction exert pressure on the fluid shut-off valve 5 under the action of centrifugal force, breaking through the fluid shut-off valve 5 and entering the second reaction tank 4 to react with the third complex. By setting the fluid shut-off valve 5, the entire detection process is physically divided into two distinct reaction steps. After the competitive reaction is completed, the fluid shut-off valve 5 is opened by increasing the rotation speed, allowing the reactants after the competitive reaction to enter the second reaction tank 4. This ensures that the reaction proceeds in the predetermined order and prevents non-specific binding or reaction interference caused by premature mixing of reagents, thereby significantly improving the accuracy and specificity of the detection. At the same time, the centrifugal force of the reactants after the competitive reaction is used to automatically open the fluid shut-off valve 5, eliminating the need for additional mechanical, electrical, or thermal drive components. This not only simplifies the chip structure and reduces manufacturing costs but also enables the entire detection process to be fully synchronized with the rotation control of the detection platform, improving the automation level and operational reliability of the system.

[0026] In a preferred embodiment, the sample introduction unit includes a sample loading groove 6, a sample quantitative groove 7, and a sample flow channel 8. The sample loading groove 6 and the sample quantitative groove 7 are arranged radially along the fan-shaped substrate 1 and are connected. The sample quantitative groove 7 is connected to the mixing groove 2 through the sample flow channel 8. The diluent unit includes a diluent storage tank 9, a diluent quantitative groove 10, and a diluent flow channel 11. The diluent storage tank 9 is provided with a diluent bladder. The diluent storage tank 9, the diluent quantitative groove 10, the diluent flow channel 11, and the mixing groove 2 are connected in sequence. An annular groove 13 is also provided between the multiple detection units and the mixing groove 2. The mixing groove 2 is connected to the annular groove 13 through the mixing flow channel 12. The annular groove 13 is connected to each of the first reaction grooves 3.

[0027] like Figure 1As shown, the sample loading slot 6 is located on the fan-shaped substrate 1 near the center. The sample loading slot 6 and the sample quantitative slot 7 are arranged radially along the fan-shaped substrate 1 and are connected. The sample quantitative slot 7 is connected to the mixing tank 2 through the sample flow channel 8. The sample loading slot 6 has a sample loading hole for adding the sample. The diluent storage tank 9 is distributed on one side of the sample loading slot 6. The diluent quantitative slot 10 is also approximately annular and is distributed on the side of the diluent storage tank 9 away from the center of the fan-shaped substrate 1. The diluent quantitative slot 10 is connected to the mixing tank 2 through the diluent flow channel 11. The diluent storage tank 9 is provided with a sealed diluent bladder. The diluent bladder can be automatically opened at the start of the detection to release the diluent. The relevant structure of the diluent bladder is prior art well known to those skilled in the art and will not be described in detail here. The annular slot 13 is located on the side of the mixing tank 2 away from the center of the fan-shaped substrate 1. The annular slot 13 is connected to each of the first reaction slots 3 and is also connected to the mixing tank 2 through the mixing flow channel 12.

[0028] Preferably, a first waste liquid tank and a second waste liquid tank are respectively provided at both ends of the fan-shaped substrate 1 in the circumferential direction. The first waste liquid tank is connected to the sample quantitative tank 7, and the second waste liquid tank is connected to the diluent quantitative tank 10 and the annular tank 13. The first waste liquid tank and the second waste liquid tank are used to store excess liquid after the sample, diluent and mixture are quantified during the detection process. Preferably, the fan-shaped substrate 1 is further provided with a vent hole 15, which is located on the side of the mixing tank 2 near the center of the fan-shaped substrate 1 and communicates with the mixing tank 2 along the radial direction of the fan-shaped substrate 1.

[0029] Furthermore, a separation groove 14 is provided on the side of the sample quantitative groove 7 away from the center of the fan-shaped substrate 1, and the separation groove 14 is connected to the sample quantitative groove 7; For details, please refer to Figure 1 The sample loading tank 6, sample quantification tank 7, and separation tank 14 are connected in sequence. The sample quantification tank 7 and separation tank 14 are distributed radially along the fan-shaped substrate 1. The sample flow channel 8 is connected between the sample quantification tank 7 and the separation tank 14. During the detection process, the blood cells in the plasma can enter the separation tank 14 through the action of centrifugal force, while the serum enters the mixing tank 2 through the sample flow channel 8. Thus, the microfluidic chip can directly detect whole blood samples.

[0030] In a preferred embodiment, the inner surfaces of the sample channel 8, the diluent channel 11, and the mixing channel 12 are treated with a hydrophilic coating so that the liquid fills the channels by relying on surface tension. like Figure 1As shown, the inner surfaces of the sample channel 8, the diluent channel 11, and the mixing channel 12 are treated with hydrophilicity. The treatment methods include, but are not limited to, oxygen plasma surface treatment, ozone radiation treatment, surface activator treatment, and graft copolymerization treatment, so that the inner surfaces of the channels achieve a hydrophilic effect. By treating the inner surfaces of the channels with hydrophilicity, the flow resistance and contact angle of the liquid in the channels are significantly reduced, so that the liquid can spontaneously, quickly and completely fill the channels by relying on its own surface tension and capillary action. Relying on the passive filling mechanism of capillary force and surface tension, the dependence on high-speed or complex external drive pumps is reduced.

[0031] Example 2 like Figure 2 The diagram shown is a flowchart illustrating a method of using a microfluidic chip for SDMA photochemiluminescence detection provided in this embodiment. The method includes: S1. Add the sample to be tested into the sample loading slot 6 and fix the microfluidic chip on the detection platform; Specifically, the sample to be tested (which can be whole blood, serum or plasma) is added into the sample loading tank 6, and a pre-packaged diluent sac is placed in the diluent storage tank 9. The microfluidic chip is placed on the detection platform, and the microfluidic chip is fixed to the drive end of the drive motor of the detection platform. The diluent sac is opened, and the diluent enters the diluent storage tank 9.

[0032] S2. Start the drive motor to make the microfluidic chip rotate at the first set speed for the first set time so that the sample to be tested enters the sample quantitative tank 7 and the diluent enters the diluent quantitative tank 10. Specifically, the drive motor is started for the first centrifugation, with a centrifugation speed of 2000rpm-6000rpm and a centrifugation time of 10s-300s, preferably 30s-200s. During the centrifugation, the sample to be tested in the sample loading tank 6 enters the sample quantification tank 7 to complete the quantification, and the diluent in the diluent storage tank 9 enters the diluent quantification tank 10 to complete the quantification. After the centrifugation is completed, the sample flow channel 8 and the diluent flow channel 11 are opened. S3. The microfluidic chip is rotated at a first set speed for a first set time so that the sample to be tested and the diluent enter the mixing tank 2 for mixing to form a test mixture. Specifically, the drive motor is started for a second centrifugation at a speed of 2000 rpm to 6000 rpm for a time of 10 s to 300 s, preferably 30 s to 200 s. During the centrifugation, the sample to be tested in the sample quantitative tank 7 enters the mixing tank 2 through the sample flow channel 8, and the diluent in the diluent quantitative tank 10 enters the mixing tank 2 through the diluent flow channel 11. The sample to be tested and the diluent are diluted and mixed in the mixing tank 2. At this time, the drive motor can drive the microfluidic chip to repeatedly perform acceleration and deceleration or forward and reverse motion, thereby generating an oscillation effect to promote the dilution and mixing of the liquid in the mixing tank 2 and form the test mixture. After the centrifugation is completed, the mixing flow channel 12 is opened.

[0033] S4. The microfluidic chip is rotated at a second set speed for a second set time so that the mixture to be tested enters the first reaction tank 3. The mixture to be tested reacts with the first complex and the second complex to form the first reactant. Specifically, the drive motor is started for the third centrifugation, with a centrifugation speed of 200rpm-2000rpm and a centrifugation time of 30s-300s. During the centrifugation process, the test mixture in the mixing tank 2 enters each of the first reaction tanks 3 through the mixing channel 12 and the annular tank 13 respectively. The test mixture competes with the first complex and the second complex to form the first reactant.

[0034] S5. The microfluidic chip is rotated at a third set speed for a third set time to open the fluid shut-off valve 5, and the first reactant enters the second reaction tank 4. The first reactant reacts with the third complex to form the second reactant. The third set speed is greater than the second set speed. Specifically, the drive motor is started to perform the fourth centrifugation at a speed of 3000-6000 rpm for 20-200 seconds. During the centrifugation, the first reactant opens the fluid shut-off valve 5 under the action of centrifugal force and enters the corresponding second reaction tank 4. The first reactant reacts with the third complex. At this time, the drive motor can drive the microfluidic chip to repeatedly perform acceleration and deceleration or forward and reverse motion, thereby generating an oscillation effect to promote the reaction between the first reactant and the third complex in the second reaction tank 4, forming the second reactant.

[0035] S5. Irradiate the second reactant with a laser and detect the chemiluminescence signal generated by the second reactant after excitation.

[0036] Specifically, the optical module on the detection platform irradiates the second reactant in the second reaction tank 4 with a laser, causing the second reactant to generate a chemiluminescence signal after being excited. The photoelectric conversion module on the detection platform converts the chemiluminescence signal into a digital signal, and then calculates the SDMA concentration in the sample to be tested through the calibration curve of absorbance and concentration.

[0037] The preparation methods of SDMA antibody-crosslinked receptor microspheres and biotin-labeled SDMA antigens are described respectively; SDMA antibody-crosslinked receptor microspheres: Add receptor microspheres to centrifuge tubes, centrifuge at 20000g for 20min, remove supernatant, add labeling buffer, sonicate, centrifuge again, remove supernatant, add microsphere activation buffer, sonicate to disperse evenly, add EDC solution, vortex mix, then add NHS solution and mix well; place centrifuge tubes on a turntable, temperature 25℃, speed 40r / min, activate in the dark for 20min; add activation buffer for washing, add SDMA antibody for conjugation in the dark for 2h, add blocking solution for blocking overnight, remove supernatant, add microsphere preservation solution, dilute for later use; Biotin-labeled SDMA antigen: Add SDMA antigen to a dialysis bag and replace it with dialysis solution; after dialysis, add biotin and incubate at room temperature in the dark for 4 hours; use PBS solution at 4°C for dialysis for 2 hours, replace three times, collect the liquid in the dialysis bag, and dilute for later use.

[0038] The sensitivity of the competitive method combined with photo-induced chemiluminescence detection method used in this application was compared with that of the existing SDMA detection method. The results are shown in Table 1 below: Table 1. Comparison of the sensitivity of the detection results of the detection method in this application and existing detection methods.

[0039] Therefore, it can be seen that the detection method of this application has higher luminescence efficiency at all concentrations compared with the prior art, and can effectively improve the sensitivity of low values ​​of samples.

[0040] The precision of the competitive method combined with photo-induced chemiluminescence detection method used in this application was compared with that of the existing SDMA detection method. The results are shown in Table 2 below: Table 2 Comparison of the precision of the detection results of the detection method in this application and existing detection methods

[0041] Therefore, the detection method of this application has higher precision than the prior art and can effectively improve the repeatability of low values ​​in samples.

[0042] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A microfluidic chip for SDMA photochemiluminescence detection, characterized in that, include: A fan-shaped substrate (1) is provided with a sample introduction unit and a diluent unit; The mixing tank (2) is located on the side of the diluent unit away from the center of the fan-shaped substrate (1) and is connected to the sample introduction unit and the diluent unit respectively. The mixing tank (2) is used to mix the sample to be tested and the diluent. The detection unit comprises multiple detection units located on the side of the mixing tank (2) away from the center of the fan-shaped substrate (1) and distributed circumferentially along the fan-shaped substrate (1). Each detection unit includes a first reaction tank (3) and a second reaction tank (4) arranged radially along the fan-shaped substrate (1) and connected to it. Multiple first reaction tanks (3) are connected to the mixing tank (2). The first reaction tank (3) contains a first complex containing SDMA antibody and a second complex containing biotin-labeled SDMA antigen. The second reaction tank (4) contains a third complex containing streptavidin. The first reaction tank (3) is used for competitive reaction, where SDMA in the sample to be tested competes fully with biotin-labeled SDMA antigen, completely eliminating the possibility of false signals due to non-specific effects or premature binding. The second reaction tank (4) is used for signal generation reaction, ensuring that only the result of specific competition can be converted into light signal, which greatly improves the signal-to-noise ratio.

2. The microfluidic chip for SDMA photochemiluminescence detection according to claim 1, characterized in that, A fluid shut-off valve (5) is provided between the first reaction tank (3) and the second reaction tank (4). The fluid shut-off valve (5) is configured such that when the pressure on the fluid shut-off valve (5) reaches a threshold, the fluid shut-off valve (5) opens, and the first reaction tank (3) connects to the second reaction tank (4).

3. A microfluidic chip for SDMA photochemiluminescence detection according to claim 1, characterized in that, The sample introduction unit includes a sample loading groove (6), a sample quantification groove (7), and a sample flow channel (8). The sample loading groove (6) and the sample quantification groove (7) are arranged and connected along the radial direction of the fan-shaped substrate (1). The sample quantification groove (7) is connected to the mixing groove (2) through the sample flow channel (8).

4. A microfluidic chip for SDMA photochemiluminescence detection according to claim 3, characterized in that, The diluent unit includes a diluent storage tank (9), a diluent metering tank (10), and a diluent flow channel (11). The diluent storage tank (9) is provided with a diluent bladder. The diluent storage tank (9), the diluent metering tank (10), the diluent flow channel (11), and the mixing tank (2) are connected in sequence.

5. A microfluidic chip for SDMA photochemiluminescence detection according to claim 4, characterized in that, An annular groove (13) is provided between the multiple detection units and the mixing tank (2). The mixing tank (2) is connected to the annular groove (13) through the mixing channel (12). The annular groove (13) is connected to each of the first reaction tanks (3).

6. A microfluidic chip for SDMA photochemiluminescence detection according to claim 3, characterized in that, The sample quantification groove (7) is provided with a separation groove (14) on the side away from the center of the fan-shaped substrate (1), and the separation groove (14) is connected to the sample quantification groove (7).

7. A microfluidic chip for SDMA photochemiluminescence detection according to claim 5, characterized in that, The inner surfaces of the sample channel (8), the diluent channel (11), and the mixing channel (12) are hydrophilic to allow the liquid to fill the channels by relying on surface tension.

8. A method of using the microfluidic chip for SDMA photochemiluminescence detection as described in claim 7, characterized in that, include: The sample to be tested is added to the sample loading slot (6), and the microfluidic chip is fixed on the detection platform; Start the drive motor to make the microfluidic chip rotate at a first set speed for a first set time so that the sample to be tested enters the sample quantitative tank (7) and the diluent enters the diluent quantitative tank (10). The microfluidic chip is rotated at a first set speed for a first set time so that the sample to be tested and the diluent enter the mixing tank (2) for mixing to form the test mixture; The microfluidic chip is rotated at a second set speed for a second set time so that the mixture to be tested enters the first reaction tank (3). The mixture to be tested reacts with the first complex and the second complex to form the first reactant. The microfluidic chip is rotated at a third set speed for a third set time so that the fluid shut-off valve (5) is opened, the first reactant enters the second reaction tank (4), and the first reactant reacts with the third complex to form the second reactant; the third set speed is greater than the second set speed. The second reactant is irradiated with a laser, and the chemiluminescence signal generated by the excited second reactant is detected.

9. A microfluidic chip for SDMA photochemiluminescence detection according to claim 8, characterized in that, The first set speed ranges from 2000 rpm to 6000 rpm, and the first set time ranges from 10 s to 300 s; the second set speed ranges from 200 rpm to 2000 rpm, and the second set time ranges from 30 s to 300 s; the third set speed ranges from 3000 rpm to 6000 rpm, and the third set time ranges from 20 s to 200 s.

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