Split type reagent disc, immunity analyzer and detection control method

Abstract

The invention provides a split type reagent disc, an immunity analyzer and a detection control method, and relates to the technical field of chemiluminescence analys.The reagent disc comprises a reagent disc body, the reagent disc body is provided with a mixing cavity, and the reagent disc body is further provided with a flow channel communicated with the mixing cavity; a plurality of reaction units are further arranged on the reagent disc body, and each reaction unit comprises a first detection cavity and a second detection cavity; the first detection cavity is communicated with the flow channel, the first detection cavity is communicated with the second detection cavity, and a reagent in the mixing cavity sequentially flows through the first detection cavity and the second detection cavity after entering the flow channel; a first freeze-drying reagent bead, a second freeze-drying reagent bead and / or a third freeze-drying reagent bead are respectively arranged in the first detection cavity and the second detection cavity. The technical problems of uncontrollable reaction and low detection result accuracy in the prior art can be effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and in particular to a split reagent disk, an immunoanalyzer and a detection control method. Background Art

[0002] Chemiluminescence is a light radiation phenomenon that accompanies a substance during a chemical reaction. It can be divided into direct luminescence and indirect luminescence. Direct luminescence is the simplest chemiluminescence reaction, which consists of two key steps: excitation and radiation. For example, when substances A and B undergo a chemical reaction to generate substance C, the energy released by the reaction is absorbed by the molecules of substance C and transitions to the excited state C. * , in the excited C * Light radiation is generated in the process of returning to the ground state. Here C* is the luminophore, and since C directly participates in the reaction, it is called direct chemiluminescence.

[0003] The invention with application number: CN202311491814.5 discloses a microfluidic fan-shaped biochemical reagent disc assembly and a method of using the same. The specification discloses a reagent disc, including a disc base with an overall fan-shaped structure and an adhesive film. The disc base is provided with a sample addition chamber, a buffer chamber, a sample quantitative chamber and a mixing chamber connected in sequence through capillary channels. A placement chamber and a liquid quantitative chamber are provided on the disc base near the sample addition chamber. The placement chamber is used to place a water cup. The placement chamber is connected to the liquid quantitative chamber, and the liquid quantitative chamber is connected to the mixing chamber. An annular flow channel is provided on the disc base near the edge of the disc base, and a plurality of detection holes connected to the annular flow channel are also provided on the disc base.

[0004] The invention patent with publication number: CN116731840A discloses a centrifugal bioreactor chip and a biodetection method, wherein the centrifugal bioreactor chip includes a chip body and a sealing structure, the chip body is provided with a reaction structure and a ventilation structure, and the ventilation structure is connected to the reaction structure to make the reaction structure self-ventilated. The reaction structure includes a sample storage chamber, a reaction liquid storage chamber, a mixing chamber, a pre-amplification chamber and a detection chamber; the sample storage chamber is provided with a sample addition port, the reaction liquid storage chamber is provided with a liquid addition port, the distal end outlet of the sample storage chamber is connected to the pre-amplification chamber inlet, the distal end outlet of the reaction liquid storage chamber is connected to the pre-amplification chamber inlet through the mixing chamber, and the pre-amplification chamber outlet is connected to the detection chamber; the sample storage chamber, the mixing chamber and the distribution chamber are all connected to the ventilation structure. In the centrifugal bioreactor chip, the first step amplification and the second step amplification are both performed in the same reaction chip, without the need for liquid transfer, which solves the system sealing and operation automation required for two-step nucleic acid amplification.

[0005] The invention patent with announcement number: CN112391280B discloses a bioreactor chip and a bioreactor device, wherein the bioreactor chip includes: a chip body provided with a reaction structure and a ventilation structure, and a sealing body; wherein the reaction structure includes: a liquid adding port, a reaction liquid storage chamber connected to the liquid adding port, a sample adding port, a preamplification chamber connected to the sample adding port, a premixing chamber that can be connected to the reaction liquid storage chamber and the preamplification chamber, a distribution channel that can be connected to the premixing chamber, and at least two reaction units that are both connected to the distribution channel; the ventilation structure is connected to the reaction structure to make the reaction structure self-ventilated; and the sealing body can seal the reaction structure and the ventilation structure. The above-mentioned bioreactor chip improves the detection sensitivity while meeting the requirements of multi-index detection.

[0006] In actual use, the sample in the mixing chamber is mixed with the reagent and then directly enters the detection hole for reaction. Since the immune reaction between antigen and antibody involves at least three substances, whether it is a sandwich method or a competitive method, although the one-step reaction of mixing the three together can use a simpler structure to get results faster, all reactions occur in the same detection hole, resulting in an uncontrollable order in the detection hole. The method in the prior art will detect, and the detection accuracy is not high. Summary of the invention

[0007] The object of the present invention is to provide a split reagent disk, an immunoanalyzer and a detection control method, which can effectively solve the technical problems of uncontrollable reactions and low accuracy of detection results in the prior art.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is: A split reagent disc comprises a reagent disc body, a mixing chamber is arranged on the reagent disc body, and a flow channel communicating with the mixing chamber is also arranged on the reagent disc body; Among them, the reagent disc body is also provided with a plurality of reaction units, and the reaction units include a first detection cavity and a second detection cavity; The first detection chamber is communicated with the flow channel, the first detection chamber and the second detection chamber are communicated with each other, and the reagent in the mixing chamber flows into the flow channel and then sequentially passes through the first detection chamber and the second detection chamber.

[0009] In one embodiment disclosed in the present invention, the first detection chamber and the second detection chamber are respectively provided with the first freeze-dried reagent beads, the second freeze-dried reagent beads and / or the third freeze-dried reagent beads.

[0010] In one embodiment disclosed in the present invention, the flow channel is located on one side of the reagent disc body, and the mixing chamber is located on the other side of the reagent disc body.

[0011] In one embodiment disclosed in the present invention, a through hole is provided on the reagent disc body, and sealing films are provided on both sides of the reagent disc body for sealing the through hole to form the first detection cavity and the second detection cavity.

[0012] In one embodiment disclosed in the present invention, the sealing film is a light-transmitting sealing film.

[0013] In one embodiment disclosed in the present invention, the first freeze-dried reagent beads are oxygen acceptor microsphere freeze-dried reagent beads coated with monoclonal antibodies, the second freeze-dried reagent beads are biotin-labeled specific antigen microsphere freeze-dried reagent beads, and the third freeze-dried reagent beads are donor microsphere freeze-dried reagent beads coated with streptavidin.

[0014] In one embodiment disclosed in the present invention, the mixing chamber is connected to the flow channel via a U-shaped microchannel.

[0015] In one embodiment disclosed in the present invention, the first detection chamber and the second detection chamber are connected via a "J"-shaped microchannel.

[0016] In an embodiment disclosed in the present invention, the flow channel is an overall arc-shaped structure.

[0017] In one embodiment disclosed in the present invention, the distance from the rotation center of the reagent disk body to the outer side wall of the flow channel increases from left to right.

[0018] In one embodiment disclosed in the present invention, the length of the connection channel between the first detection cavity and the flow channel decreases from left to right.

[0019] In one embodiment disclosed in the present invention, the distance from the rotation center of the reagent disk body to the inner wall of the flow channel decreases from left to right.

[0020] In one embodiment disclosed in the present invention, the lengths of the connecting channels between the first detection chamber and the flow channel are the same.

[0021] In one embodiment disclosed in the present invention, the connecting channel is a straight connecting channel or an arc-shaped connecting channel, one end of the arc-shaped connecting channel is tangent to the flow channel, and the other end is located at the position of the first detection cavity closest to the flow channel.

[0022] In addition, the present invention also discloses an immunoassay analyzer, comprising an analyzer body and a reagent disk, wherein the reagent disk is the split reagent disk described above.

[0023] The present invention also discloses a detection control method, which includes using the above-mentioned immunoassay analyzer, and specifically comprises the following steps: Step 1: Add the whole blood sample into the sample adding cavity of the reagent disk body; Step 2: Start the first centrifugation, the centrifugal speed is 4000rpm-6000rpm, and the centrifugal time is 30 seconds-60 seconds; during the first centrifugation, the whole blood sample enters the sample buffer chamber of the reagent disc body under the action of centrifugal force for separation, the plasma enters the sample quantitative chamber of the reagent disc body, the excess plasma enters the waste liquid chamber of the reagent disc body, and the blood cells are blocked in the blood cell collection chamber of the reagent disc body; At the same time, the water cup placed in the diluent placement cavity of the reagent disc body is pierced by the piercing structure, and the diluent flows out synchronously, and enters the diluent quantitative cavity of the reagent disc body under the action of centrifugal force to complete the quantitative determination; Step 3: The plasma and diluent enter the mixing chamber through various capillaries; In the second high and low speed of 1000rpm-5000rpm, the time is 30 seconds-60 seconds, and the mixed sample is fully mixed under the action of cross centrifugation; Step 4: Start the third centrifugation, the centrifugal speed is 4000rpm-6000rpm, and the centrifugal time is 30 seconds-60 seconds; During the third centrifugation, the mixed sample flows into the flow channel through the capillary; Step 5: Two-step reaction: 1) After the mixed sample enters the flow channel, it enters the first detection chamber in turn and undergoes a reaction in the first detection chamber; 2) After the reaction is completed, the sample enters the second detection chamber for a secondary reaction, achieving the purpose of step-by-step reaction.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The reagent disc of the present invention is mainly used for being installed on a reagent disc seat, and is used for being placed in a detection device for analysis and detection. After the sample is mixed with the diluent, it flows into the flow channel through the mixing chamber on the front side of the reagent disc, enters the first detection chamber for the first reaction, dissolves the oxygen acceptor microspheres (first freeze-dried reagent beads) coated with monoclonal antibodies, mixes and incubates, and on the basis of completing the first step reaction, the reaction-completed mixed solution is centrifuged to reach the second detection chamber, dissolves the second freeze-dried reagent beads (biotin-labeled specific antigens) and / or the third reagent beads (donor microspheres coated with streptavidin); the purpose of distributed reaction is achieved, independent reactions are carried out in the first detection chamber and the second detection chamber, the controllability of the reaction system is improved, so as to ensure that the reactions in the first detection chamber and the second detection chamber are normal and fully reacted, and the accuracy of the detection results is improved.

[0025] The distance from the rotation center of the reagent disk body of the present invention to the outer wall of the flow channel increases from left to right. That is, the radius of curvature of the outer wall line gradually increases, which enables the mixed sample to move from the entrance to the exit of the flow channel without remaining on the outer wall surface. At the same time, the length of the connecting channel between the first detection cavity and the flow channel decreases from left to right. This arrangement increases the liquid flow resistance at the entrance and reduces the liquid flow resistance at the exit, which helps to balance the liquid flow resistance of different connecting channels, thereby realizing "same-process transportation of mixed liquids" and avoiding poor circulation and low circulation efficiency due to uneven distribution of liquid resistance concentrated in a certain area.

[0026] The distance from the rotation center of the reagent disk body to the inner wall of the flow channel decreases from left to right. That is, the radius of curvature of the inner wall line gradually decreases; in actual use, the liquid resistance near the inner wall will increase, forcing the liquid to flow to the outer wall where the centrifugal force is larger. This method is conducive to exhaust. From left to right, the exhaust will become smoother and smoother, improving the exhaust effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 It is a schematic diagram of the overall structure of the front side of the present invention.

[0029] Figure 2 It is a schematic diagram of the overall structure of the back side of the present invention.

[0030] Figure 3 This is a schematic diagram of the flow channel structure in Example 2 of the present invention.

[0031] Figure 4 This is one of the schematic diagrams of the connection relationship between the arc-shaped connecting channel and the flow channel of the present invention.

[0032] Figure 5 This is the second schematic diagram of the connection relationship between the arc-shaped connecting channel and the flow channel of the present invention.

[0033] Reference numerals: 101 reagent disc body, 102 mixing chamber, 103 flow channel, 104 reaction unit, 105 first detection chamber, 106 second detection chamber, 107 U-shaped microfluidic channel, 108 "J"-shaped microfluidic channel, 109 straight connecting channel, 110 arc-shaped connecting channel, 111 first filtering unit, 112 second filtering unit, 113 sample adding chamber, 114 sample buffer chamber, 115 sample quantification chamber, 116 microfluidic channel inlet. DETAILED DESCRIPTION

[0034] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0035] In the description of the embodiments of the present invention, it should be understood that the terms "length", "vertical", "horizontal", "top", "bottom", etc. indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present invention.

[0036] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0037] In the embodiments of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or a communication; it can be a direct connection, or an indirect connection through an intermediate medium, and it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0038] In the embodiments of the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes the first feature being directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes the first feature being directly above and obliquely above the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0039] The disclosure below provides many different embodiments or examples to implement different structures of the embodiments of the present invention. In order to simplify the disclosure of the embodiments of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the embodiments of the present invention. In addition, the embodiments of the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed.

[0040] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0041] Embodiment 1: This embodiment discloses a split reagent disc, including a reagent disc body 101, a mixing chamber 102 is provided on the reagent disc body 101, and a flow channel 103 communicating with the mixing chamber 102 is also provided on the reagent disc body 101; The reagent disc body 101 is also provided with a plurality of reaction units 104, and the reaction units 104 include a first detection chamber 105 and a second detection chamber 106; The first detection chamber 105 is connected to the flow channel 103 , and the first detection chamber 105 and the second detection chamber 106 are connected to each other. After the reagent in the mixing chamber 102 enters the flow channel 103 , it flows through the first detection chamber 105 and the second detection chamber 106 in sequence.

[0042] Furthermore, the first detection chamber 105 and the second detection chamber 106 are respectively provided with the first freeze-dried reagent beads, the second freeze-dried reagent beads and / or the third freeze-dried reagent beads.

[0043] In actual use, the first freeze-dried reagent beads are oxygen acceptor microsphere freeze-dried reagent beads coated with monoclonal antibodies, the second freeze-dried reagent beads are biotin-labeled specific antigen microsphere freeze-dried reagent beads, and the third freeze-dried reagent beads are donor microsphere freeze-dried reagent beads coated with streptavidin.

[0044] The reagent disc of the present invention is mainly used to be installed on a reagent disc seat and to be placed in a detection device for analysis and detection. In the present application, after the sample is mixed with the diluent, it flows into the flow channel 103 through the mixing chamber 102 on the front of the reagent disc, enters the first detection chamber 105 for the first reaction, dissolves the oxygen acceptor microspheres (first freeze-dried reagent beads) coated with monoclonal antibodies, mixes and incubates, and on the basis of completing the first reaction, the reaction-completed mixed solution is centrifuged to reach the second detection chamber 106 to dissolve the second freeze-dried reagent beads (biotin-labeled specific antigens) and / or the third reagent beads (donor microspheres coated with streptavidin); the purpose of step-by-step reaction is achieved, independent reactions are carried out in the first detection chamber 105 and the second detection chamber 106, and the controllability of the reaction system is improved to ensure that the reactions in the first detection chamber 105 and the second detection chamber 106 are normal and fully reacted, thereby improving the accuracy of the detection results.

[0045] In the present invention, the biotin-labeled specific antigen competes with the specific antigen to be detected for binding to the specific antibody on the acceptor microsphere to form a complex of the "acceptor microsphere-antibody-antigen-antibody-biotin-streptavidin-donor microsphere" type. Under 680nm laser irradiation, the photosensitizer in the donor microsphere converts the oxygen molecules in the surrounding environment into singlet oxygen. Since the distance between the acceptor microsphere and the donor microsphere in the formed immune complex is less than 200nm, the singlet oxygen can diffuse to the acceptor microsphere and transfer energy to activate the luminescent substance in the acceptor microsphere, and finally generate a light signal at 615nm. The luminescence intensity is measured using a light detection device, and the measured specific antigen concentration is inversely proportional to the luminescence intensity.

[0046] Furthermore, the flow channel 103 is located on one side of the reagent disc body 101 , and the mixing chamber 102 is located on the other side of the reagent disc body 101 .

[0047] In this embodiment, a through hole is provided on the reagent disc body 101 , and sealing films are provided on both sides of the reagent disc body 101 for sealing the through hole to form the first detection cavity 105 and the second detection cavity 106 .

[0048] It is further defined that the sealing film is a light-transmitting sealing film, which can not only play a sealing effect, but also allow the detection light to be transmitted into the first detection cavity 105 and the second detection cavity 106. The reagent disk body is made of opaque material. The combination of these two materials can reduce the interference of ambient light while ensuring the accuracy of laser detection, thereby further improving the detection sensitivity and the accuracy of the detection results.

[0049] The mixing chamber 102 and the flow channel 103 are connected via a U-shaped microchannel 107. The highest point of the U-shaped microchannel 107 can prevent the liquid from overflowing prematurely during the mixing process of the mixing chamber 102, and provide the siphon force required for the next step of liquid flow.

[0050] Furthermore, the first detection chamber 105 and the second detection chamber 106 are connected via a "J"-shaped microfluidic channel 108, the highest point of which needs to be higher than the first detection chamber 105 to prevent the liquid from entering the second detection chamber 106 prematurely during the first reaction; when the first step of the reaction is completed, the siphon force in the "J"-shaped microfluidic channel 108 fills the entire microfluidic channel with liquid, making it easier to draw the liquid in the first detection chamber into the second detection chamber during subsequent high-speed centrifugation.

[0051] The flow channel 103 is an arc-shaped structure as a whole.

[0052] Embodiment 2: See also Figure 3 and Figure 4 This embodiment is further optimized on the basis of the embodiment 1. In this embodiment, the distance from the rotation center of the reagent disc body 101 to the outer wall of the flow channel 103 increases from left to right. That is, the radius of curvature of the outer wall line gradually increases. According to the centrifugal force formula (F = m * ω² * r), when the rotation speed is constant, the greater the radius of curvature r, the greater the centrifugal force F. The liquid at the outer wall has an acceleration along the tangent direction of the outer wall. Therefore, the mixed sample can move quickly from the inlet of the flow channel 103 to the outlet without remaining on the outer wall surface.

[0053] Furthermore, the length of the connecting channel between the first detection cavity 105 and the flow channel 103 decreases from left to right. This arrangement increases the liquid flow resistance at the inlet and reduces the liquid flow resistance at the outlet, which helps to balance the liquid flow resistance of different connecting channels, thereby achieving "mixed liquid transportation in the same way" and avoiding poor circulation and low circulation efficiency due to uneven distribution of liquid resistance concentrated in a certain area.

[0054] See also Figure 3 In this embodiment, the connecting channel is a straight connecting channel 109 .

[0055] Example 3 This embodiment is further optimized on the basis of the embodiment 1. In this embodiment, the distance from the rotation center of the reagent disk body 101 to the inner wall of the flow channel 103 decreases from left to right.

[0056] That is, the radius of curvature of the inner wall line gradually decreases. According to the centrifugal force formula (F = m * ω² * r), when the rotation speed is constant, the smaller the radius of curvature r, the smaller the centrifugal force F. The liquid resistance near the inner wall will increase, forcing the liquid to flow to the outer wall where the centrifugal force is larger. This method is conducive to exhaust, and the exhaust will become smoother and smoother from left to right.

[0057] Furthermore, the length of the connecting channel between the first detection chamber 105 and the flow channel 103 is the same. This arrangement helps the mixed samples to enter the detection chamber one by one and react one by one, thereby avoiding cross contamination between different samples.

[0058] In some preferred implementation cases, the connecting channel is a straight connecting channel 109. When the straight connecting channel 109 is used, exhaust can be performed while liquid is being introduced.

[0059] Example 4 See also Figure 4 or Figure 5 This embodiment is basically the same as Embodiment 2 or Embodiment 3, except that, in this embodiment, the connecting channel is an arc-shaped connecting channel 110, one end of the arc-shaped connecting channel 110 is tangent to the flow channel 103, and the other end is located at the position of the first detection cavity 105 closest to the flow channel 103. The connecting channel is designed to be arc-shaped and tangent to the outer wall line, which is conducive to reducing the liquid inlet resistance and improving the flow rate.

[0060] Meanwhile, the other end of the arc-shaped connecting channel 110 is located at the position of the first detection chamber 105 closest to the flow channel 103 to facilitate exhaustion.

[0061] As an option, in actual use, the lower end of the arc-shaped connecting channel 110 is tangent to the first detection chamber 105 . The arc-shaped connecting channel 110 is arranged in a tangent manner to further reduce the flow resistance.

[0062] When exhausting, air is exhausted from the exhaust passage connected to the second detection chamber 106 .

[0063] Furthermore, in some preferred implementation cases, a first filtering unit 111 is disposed on the reagent disc body 101 between the sample adding cavity 113 and the sample buffer cavity 114 .

[0064] Furthermore, a second filtering unit 112 is provided at the microchannel inlet 116 where the sample quantification chamber 115 of the reagent disc body 101 is connected to the mixing chamber 102 .

[0065] Since there are some impurities such as hair in the sample, the microchannel 103 on the reagent disc body 101 may be easily blocked. Therefore, the first filter unit 111 and the second filter unit 112 are provided to effectively filter the sample to prevent the impurities from entering the microchannel 103 .

[0066] Furthermore, in actual use, the first filter unit 111 and the second filter unit 112 are filter cotton or molecular sieves.

[0067] Furthermore, a card slot is provided on the reagent disc body 101, and the first filter unit 111 and the second filter unit 112 are mounted on the reagent disc body 101 through the card slot, and are covered and sealed by a sealing film, thereby reducing the difficulty of assembly.

[0068] Example 5 This embodiment mainly discloses a light-excited chemiluminescence immunoassay analyzer, including an analyzer body and a reagent disk, wherein the reagent disk is the reagent disk described in any one of the above embodiments 1-5.

[0069] The detection and control method specifically includes the following steps: Step 1: Add the whole blood sample into the sample adding chamber 113 of the reagent disk body 101; Step 2: Start the first centrifugation, the centrifugal speed is 4000rpm-6000rpm, and the centrifugal time is 30 seconds-60 seconds; during the first centrifugation, the whole blood sample enters the sample buffer chamber 114 of the reagent disc body 101 under the action of centrifugal force for separation, the plasma enters the sample quantitative chamber 115 of the reagent disc body 101, the excess plasma enters the waste liquid chamber of the reagent disc body 101, and the blood cells are blocked in the blood cell collection chamber of the reagent disc body 101; At the same time, the water cup placed in the diluent placement chamber of the reagent disc body 101 is pierced by the piercing structure, and the diluent flows out synchronously, and enters the diluent quantitative chamber of the reagent disc body 101 under the action of centrifugal force to complete the quantitative determination; Step 3: The plasma and diluent enter the mixing chamber 102 through various capillaries; In the second high and low speed of 1000rpm-5000rpm, the time is 30 seconds-60 seconds, and the mixed sample is fully mixed under the action of cross centrifugation; Step 4: Start the third centrifugation, the centrifugal speed is 4000rpm-6000rpm, and the centrifugal time is 30 seconds-60 seconds; During the third centrifugation process, the mixed sample flows into the flow channel 103 through the capillary tube; Step 5: Two-step reaction: 1) After the mixed sample enters the flow channel 103, it enters the first detection chamber 105 in sequence and undergoes a reaction in the first detection chamber 105; 2) After the reaction is completed, the sample enters the second detection chamber 106 for a secondary reaction, thereby achieving the purpose of step-by-step reaction.

[0070] In addition, in this embodiment, a method for preparing freeze-dried reagent beads is also disclosed, which is as follows: (1) Preparation process of reagent 1: 1. Antigen / antibody desalination: 1) Confirm the batch number, amount, volume and concentration of the antigen / antibody required for desalting and fill in the record.

[0071] 2) Take 0.5mL of antigen / antibody, read the absorbance at 280nm, and calculate: protein concentration = ABS 280 / extinction coefficient, protein concentration multiplied by volume 0.5mL, to obtain the total amount of antigen / antibody.

[0072] 3) Yield = (total amount of antigen / antibody obtained) / (amount of antigen / antibody added). When the yield is lower than 60%, add the same amount of antigen / antibody, re-test the absorbance and calculate the total amount of antigen / antibody.

[0073] 4) Use ultrafiltration concentrators to remove salt, use 10-100Mm PBS as eluent, add buffer to 0.5mL, centrifuge at 13000rpm, 4℃ for 10min, and determine the retention volume is not higher than 0.05mL; add buffer to 0.5mL, perform a second centrifugation at 13000rpm, 4℃ for 10min.

[0074] 5) If the retention volume is not higher than 0.05 mL, add 0.5 mL of buffer, read the absorbance at 280 nm, and calculate the protein concentration: =ABS 280 / extinction coefficient.

[0075] 6) Multiply the protein concentration by the volume 0.5 mL to get the total protein amount, and divide it by the feed amount to calculate the yield, which is generally not less than 80%.

[0076] 7) Centrifuge the antibody for the third time after absorbance measurement at 13,000 rpm, 4°C for 10 min, and measure the retention volume. Ensure that the protein concentration is between 2 and 4 mg / mL.

[0077] 2. Biotin connection reaction and desalting and collection process: 1) Record the mass, volume, and concentration of antigen / antibody to be used for activation.

[0078] 2) Weigh 0.3±0.1 mg of biotin and dissolve it in DMSO to 4.6 mg / mL (this solution must be used for coupling within 2 minutes).

[0079] 3) Pre-add the antigen / antibody solution into a conical test tube, and add the biotin working solution at a volume (ul) ratio of 13.3 times the mass of the antigen / antibody for coupling.

[0080] 4) Mix well and react at room temperature for 0.5-4 hours.

[0081] 5) Add Tris buffer at a ratio of 3 times the amount of biotin working solution, mix well to terminate the reaction, and leave at room temperature for at least 5 minutes.

[0082] 6) Use 0.5mL ultrafiltration concentrator to desalt, use 10-100Mm PBS as eluent, add buffer to 0.5mL, centrifuge at 13000rpm, 4℃ for 10min, determine the retention volume is not higher than 0.05mL, add buffer to 0.5mL, and perform a second centrifugation at 13000rpm, 4℃ for 10min.

[0083] 7) Determine that the retention volume is not higher than 0.05 mL, make up to 0.5 mL with buffer and read the absorbance at 280 nm.

[0084] 8) Calculate protein concentration: =ABS 280 / Extinction coefficient 2.9 protein concentration multiplied by volume 0.5mL, the total amount of protein after activation was divided by the feed amount to obtain the yield.

[0085] 9) After measuring the absorbance, centrifuge the antigen / antibody for the third time at 13,000 rpm and 4°C for 10 min. The retention volume should be no higher than 0.1 mL. Add an equal amount of glycerol for storage.

[0086] (2) Preparation process of reagent 2: 1) Take 1 mg of receptor microspheres (100 μL), place in a 1.5 mL centrifuge tube, add 10 mM MES buffer (pH=6.0) to 800 μL and mix well.

[0087] 2) Take 100 μL of 20 mg / mL NHS-sulfo, add it to the microspheres, and vortex to mix; then take 100 μL of 10 mg / mL EDC, add it to the microspheres, vortex to mix, and disperse by ultrasound; mix and incubate at room temperature for 20 min.

[0088] 3) Centrifuge the activated microspheres at 20,000 g (or maximum speed) for 25 min; remove the supernatant, resuspend the microspheres with 1000 μL of 10 mM MES buffer (pH = 6.0), and disperse them by ultrasound; centrifuge at 20,000 g (or maximum speed) for 25 min; remove the supernatant, resuspend the microspheres with MES coupling buffer, and disperse them by ultrasound for later use.

[0089] 4) Add 0.1 mg of antibody solution, vortex to mix, and place in a 37°C incubator on a rotating mixer in the dark for 2 h.

[0090] 5) After the reaction, add 50 µL of blocking solution (BSA: 20 mg / mL, 100 mM glycine, 25 mM HEPES, 150 mM NaCl, pH = 7.4) and place in a 37°C incubator on a rotating mixer in the dark for 1 h.

[0091] 6) Centrifuge at 20,000 g (or maximum speed) for 15 min, first time; remove the supernatant, resuspend the microspheres with 500 μL microsphere washing solution, and vortex ultrasonic dispersion; centrifuge at 20,000 g (or maximum speed) for 15 min, second time; remove the supernatant; 7) Add 100 μL storage solution (0.03% Proclin-300, 1% BSA, 25 mM HEPES, 150 mM NaCl, pH=7.4) to resuspend the microspheres, vortex to mix, and ultrasonically disperse. Place in a dark place at 4°C until ready for use.

[0092] (3) Preparation process of reagent 3: 1) Take 1 mg of donor microspheres (100 μL), place it in a 1.5 mL centrifuge tube, add 10 mM MES buffer (pH=6.0) to 800 μL and mix well.

[0093] 2) Take 100 μL of 20 mg / mL NHS-sulfo, add it to the microspheres, and vortex to mix; then take 100 μL of 10 mg / mL EDC, add it to the microspheres, vortex to mix, and disperse by ultrasound; mix and incubate at room temperature for 20 minutes.

[0094] 3) Centrifuge the activated microspheres at 20,000 g (or maximum speed) for 25 min; remove the supernatant, resuspend the microspheres with 1000 μL 10 mM MES buffer (pH = 6.0), and disperse them by ultrasound; centrifuge at 20,000 g (or maximum speed) for 25 min; remove the supernatant, resuspend the microspheres with MES coupling buffer, and disperse them by ultrasound for later use.

[0095] 4) Add 0.1 mg of antibody solution, vortex to mix, and place in a 37°C incubator on a rotating mixer in the dark for 2 h.

[0096] 5) After the reaction, add 50 μL of blocking solution (BSA: 20 mg / mL, 100 mM glycine, 25 mM HEPES, 150 mM NaCl, pH = 7.4) and place in a 37°C incubator on a rotating mixer in the dark for 1 hour.

[0097] 6) Centrifuge at 20,000 g (or maximum speed) for 15 min, first time; remove the supernatant, resuspend the microspheres in 500 μL microsphere washing solution, and vortex and ultrasonically disperse; centrifuge at 20,000 g (or maximum speed) for 15 min, second time; remove the supernatant.

[0098] 7) Add 100 μL storage solution (0.03% Proclin-300, 1% BSA, 25 mM HEPES, 150 mM NaCl, pH=7.4) to resuspend the microspheres, vortex to mix, and ultrasonically disperse. Place at 4°C in the dark until ready for use.

[0099] Among them, the preparation of freeze-drying buffer: The lyophilization buffer was combined according to the following base buffer and excipient ingredients; Buffer: 10-50mM PBS, 10-50mM HEPES, 10-50mM Tris, 10-50mM Mops, 10-50mM PPB, one of them; Component 1: During the freeze-drying process, the removal of water may cause structural damage to biological macromolecules such as proteins, such as denaturation or aggregation. Polyhydroxy compounds may interact with these macromolecules through hydrogen bonds, replacing water molecules, maintaining their three-dimensional structure and preventing denaturation.

[0100] Polyhydroxy compounds: 0.5%-1% glycerol, 2%-5% mannitol, 2%-5% inositol, 2%-5% sorbitol, 2%-5% thiol, 2%-5% polyethylene glycol, one of them.

[0101] Component 2: During the freezing and drying process, sugars form an amorphous glass structure, encapsulate biomolecules, inhibit molecular motion and chemical reactions (such as oxidation and hydrolysis), and can lower the freezing point of the solution and reduce the mechanical damage of ice crystal growth to cells or proteins. Sugar: 2%-6% glucose, 2%-6% sucrose, 2%-6% lactose, 2%-6% trehalose, 2%-6% α-D-pyranose, one of them.

[0102] Component 3: Charged amino acids (such as arginine) reduce the hydrophobic interactions between protein molecules and prevent aggregation through electrostatic repulsion. Amino acids: 0.5%-1.5% proline, 0.5%-1.5% tryptophan, 0.5%-1.5% alanine, 0.5%-1.5% glycine, one of them.

[0103] Component 4, polymer: forms a continuous mesh or porous matrix during the freeze-drying process to encapsulate active ingredients (such as proteins, cells) to prevent structural collapse or mechanical stress damage during the drying process. 0.5%-1% polyethylene glycol (PEG), 0.5%-1% polyvinyl pyrrolidone (PVP), 0.5%-1% gelatin, 0.5%-1% polyethyleneimine, one of them.

[0104] Component 5: Protein: 1-5% bovine serum albumin, 1-5% recombinant human albumin, or one of the above.

[0105] Component 6, surfactant: Surfactants can reduce protein aggregation during freeze-thaw and rehydration and help inhibit protein unfolding during the pre-freezing stage. Examination of specific infrared bands of aggregates in dried solids shows that surfactants can inhibit aggregation produced during freeze-drying. During the rehydration process, the aggregation of folded molecules can be inhibited by surfactants. It is speculated that it is through molecular interactions and / or as a wetting agent that accelerates the dissolution of the freeze-dried product. This product contains 0.1%-1% Tween 20, 0.1%-1% MERPOL SE, 0.1%-1% Triton X-305, 0.1%-1% Triton X-100, 0.1%-1% sodium dodecyl sulfate, 0.1%-1% and ascorbic acid, one of them.

[0106] Lyophilized Reagent Beads: The reagent beads were dropped into round beads in liquid nitrogen at a volume of 3.2 ul each, and then transferred to a freeze-vacuum freeze dryer for freeze drying.

[0107] The temperature setting of freeze-drying process is as follows: -40℃, 10min; -35℃, 10min; -30℃, 10min; -25℃, 120min; -20℃, 10min; -12℃, 120min; -10℃, 30min; 0℃, 10min; 10℃, -30min; 20℃, 200min; 30℃,1200min.

[0108] Further, in the present invention Figure 3 , Figure 4 and Figure 5 In the figure, the dotted line is a reference line drawn with the center position of the reagent disk body, which is used to reflect the size of the curvature radius.

[0109] In the present invention: PBS: phosphate buffered saline; DMSO: dimethyl sulfoxide; Tris: tris(hydroxymethyl)aminomethane hydrochloride; MES: 2-(N-morpholino)ethanesulfonic acid; EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide; HEPES: 4-hydroxyethylpiperazineethanesulfonic acid; Proclin-300: water-soluble preservative; BSA: bovine serum albumin; NHS-sulfo: N-hydroxysulfosuccinimide; Mops: 3-(N-morpholine)propanesulfonic acid; PB: phosphate buffer; MERPOL SE: Surfactant.

[0110] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A split reagent disc, comprising a reagent disc body, on which a mixing chamber is provided, characterized in that: The reagent disc body is also provided with a flow channel communicating with the mixing chamber; Among them, the reagent disc body is also provided with a plurality of reaction units, and the reaction units include a first detection cavity and a second detection cavity; The first detection chamber is communicated with the flow channel, the first detection chamber and the second detection chamber are communicated with each other, and the reagent in the mixing chamber flows into the flow channel and then sequentially passes through the first detection chamber and the second detection chamber.

2. A split reagent disc according to claim 1, characterized in that: The first detection chamber and the second detection chamber are respectively provided with the first freeze-dried reagent beads, the second freeze-dried reagent beads and / or the third freeze-dried reagent beads.

3. A split reagent disc according to claim 1, characterized in that: The flow channel is located on one side of the reagent disk body, and the mixing chamber is located on the other side of the reagent disk body.

4. A split reagent disc according to claim 1, characterized in that: A through hole is arranged on the reagent disc body, and sealing films are arranged on both sides of the reagent disc body for sealing the through hole to form the first detection cavity and the second detection cavity.

5. A split reagent disc according to claim 4, characterized in that: The sealing film is a light-transmitting sealing film.

6. A split reagent disc according to claim 2, characterized in that: The first freeze-dried reagent beads are oxygen acceptor microsphere freeze-dried reagent beads coated with monoclonal antibodies, the second freeze-dried reagent beads are biotin-labeled specific antigen microsphere freeze-dried reagent beads, and the third freeze-dried reagent beads are donor microsphere freeze-dried reagent beads coated with streptavidin.

7. A split reagent disc according to claim 1, characterized in that: The mixing chamber is communicated with the flow channel through a U-shaped microchannel.

8. A split reagent disc according to claim 1, characterized in that: The first detection chamber and the second detection chamber are connected via a "J"-shaped microchannel.

9. A split reagent disc according to claim 1, characterized in that: The flow channel has an overall arc-shaped structure.

10. A split reagent disc according to claim 9, characterized in that: The distance from the rotation center of the reagent disk body to the outer side wall of the flow channel increases from left to right.

11. A split reagent disc according to claim 10, characterized in that: The length of the connection channel between the first detection cavity and the flow channel decreases from left to right.

12. A split reagent disc according to claim 9, characterized in that: The distance from the rotation center of the reagent disk body to the inner wall of the flow channel decreases from left to right.

13. A split reagent disc according to claim 12, characterized in that: The length of the connecting channel between the first detection cavity and the flow channel is the same.

14. A split reagent disc according to claim 11, characterized in that: The connecting channel is a straight connecting channel or an arc-shaped connecting channel. One end of the arc-shaped connecting channel is tangent to the flow channel, and the other end is located at a position of the first detection cavity closest to the flow channel.

15. A light-excited chemiluminescent immunoassay analyzer, comprising an analyzer body and a reagent disk, characterized in that: The reagent disc is a split reagent disc as described in any one of claims 1-14.

16. A two-step reaction detection control method, characterized in that: The method comprises using the light-excited chemiluminescent immunoassay analyzer described in claim 15, and specifically comprises the following steps: Step 1: Add the whole blood sample into the sample adding cavity of the reagent disk body; Step 2: Start the first centrifugation, the centrifugal speed is 4000rpm-6000rpm, and the centrifugal time is 30 seconds-60 seconds; during the first centrifugation, the whole blood sample enters the sample buffer chamber of the reagent disc body under the action of centrifugal force for separation, the plasma enters the sample quantitative chamber of the reagent disc body, the excess plasma enters the waste liquid chamber of the reagent disc body, and the blood cells are blocked in the blood cell collection chamber of the reagent disc body; At the same time, the water cup placed in the diluent placement cavity of the reagent disc body is pierced by the piercing structure, and the diluent flows out synchronously, and enters the diluent quantitative cavity of the reagent disc body under the action of centrifugal force to complete the quantitative determination; Step 3: The plasma and diluent enter the mixing chamber through various capillaries; In the second high and low speed of 1000rpm-5000rpm, the time is 30 seconds-60 seconds, and the mixed sample is fully mixed under the action of cross centrifugation; Step 4: Start the third centrifugation, the centrifugal speed is 4000rpm-6000rpm, and the centrifugal time is 30 seconds-60 seconds; During the third centrifugation, the mixed sample flows into the flow channel through the capillary; Step 5: Two-step reaction: 1) After the mixed sample enters the flow channel, it enters the first detection chamber in turn and undergoes a reaction in the first detection chamber; 2) After the reaction is completed, the sample enters the second detection chamber for a secondary reaction, achieving the purpose of step-by-step reaction.

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