Biochemical reagent kit and detection method for resisting interference of hemolytic lipemia

Abstract

The application provides a biochemical detection kit and a detection method for resisting hemolytic lipid blood interference, relates to the technical field of kits, and comprises a kit body, a protective support frame is installed on the inner side wall of the kit body, multiple partition plates are installed side by side in the interior of the protective support frame, a gas storage bag is installed on the inner side bottom of the partition plate, three inflation gas bags are installed on the inner side of the partition plate, the top of the multiple gas storage bags and the inner side of the inflation gas bags are respectively inserted into reagent tube one and reagent tube two. The front and rear double-wavelength differences of the same reaction cup take each sample itself as a background reference, the errors caused by the color and lipoprotein composition differences of different patient samples are reduced, the technical units are all conventional liquid reagents, common double-wavelength reading and simple algebraic operation, manufacturing, quality control and instrument parameter transplantation are facilitated, the problem that the proportion between the main wavelength and the reference wavelength changes is solved, and the problem that special high-end biochemical instruments are relied on and the general popularity is low is solved.

Description

Technical Field

[0001] This invention relates to the field of reagent kit technology, and in particular to a biochemical detection kit and detection method that is resistant to interference from hemolysin. Background Technology

[0002] Biochemical assay kits are a set of pre-prepared, standardized reagents that rely on enzyme-catalyzed reactions and chemical colorimetric reactions to quantitatively detect metabolites, enzyme activity, proteins, ions, and other substances in biological samples using spectrophotometry. Existing hemolysis correction methods include sample blank, subtraction of dominant and secondary wavelengths, independent hemolysis index determination, and correction based on pre-reaction or regression models. Ordinary dual-wavelength methods usually treat the sample background as a fixed value, but when lipemia particles rearrange during the reaction, the ratio between the dominant wavelength and the reference wavelength will change. Complex multi-wavelength models often rely on specific instruments and databases, and depend on dedicated high-end biochemical instruments, resulting in low universality and widespread adoption. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a biochemical detection kit and method for detecting blood lipids that resist interference from hemolysis, thereby solving the problems mentioned in the background section.

[0004] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: This invention relates to a biochemical detection kit and method for detecting blood lipids that are resistant to interference from lipolysis. Specifically, it includes: a kit body with a rectangular structure and a protective support frame installed on its inner sidewall; multiple partitions arranged side-by-side inside the protective support frame; an air-filled bladder installed at the bottom inner side of each partition, and three inflatable air bladders installed inside the partition, with the inflatable air bladders positioned above the air-filled bladders; reagent tube one and reagent tube two are inserted into the top of each air-filled bladder and the inner side of each inflatable air bladder, respectively. The first reagent tube contains a first reagent R1, and the second reagent tube contains a second reagent R2. The first reagent R1 includes: a buffer solution, a lipid particle optical state prestabilization composition, ascorbic acid oxidase, a chelating agent, and a preservative. The lipid particle optical state prestabilization composition includes: a first nonionic surfactant, a polyoxyethylene-polyoxypropylene block polymer, and a water-soluble polymer. The second reagent R2 includes: a specific reaction component for directly or indirectly generating hydrogen peroxide from the analyte, a peroxidase, a far-infrared colorimetric agent with a maximum absorption wavelength in the range of 620–680 nm, a first nonionic surfactant, a polyoxyethylene-polyoxypropylene block polymer, and at least the first two of the water-soluble polymer.

[0005] Furthermore, the bottom of the air sump is installed on the inner bottom of the protective support frame; four main flow pipes are installed on the outer side of the air sump.

[0006] Furthermore, the main flow pipe is located at the inner angle of the partition plate, and a diversion pipe is connected to the side of the main flow pipe; the side end of the diversion pipe at the same height is connected to the air storage bag at the same height.

[0007] Furthermore, a positioning seat is installed on the inner bottom of the air sump; a groove is provided on the top of the positioning seat, and a spring is connected to the top of the groove; a force-bearing plate is installed on the inner top of the air sump.

[0008] Furthermore, a fixing cone is installed at the bottom of the force-bearing plate; both the upper and lower ends of the fixing cone are tapered structures, and the fixing cone penetrates the spring piece downward and is inserted into the groove at the top of the positioning seat.

[0009] Furthermore, the first nonionic surfactant is selected from one or more of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, alkyl glycosides, and polyoxyethylene fatty acid esters; the polyoxyethylene-polyoxypropylene block polymer is selected from one or both of poloxamer 188 and poloxamer 407; and the water-soluble polymer is selected from one or more of polyvinylpyrrolidone, polyethylene glycol, hydroxypropyl methylcellulose, and polyvinyl alcohol.

[0010] Furthermore, in the first reagent R1, the concentration of the first nonionic surfactant is 0.10–1.50 g / L, the concentration of the polyoxyethylene-polyoxypropylene block polymer is 0.05–1.20 g / L, the concentration of the water-soluble polymer is 0.10–2.00 g / L, and the mass ratio of the first nonionic surfactant to the block polymer is 1:0.2–3.0.

[0011] Furthermore, this includes the following steps: First, mix the sample with the first reagent R1 in reagent tube one, and allow the optical state of the lipid particles to reach a stable reading window within 30 to 180 seconds. Second, the absorbance A at the first dominant wavelength λp and the reference wavelength λr was read respectively. 0，p Absorbance A at the first reference wavelength 0，r ; Third, add the second reagent R2 from reagent tube two into the same reaction vessel to initiate the specific reaction of the analyte and form a far-infrared colored product. Fourth, the absorbance A at the second dominant wavelength was read at both the dominant wavelength λp and the reference wavelength λr. 1，p Absorbance A at the second reference wavelength 1，r ; Fifth, according to A 0，p A 0，r A 1，p and A 1，r The concentration of the analyte is obtained by analyzing the absorbance difference signal before and after the two wavelengths and the calibration curve.

[0012] Furthermore, within the stable reading window of step one, the rate of change of the absorbance of the reference wavelength is no greater than 0.005 A / min, and after adding the second reagent R2, the instantaneous change in the absorbance of the reference wavelength is no greater than 0.015 A.

[0013] Furthermore, the absorbance difference signal ΔD between the two wavelengths in step S5 is obtained according to the following formula: ΔD=(A 1，p -kA 1，r )-(A 0，p -kA 0，r )-B, where k is the lipid scattering correction coefficient and B is the reagent blank.

[0014] This invention provides a biochemical detection kit and method for detecting lipolysis, which has the following beneficial effects: When this invention is used, the difference between the two wavelengths before and after the same reaction cup uses each sample itself as a baseline reference, reducing the error caused by differences in color and lipoprotein composition of different patient samples. The technical units are all conventional liquid reagents, ordinary dual-wavelength readings and simple algebraic calculations, which facilitates manufacturing, quality control and instrument parameter transfer.

[0015] In addition, the compound prestabilizing component does not simply reduce the initial turbidity, but maintains the lipid particles in a repeatable optical state within the reading window. It also reduces the dynamic turbidity drift after adding the second reagent R2 by matching the concentrations of the first reagent R1 and the second reagent R2. The far-infrared color development reduces the overlap of the main visible absorption peaks of hemoglobin, while the long wavelength band is relatively insensitive to lipid scattering.

[0016] Furthermore, it eliminates the need for high-speed centrifugation, organic solvent extraction, precipitation separation, or additional sample blank reaction cups, and can be directly adapted to conventional liquid dual-reagent fully automated biochemical analyzers. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly described below.

[0018] The accompanying drawings described below are only related to some embodiments of the invention and are not intended to limit the invention.

[0019] In the attached diagram: Figure 1 A schematic diagram of the overall structure of the present invention is shown; Figure 2 A schematic diagram of the kit body in the unfolded state of the present invention is shown; Figure 3 A three-dimensional structural diagram of the protective support frame of the present invention is shown; Figure 4 A schematic cross-sectional view of the partition plate of the present invention is shown; Figure 5 A schematic cross-sectional view of the inflatable airbag of the present invention is shown; Figure 6 A schematic cross-sectional view of the air bladder structure of the present invention is shown; Figure 7 A schematic diagram of the two-stage detection process of the same reaction vessel of the present invention is shown; Figure 8 A schematic diagram of the absorbance-time curves of the addition of the first reagent R1, the addition of the second reagent R2, and two reading windows is shown. Figure 9 A schematic diagram of the calculation process for the dual-wavelength differential signals of the present invention is shown.

[0020] List of reference numerals 1. Reagent kit; 101. Protective support frame; 102. Divider plate; 103. Air accumulator; 104. Inflatable air accumulator; 105. Main flow tube; 106. Diverter tube; 107. Positioning seat; 108. Spring; 109. Force plate; 1010. Fixing cone; 1011. Reagent tube one; 1012. Reagent tube two. Detailed Implementation

[0021] Please refer to Figures 1 to 9 : Example 1: This invention proposes a biochemical detection kit and method for detecting interference from hemolyzed lipids, comprising: a kit body 1, which has a rectangular structure, and a protective support frame 101 installed on the inner wall of the kit body 1; multiple partition plates 102 are installed side by side inside the protective support frame 101; an air storage bag 103 is installed at the bottom inner side of the partition plate 102, and three inflatable air bags 104 are installed inside the partition plate 102, with the inflatable air bags 104 positioned above the air storage bag 103; reagent tube one 1011 and reagent tube two 1012 are respectively inserted into the top of the multiple air storage bags 103 and the inner side of the inflatable air bags 104; Reagent tube 1011 contains a first reagent R1, and reagent tube 2 1012 contains a second reagent R2. The first reagent R1 includes: a buffer solution, a lipid particle optical state prestabilization composition, ascorbic acid oxidase, a chelating agent, and a preservative. The lipid particle optical state prestabilization composition includes: a first nonionic surfactant, a polyoxyethylene-polyoxypropylene block polymer, and a water-soluble polymer. The second reagent R2 includes: a specific reaction component for directly or indirectly generating hydrogen peroxide from the analyte, a peroxidase, a far-infrared colorimetric agent with a maximum absorption wavelength in the range of 620–680 nm, and at least two of the first nonionic surfactant, the polyoxyethylene-polyoxypropylene block polymer, and the water-soluble polymer. The bottom of the air accumulator 103 is installed on the inner bottom of the protective support frame 101; four main flow pipes 105 are installed on the outer side of the air accumulator 103; the main flow pipes 105 are located at the inner angle of the partition plate 102, and the sides of the main flow pipes 105 are connected to diversion pipes 106; the side ends of the diversion pipes 106 at the same height are connected to the air accumulator 103 at the same height; a positioning seat 107 is installed on the inner bottom of the air accumulator 103; the top of the positioning seat 107 has a groove, and the top of the groove is connected to a spring piece 108; a force-bearing plate 109 is installed on the inner top of the air accumulator 103; a fixing cone 1010 is installed on the bottom of the force-bearing plate 109; the upper and lower ends of the fixing cone 1010 are both conical structures, and the fixing cone 1010 penetrates downward through the spring piece 108 and is inserted into the top of the positioning seat 107. Within the groove; the first nonionic surfactant is selected from one or more of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, alkyl glycosides, and polyoxyethylene fatty acid esters; the polyoxyethylene-polyoxypropylene block polymer is selected from one or two of poloxamer 188 and poloxamer 407; the water-soluble polymer is selected from one or more of polyvinylpyrrolidone, polyethylene glycol, hydroxypropyl methylcellulose, and polyvinyl alcohol; the concentration of the first nonionic surfactant in the first reagent R1 is 0.10–1.50 g / L, the concentration of the polyoxyethylene-polyoxypropylene block polymer is 0.05–1.20 g / L, the concentration of the water-soluble polymer is 0.10–2.00 g / L, and the mass ratio of the first nonionic surfactant to the block polymer is 1:0.2–3.0; A biochemical detection method for lipemia interference, comprising the following steps: First, mixing the sample with reagent R1 in reagent tube 1011, allowing the optical state of lipemia particles to reach a stable reading window within 30–180 s. Within this stable reading window, the rate of change of absorbance at the reference wavelength is no greater than 0.005 A / min. After adding reagent R2, the instantaneous change in absorbance at the reference wavelength is no greater than 0.015 A. Second, reading the absorbance A at the first dominant wavelength λp and the reference wavelength λr, respectively. 0，p Absorbance A at the first reference wavelength 0，r Third, add the second reagent R2 from reagent tube 1012 into the same reaction vessel to initiate the analyte-specific reaction and form a far-infrared colored product; Fourth, read the absorbance A at the second dominant wavelength λp and the reference wavelength λr, respectively. 1，p Absorbance A at the second reference wavelength 1，r 5. According to A 0，p A 0，r A 1，p and A 1，r The absorbance difference signal before and after the calibration curve is used to obtain the concentration of the analyte. The absorbance difference signal ΔD before and after the calibration curve is obtained according to the following formula: ΔD = (A 1，p -kA1，r )-(A 0，p -kA 0，r )-B, where k is the lipid scattering correction coefficient and B is the reagent blank.

[0022] In this embodiment of the invention, reagent tube 1011 stores a first reagent R1, and reagent tube 1012 stores a second reagent R2. Reagent tubes 1011 and 1012 are inserted inside a partition plate 102. As reagent tubes 1011 and 1012 move downwards, they push the top of the gas storage bladder 103. The gas storage bladder 103 stores airflow. When compressed, the airflow travels along the main flow pipe 105 and through the branch pipe 106 into the expansion bladder 104, causing the expansion bladder 104 to expand outside the reagent tubes 1011 and 1012. The top of the gas storage bladder 103 continues to move downwards. The movement causes the force plate 109 to move, which in turn drives the fixed cone block 1010 to penetrate the interior of the spring piece 108 and insert into the groove at the top of the positioning seat 107, thus fixing the position of the force plate 109 and the fixed cone block 1010. The expansion airbag 104 expands outward and squeezes onto the outside of the first reagent tube 1011 and the second reagent tube 1012, thereby fixing the position of the first reagent tube 1011 and the second reagent tube 1012. At the same time, there is a certain airflow inside the expansion airbag 104, which can buffer the external vibration and ensure the stability of the first reagent R1 and the second reagent R2 stored inside.

[0023] Example 2: Based on Example 1, a general lipid particle optical state pre-stabilization system was prepared. The first reagent R1 consisted of: MES buffer 60 mmol / L, sodium chloride 50 mmol / L, polyoxyethylene lauryl ether 0.40 g / L, poloxamer 188 0.60 g / L, PVPK 30 0.50 g / L, disodium EDTA 0.20 mmol / L, ascorbic acid oxidase 1000 U / L, and ProClin 300 0.05%, and the pH was adjusted to 6.8. The second reagent R2 is prepared as follows: 60 mmol / L MES buffer, 50 mmol / L sodium chloride, 0.39–0.43 g / L polyoxyethylene lauryl ether, 0.58–0.64 g / L poloxamer 188, 0.48–0.53 g / L PVPK30, and 0.05% ProClin300, adjusting the pH to 6.8. After mixing the lipemia sample with the reagent, incubate at 37°C for 60–120 s. Select 660 nm as the main wavelength and 750 nm or 800 nm as the reference wavelength to observe the change in reference absorbance. When the reference absorbance drifts significantly, the ratio of polyoxyethylene lauryl ether to poloxamer 188 can be adjusted without reducing the target enzyme activity below 85%. When a transient turbidity jump occurs after adding the second reagent R2, prioritize adjusting the concentration of the corresponding component in the second reagent R2 rather than continuing to increase the total surfactant concentration of the first reagent R1.

[0024] Example 3: Based on Example 1, a glucose detection kit resistant to lipolysis interference was developed. The first reagent R1 used the formulation of Example 2. The second reagent R2 was based on the general matrix of Example 2, with the addition of glucose oxidase 15–25 kU / L, peroxidase 2–6 kU / L, and DA-67 0.05–0.12 mmol / L. 3 μL of sample was mixed with 240 μL of the first reagent R1 and incubated at 37°C for 90 s. A was read at 660 nm and 750 nm. 0，p A 0，r Add 260 μL of the second reagent R, react at 37°C for 180 s; read A at the same wavelength. 1，p and A 1，r Using ΔD=(A) 1，p -kA 1，r )-(A 0，p -kA 0，r The net signal is calculated using )-B, and a linear or piecewise linear calibration curve is established using glucose calibrator; glucose oxidase oxidizes glucose to generate hydrogen peroxide; peroxidase catalyzes the oxidation of DA-67 with hydrogen peroxide to form a far-red product; ascorbic acid oxidase in the first reagent R1 pre-reduces the negative interference of ascorbic acid; far-red detection and sample background difference mainly reduce hemolysis color interference; pre-stabilized components and concentration matching mainly reduce lipemia scattering and dynamic turbidity changes after the addition of the second reagent R2.

[0025] The working principle of this embodiment is as follows: Reagent tube 1011 contains a first reagent R1, and reagent tube 1012 contains a second reagent R2. Reagent tubes 1011 and 1012 are inserted into the separator plate 102, compressing the air accumulator 103. This causes the airflow inside to flow along the main stream pipe 105 through the diversion pipe 106 into the expansion accumulator 104. The expansion accumulator 104 expands outside the reagent tubes 1011 and 1012. The continuous compression of the air accumulator 103 causes the fixing cone 1010 to insert into the groove at the top of the positioning seat 107. The expansion bladder 104 is fixed in place, expanding outward to fix reagent tube 1011 and reagent tube 1012. At the same time, the expansion bladder 104 buffers external vibrations. During detection, the first reagent R1 is added to the sample to be tested and mixed to stabilize the optical state of the lipid particles. Then, the second reagent R2 is added to the same reaction vessel to trigger a specific enzymatic reaction of the analyte, generating a far-infrared chromogenic substance. The total signal after the reaction is collected, and the net reaction difference signal is calculated. The concentration of the analyte in the sample is then calculated by combining the results with the calibration curve.

Claims

1. A biochemical detection kit for detecting lipolysis, comprising: The reagent kit (1) has a protective support frame (101) installed on its inner sidewall; characterized in that multiple partition plates (102) are installed side by side inside the protective support frame (101); an air sac (103) is installed at the bottom inner side of the partition plate (102), and three expansion air sacs (104) are installed inside the partition plate (102), with the expansion air sacs (104) positioned above the air sacs (103); reagent tube one (1011) and reagent tube two (1012) are respectively inserted into the top of the multiple air sacs (103) and the inner side of the expansion air sacs (104). The first reagent tube (1011) contains a first reagent R1, and the second reagent tube (1012) contains a second reagent R2. The first reagent R1 includes: a buffer solution, a lipid particle optical state prestabilization composition, ascorbic acid oxidase, a chelating agent, and a preservative. The lipid particle optical state prestabilization composition includes: a first nonionic surfactant, a polyoxyethylene-polyoxypropylene block polymer, and a water-soluble polymer. The second reagent R2 includes: an analyte-specific reaction component, a peroxidase, a far-infrared colorimetric agent, a first nonionic surfactant, a polyoxyethylene-polyoxypropylene block polymer, and a water-soluble polymer.

2. The biochemical detection kit for resisting interference from hemolysis and lipolysis according to claim 1, characterized in that, The bottom of the air sac (103) is installed on the inner bottom of the protective support frame (101); four main flow pipes (105) are installed on the outer side of the air sac (103).

3. The biochemical detection kit for resisting interference from hemolysis and lipolysis according to claim 2, characterized in that, The main pipe (105) is located at the inner angle of the partition plate (102), and the side of the main pipe (105) is connected to the diversion pipe (106); the side end of the diversion pipe (106) at the same height is connected to the air sac (103) at the same height.

4. The biochemical detection kit for resisting interference from hemolysis and lipolysis according to claim 3, characterized in that, A positioning seat (107) is installed on the inner bottom of the air sac (103); a groove is provided on the top of the positioning seat (107), and a spring piece (108) is connected to the top of the groove; a force plate (109) is installed on the inner top of the air sac (103).

5. The biochemical detection kit for resisting interference from hemolysis and lipolysis according to claim 4, characterized in that, The bottom of the force plate (109) is equipped with a fixed cone block (1010); the upper and lower ends of the fixed cone block (1010) are both cone-shaped structures, and the fixed cone block (1010) penetrates the spring piece (108) downward and is inserted into the groove at the top of the positioning seat (107).

6. A biochemical detection kit for resisting interference from hemolysis and lipolysis according to claim 5, characterized in that, The first nonionic surfactant is selected from one or more of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, alkyl glycosides and polyoxyethylene fatty acid esters; the polyoxyethylene-polyoxypropylene block polymer is selected from poloxamer 188 and poloxamer 407; the water-soluble polymer is selected from polyvinylpyrrolidone, polyethylene glycol, hydroxypropyl methylcellulose and polyvinyl alcohol.

7. A biochemical detection kit for resisting interference from hemolysis and lipolysis according to claim 6, characterized in that, The concentration of the first nonionic surfactant in the first reagent R1 is 0.10–1.50 g / L, the concentration of the polyoxyethylene-polyoxypropylene block polymer is 0.05–1.20 g / L, the concentration of the water-soluble polymer is 0.10–2.00 g / L, and the mass ratio of the first nonionic surfactant to the block polymer is 1:0.2–3.

0.

8. A biochemical detection method for detecting lipid-lowering interference as described in any one of claims 1 to 7, characterized in that, Includes the following steps: First, mix the sample with the first reagent R1 in reagent tube one (1011) and allow the optical state of the lipid particles to reach a stable reading window within 30 to 180 seconds. Second, the absorbance A at the first dominant wavelength λp and the reference wavelength λr was read respectively. 0，p Absorbance A at the first reference wavelength 0，r ; Third, add the second reagent R2 from reagent tube two (1012) into the same reaction vessel to initiate the specific reaction of the analyte and form a far-infrared colored product; Fourth, the absorbance A at the second dominant wavelength was read at both the dominant wavelength λp and the reference wavelength λr. 1，p Absorbance A at the second reference wavelength 1，r ; Fifth, according to A 0，p A 0，r A 1，p and A 1，r The concentration of the analyte is obtained by analyzing the absorbance difference signal before and after the two wavelengths and the calibration curve.

9. The biochemical detection method for resisting interference from hemolysis and lipolysis according to claim 8, characterized in that, Within the stable reading window of step one, the rate of change of absorbance at the reference wavelength is no greater than 0.005 A / min, and after adding the second reagent R2, the instantaneous change in absorbance at the reference wavelength is no greater than 0.015 A.

10. The biochemical detection method for resisting lipolysis interference according to claim 9, characterized in that, The absorbance difference signal ΔD between the front and rear wavelengths in step S5 is obtained according to the following formula: ΔD=(A 1，p -kA 1，r )-(A 0，p -kA 0，r )-B, where k is the lipid scattering correction coefficient and B is the reagent blank.

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