A microfluidic chip for latex immunoturbidimetric detection and its usage method

Abstract

This invention relates to the field of microfluidic chip technology and provides a microfluidic chip for latex immunoturbidimetric detection and its usage method. The method is as follows: A sample is added, the chip is placed on the detection platform, the diluent sac is opened, and the motor is started to rotate, allowing the sample to enter the sample quantitative sac and the diluent to enter the diluent quantitative sac. The motor stops rotating, and siphon channels one and two are opened. The motor starts rotating again, allowing the sample in the sample quantitative sac to enter the mixing sac, and the diluent in the diluent quantitative sac to enter the mixing sac, where the two are diluted and mixed. The motor stops rotating, siphon channel three is opened, and the sample mixture sequentially enters detection well one and detection well two. This invention enables the detection of the tested sample on a microfluidic chip, requiring only a fraction of the sample volume and reaction reagent volume of traditional large-scale biochemical assays, significantly shortening the detection time. Furthermore, it can improve both the reactivity of low values ​​and the resistance to hooking.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, and particularly relates to a microfluidic chip for latex immunoturbidimetric detection and its usage method. Background Technology

[0002] Latex immunoturbidimetry is a relatively stable and accurate immunoassay method that has emerged in recent years. Its principle involves cross-linking specific antibodies onto the surface of latex microspheres. When the antibodies on the cross-linked microspheres specifically bind to the antigen, they rapidly aggregate, altering the turbidity of the reaction solution. This change in turbidity is strongly correlated with the concentration of the antigen being tested in the sample, reflecting the antigen concentration within a certain range. Currently, latex immunoturbidimetry is primarily performed using large-scale biochemical analyzers, which leads to drawbacks such as high sample consumption, complex procedures, and high instrument costs. Furthermore, it often requires a high-level laboratory environment and highly skilled technicians, significantly hindering the widespread adoption and application of timely and rapid detection. Additionally, in clinical applications, high levels of the target protein in the blood of some animals can cause a hook effect, easily leading to false negatives and ultimately misdiagnosis by physicians. The hook effect is usually avoided or delayed by serially diluting samples or reducing the amount of coagulant. However, this can lead to a decrease in the reactivity of low-value samples, which in turn reduces the precision of low-value detection and can easily affect the doctor's judgment. Therefore, developing a rapid, portable, intuitive, and easy-to-operate miniature detection system has become a key research focus both domestically and internationally.

[0003] Microfluidic chip technology is a new technology that precisely manipulates and controls nanoliter and picoliter-level fluids (biological sample fluids) in micrometer-scale channels. This technology allows for the integration or near-integration of basic operational units involved in sample preparation, reaction, separation, detection, cell culture, sorting, and lysis from fields such as chemistry and biology onto a chip of a few square centimeters (or even smaller). A network of microchannels forms the core, enabling controllable fluid flow throughout the system, serving as a technological platform to replace various functions in conventional chemical or biological laboratories. The fundamental characteristic and greatest advantage of microfluidic chip laboratories is the flexible combination and large-scale integration of multiple unit technologies on a controllable micro-platform. Summary of the Invention

[0004] The purpose of this invention is to provide a microfluidic chip for latex immunoturbidimetric detection and its usage method, aiming to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for using a microfluidic chip for latex immunoturbidimetric detection, the method being based on a microfluidic chip for latex immunoturbidimetric detection, the microfluidic chip for latex immunoturbidimetric detection comprising a chip, wherein the chip is provided radially along its center with a diluent storage tank, a sample loading tank, a diluent transition tank, a sample quantification tank, a diluent quantification tank, a mixing tank and a detection port, and the chip is provided with a microchannel one, a microchannel two, a siphon channel one, a siphon channel two and a siphon channel three;

[0007] The diluent storage tank is connected to the diluent transition tank via microchannel one; the sample addition tank is connected to the sample quantitative tank via microchannel two; the diluent quantitative tank is connected to the mixing tank via siphon channel one; the sample quantitative tank is connected to the mixing tank via siphon channel two; and the mixing tank is connected to the detection orifice via siphon channel three.

[0008] The detection holes include detection hole one, detection hole two, detection hole three, and detection hole four;

[0009] The detection well contains a lyophilized reagent, which is an antigen detection reagent and consists of two lyophilized microspheres, R1 and R2.

[0010] The detection well two contains lyophilized reagent two, which is another antigen detection reagent, and consists of two lyophilized microspheres R1 and R3.

[0011] The usage method includes the following steps:

[0012] Step S1: Add the test sample into the sample loading tank, place the pre-packaged diluent sac in the diluent storage tank, place the chip on the detection platform and fix it, open the diluent sac, start the motor on the detection platform to rotate, at this time the sample enters the sample quantification tank to complete the sample quantification, and the diluent enters the diluent quantification tank to complete the quantification.

[0013] Step S2: The motor stops rotating, and siphon channel one and siphon channel two are connected; the motor starts rotating, and the sample in the sample quantitative cell enters the mixing tank through siphon channel two, and the diluent in the diluent quantitative cell enters the mixing tank through siphon channel one.

[0014] Step S3: The sample and diluent are diluted and mixed in a mixing tank to obtain a sample mixture;

[0015] Step S4: The motor stops rotating, the siphon channel three is opened, and the sample mixture enters the detection hole one and detection hole two in sequence through the siphon channel three.

[0016] Furthermore, the components of R1 include Tris buffer, sodium chloride, guanidine isothiocyanate, and Tween-20.

[0017] Furthermore, the components of R2 include latex microspheres cross-linked with specific antibodies, bovine serum albumin, and sucrose.

[0018] Furthermore, the components of R3 include latex microspheres cross-linked with specific antibodies, free antibodies, bovine serum albumin, and sucrose.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] The microfluidic chip provided by this invention has a compact and simple structure and reliable performance. It realizes the detection of the sample to be tested on the microfluidic chip. The required sample volume and reaction reagent volume are only a fraction of those of traditional large-scale biochemical detection, which greatly shortens the detection time. Furthermore, the use of high-concentration antigen-assisted detection wells can improve both the reactivity of low values ​​and the anti-hooking ability. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the microfluidic chip in this invention.

[0022] Figure 2 The "concentration-absorbance" calibration curves were prepared using six CRP calibrators of different concentrations in Example 1 of this invention.

[0023] Figure 3 The "concentration-absorbance" calibration curves were prepared using four CRP calibrators of different concentrations in Example 1 of this invention.

[0024] Figure 4 This is a single-well detection calibration curve established by directly adding free antibody to detection well one in Embodiment 1 of the present invention.

[0025] In the diagram: Chip 01, Diluent Storage Tank 11, Diluent Transition Tank 12, Diluent Quantitative Tank 13, Sample Addition Tank 21, Sample Quantitative Tank 22, Mixing Tank 03, Detection Hole 1 41, Detection Hole 2 42, Detection Hole 3 43, Detection Hole 4 44, Microchannel 1 51, Microchannel 2 52, Suction Channel 1 53, Siphon Channel 2 54, Siphon Channel 3 55. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0028] like Figure 1As shown, a microfluidic chip for latex immunoturbidimetric detection is provided in one embodiment of the present invention. The chip includes a chip 01, on which, along a radial direction from the center, are arranged a diluent storage tank 11, a sample loading tank 21, a diluent transition tank 12, a sample quantification tank 22, a diluent quantification tank 13, a mixing tank 03, and a detection hole. The chip 01 also has a first microchannel 51, a second microchannel 52 (the width of the microchannel is 0.01-1 mm, preferably 0.05-0.3 mm; the depth of the microchannel is 0.01-1 mm, preferably 0.05-0.3 mm), a first siphon channel 53, a second siphon channel 54, and a third siphon channel 55.

[0029] The diluent storage tank 11 is connected to the diluent transition tank 12 via microchannel 1 51; the sample addition tank 21 is connected to the sample quantification tank 22 via microchannel 2 52; the diluent quantification tank 13 is connected to the mixing tank 03 via siphon channel 1 53; the sample quantification tank 22 is connected to the mixing tank 03 via siphon channel 2 54; and the mixing tank 03 is connected to the detection port via siphon channel 3 55.

[0030] In this embodiment of the invention, chip 01 has a fan-shaped structure. This structure design allows multiple fan-shaped chips 01 to be freely combined on the detection platform during a single detection process. Since one chip 01 can detect one sample, it is possible to achieve simultaneous detection of multiple samples and multiple detection items in a single detection.

[0031] Since the diluent bladder placed in the diluent storage tank 11 generally stores a large volume of diluent, the structure of adding a transition tank in the middle can control the flow rate of the fluid entering the diluent metering tank 13, and avoid the generation of air bubbles in the diluent during the metering process, which would lead to inaccurate metering volume.

[0032] In a preferred embodiment of the present invention, the detection holes include a first detection hole 41, a second detection hole 42, a third detection hole 43, and a fourth detection hole 44;

[0033] The detection well 41 contains a lyophilized reagent, which is an antigen detection reagent and consists of two lyophilized microspheres R1 and R2.

[0034] The detection well 42 contains a second lyophilized reagent, which is another antigen detection reagent composed of two lyophilized microspheres R1 and R3.

[0035] In a preferred embodiment of the present invention, the main components of R1 are Tris (tris(hydroxymethyl)aminomethane) buffer, sodium chloride, guanidine isothiocyanate, TWEEN-20, etc., and also include excipients and plasticizers, including but not limited to sugars, amino acids, proteins, polyols, surfactants, polymers, etc.

[0036] In this embodiment of the invention, the preparation method of R1 is as follows:

[0037] R1 is prepared from 50 mM Tris, guanidine isothiocyanate, sodium chloride, and Tween-20.

[0038] In a preferred embodiment of the present invention, the main components of R2 are latex microspheres cross-linked with specific antibodies, bovine serum albumin (BSA), and sucrose, including but not limited to sugars, amino acids, proteins, polyols, surfactants, and polymers.

[0039] In this embodiment of the invention, the preparation method of R2 is as follows:

[0040] 1) Dilute the surface carboxylated polystyrene latex particles (80nm) with 50mM pH 6.0 MES buffer to 5ml, with a final concentration of 2.5%;

[0041] 2) Add 0.25 mg of EDC to the above solution, activate for 30 min, add 0.6 mg of canine CRP antibody, and mix at room temperature for 1.5 h;

[0042] 3) Add 100 μL of 1% BSA to the above solution and react for 0.5 h to seal the excess active groups on the surface of the latex particles;

[0043] 4) Centrifuge at 12000 rpm for 10 min and remove the supernatant;

[0044] 5) Dilute the labeled CRP antibody latex particles to the working concentration using R2 storage buffer.

[0045] R2 storage buffer is prepared from 80 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 4 mg / mL BSA and 351 mM sucrose.

[0046] In a preferred embodiment of the present invention, the main components of R3 are latex microspheres cross-linked with specific antibodies, free antibodies, BSA and sucrose, etc., and also include related excipients and plasticizers, including but not limited to sugars, amino acids, proteins, polyols, surfactants, polymers, etc.

[0047] In this embodiment of the invention, the preparation method of R3 is as follows:

[0048] Adding 0.4 mg / mL of free antibody to R2 results in reagent R3.

[0049] An embodiment of the present invention provides a method for using a microfluidic chip for latex immunoturbidimetric detection as described above, comprising the following steps:

[0050] Step S1: Add the test sample (whole blood / serum or plasma) into the sample loading tank 21. Place the pre-packaged diluent capsule in the diluent storage tank 11. Place the chip 01 on the detection platform and fix it. The diluent capsule opens automatically (see patent CN201910313138.X for the opening method). The motor on the detection platform starts to rotate. At this time, the sample enters the sample quantification tank 22 to complete the sample quantification. The diluent enters the diluent quantification tank 13 to complete the quantification. At this time, the rotation time of the motor can be set to 10-300s, preferably 30-200s.

[0051] Step S2: The motor stops rotating, and siphon channels 1 (53) and siphon channels 2 (54) are opened (siphon channels 1 (53) and siphon channels 2 (54) can be treated with hydrophilic surfaces, including but not limited to oxygen plasma surface treatment, ozone radiation treatment, surface activator treatment, graft copolymerization treatment, etc., to make the surface of the siphon channels hydrophilic. At this time, the motor stops rotating, and the hydrophilic channels are automatically wetted and opened due to surface tension). The motor starts rotating, and the sample in the sample metering tank 22 enters the mixing tank 03 through siphon channel 2 (54), and the diluent in the diluent metering tank 13 enters the mixing tank 03 through siphon channel 1 (53).

[0052] Step S3: The sample and diluent are diluted and mixed in mixing tank 03 to obtain a sample mixture; at this time, the motor can change its acceleration and deceleration to generate oscillation, which promotes the dilution and mixing of the sample.

[0053] Step S4: The motor stops rotating, the siphon channel 3 55 is opened, and the sample mixture enters the detection well 1 41 and detection well 2 42 sequentially through the siphon channel 3 55 (the liquid enters the detection well 1 41 and detection well 2 42 at a time under the action of centrifugal force). The motor can accelerate or decelerate or reverse, thereby generating oscillation and promoting the reaction between the sample and the reagent in the detection well.

[0054] After the reaction is complete, the optical module on the detection platform detects the detection wells, and the photoelectric conversion module converts the optical signals within the detection wells into digital signals. The absorbance of detection well 41 is A1, and the absorbance of detection well 42 is A2. Then, the sample concentration is calculated using a calibration curve of absorbance versus concentration. This dual-well detection method, with detection well 41 detecting low values, ensures the precision of low-value detection items. The addition of uncrosslinked antibody to detection well 42 provides better linearity and precision, effectively reducing false negatives caused by the hook effect and meeting clinical application requirements.

[0055] Example 1: C-reactive protein (CRP) is an acute protein synthesized by liver cells when an animal is infected by pathogenic microorganisms or suffers tissue damage. CRP is a major acute reaction phase protein in dogs, characterized by rapid synthesis and rapid decline. In healthy dogs, serum CRP levels are far below 10 mg / L, rising within 4-6 hours after inflammatory stimulation, reaching a peak at 48 hours, at which point serum CRP concentration is approximately 100-1000 times higher than normal. When systemic inflammation subsides, CRP concentration decreases by 50% within 24 hours. Therefore, canine CRP is highly sensitive to disease and can promptly reflect the condition.

[0056] An embodiment of the present invention provides a microfluidic chip for canine CRP detection and a method for using the same, the detection steps including:

[0057] Establishment of the dual-hole detection calibration curve:

[0058] Six different CRP concentrations (65 mg / L, 40 mg / L, 20 mg / L, 10 mg / L, 5 mg / L, and 0 mg / L) were used to calibrate well 41 in a reagent tray, with a primary wavelength of 600 nm and a secondary wavelength of 800 nm as the measurement wavelength. A concentration-absorbance calibration curve was created by plotting the absorbance of each calibration tube on the ordinate and the corresponding concentration on the abscissa. Figure 2 As shown, well 42 was calibrated using four different concentrations of CRP calibrators (65 mg / L, 110 mg / L, 155 mg / L, and 200 mg / L) in a reagent tray, with a primary wavelength of 600 nm and a secondary wavelength of 800 nm as the measurement wavelength. A concentration-absorbance calibration curve was created by plotting the absorbance of each calibration tube on the ordinate and the corresponding concentration on the abscissa. Figure 3 As shown.

[0059] Repeatability assessment:

[0060] The same batch of reagents was used to perform 10 repeated determinations at target values ​​of 5 mg / L and 10 mg / L. The coefficient of variation (CV) for 5 mg / L and 10 mg / L was <3%. The results are shown in Table 1.

[0061] Table 1. Results of Repeatability Assessment

[0062]

[0063] If free antibody is directly added to well 41 to establish a single-well detection calibration curve, the result is as follows: Figure 4 As shown in Table 2, although the high linear value increased to 100 mg / L, the linear variation decreased from 0.9991 to 0.981, the precision CV increased, and the detection of low values ​​was inaccurate.

[0064] Table 2. Repeatability Assessment Results

[0065]

[0066]

[0067] Therefore, the method of using a dual-well detection, where well 41 detects low values ​​and well 42 contains uncrosslinked resistance, exhibits good linearity and precision, meeting the needs of clinical use.

[0068] Three actual serum samples with known concentrations were measured using both two-well and single-well detection methods. The results are shown in Table 3. The two-well detection results are more accurate. Due to the hook effect, the single-well detection may mistake inflammatory blood samples for normal blood samples, which could lead to misdiagnosis by doctors.

[0069] Table 3. Results of Replacing Sample Testing

[0070]

[0071] The microfluidic chip for canine CRP detection and its usage method proposed in this invention have a simple structure and are easy to implement. Detection well 41 can detect low concentrations of canine CRP, and detection well 42 can detect high concentrations of canine CRP by adding uncrosslinked CRP polyclonal antibody, which can improve both the reactivity of low values ​​and the anti-HOOK ability.

[0072] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for using a microfluidic chip for detecting canine C-reactive protein by latex immunoturbidimetric assay, characterized in that, The method of use is based on a microfluidic chip for latex immunoturbidimetric detection. The microfluidic chip for latex immunoturbidimetric detection includes a chip, on which diluent storage tank, sample loading tank, diluent transition tank, sample quantification tank, diluent quantification tank, mixing tank and detection hole are arranged in sequence along the radial direction from the center. The chip is provided with microchannel one, microchannel two, siphon channel one, siphon channel two and siphon channel three. The diluent storage tank is connected to the diluent transition tank via microchannel one; the sample addition tank is connected to the sample quantitative tank via microchannel two; the diluent quantitative tank is connected to the mixing tank via siphon channel one; the sample quantitative tank is connected to the mixing tank via siphon channel two; and the mixing tank is connected to the detection orifice via siphon channel three. The detection holes include detection hole one, detection hole two, detection hole three, and detection hole four; The detection well contains a lyophilized reagent, which is an antigen detection reagent and consists of two lyophilized microspheres, R1 and R2. The detection well two contains lyophilized reagent two, which is another antigen detection reagent, and consists of two lyophilized microspheres R1 and R3. The components of R1 include Tris buffer, sodium chloride, guanidine isothiocyanate and Tween-20; The components of R2 include latex microspheres cross-linked with specific antibodies, bovine serum albumin, and sucrose; The components of R3 include latex microspheres cross-linked with specific antibodies, free antibodies, bovine serum albumin, and sucrose; The usage method includes the following steps: Step S1: Add the test sample into the sample loading tank, place the pre-packaged diluent sac in the diluent storage tank, place the chip on the detection platform and fix it, open the diluent sac, start the motor on the detection platform to rotate, at this time the sample enters the sample quantification tank to complete the sample quantification, and the diluent enters the diluent quantification tank to complete the quantification. Step S2: The motor stops rotating, and siphon channel one and siphon channel two are connected; the motor starts rotating, and the sample in the sample quantitative cell enters the mixing tank through siphon channel two, and the diluent in the diluent quantitative cell enters the mixing tank through siphon channel one. Step S3: The sample and diluent are diluted and mixed in a mixing tank to obtain a sample mixture; Step S4: The motor stops rotating, the siphon channel three is opened, and the sample mixture enters the detection hole one and detection hole two in sequence through the siphon channel three.

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