96-hole preassembled plate fat-soluble vitamin pretreatment kit

Abstract

The invention discloses a 96-hole preassembled plate fat-soluble vitamin pretreatment kit, and belongs to the technical field of in-vitro diagnostic reagents. Comprising magnetic bead suspension liquid, equilibrium liquid, leacheate 1, leacheate 2 and eluent which are carried on a preassembled plate, and the magnetic bead suspension liquid comprises absolute ethyl alcohol and magnetic beads; the preparation process of the magnetic beads comprises the following steps: activating carboxyl of the carboxyl magnetic beads through EDC and NHS; then nitrochlorobenzene and triethylamine are added for a coupling reaction; mixing the coupling reaction product with pyrrolidone, ammonium persulfate and a pore-foaming agent to obtain target magnetic beads; the magnetic beads can be specifically combined with fat-soluble vitamins in human serum or plasma, efficient separation and release of an object to be detected are achieved through a magnetic separation technology, and the magnetic beads are suitable for follow-up mass spectrum quantitative analysis. The kit disclosed by the invention has the characteristics of simplicity and convenience in operation, high extraction efficiency and good stability, the pretreatment time can be remarkably shortened, the detection accuracy is improved, and an efficient solution is provided for clinical detection of fat-soluble vitamins.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically relating to a 96-well pre-packed plate fat-soluble vitamin pretreatment kit. Background Technology

[0002] Fat-soluble vitamins (such as vitamins A, D, E, and K) are essential micronutrients for the human body, and abnormal concentrations of these vitamins are closely related to various diseases (such as rickets, osteoporosis, and cardiovascular diseases). In clinical testing, fat-soluble vitamins are often bound to plasma proteins, requiring pretreatment to release them from their bound state and purify them for accurate quantitative analysis.

[0003] Traditional pretreatment methods (such as liquid-liquid extraction and solid-phase extraction) suffer from problems such as cumbersome operation, long processing time, low extraction efficiency, and significant matrix interference. For example, liquid-liquid extraction requires multiple centrifugations, which can easily lead to analyte loss; traditional solid-phase extraction columns have limited adsorption capacity and exhibit significant batch-to-batch variability. Therefore, developing an efficient, stable, and easy-to-operate pretreatment tool is of great significance for improving the accuracy and efficiency of fat-soluble vitamin detection. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a 96-well pre-packed plate fat-soluble vitamin pretreatment kit, which solves the problems in the prior art.

[0005] The objective of this invention can be achieved through the following technical solutions: A method for preparing magnetic beads includes the following steps: S1. After washing the carboxyl magnetic beads, disperse them in a buffer solution; then add EDC and NHS to activate the carboxyl groups; then add nitrochlorobenzene and triethylamine for a coupling reaction, and terminate the reaction with ethanolamine. S2, the coupling reaction product is washed and dispersed in deionized water, pyrrolidone, ammonium persulfate and porogen are added, and a polymerization reaction is carried out to form a porous polymer; finally, after washing and drying, magnetic beads are obtained.

[0006] Furthermore, the average particle size of the carboxyl magnetic beads is 10 μm.

[0007] Furthermore, the pore-forming agent is a mixed solution of ethanol and water, with a volume ratio of 2:1.

[0008] A magnetic bead is prepared using the above-described method for preparing a magnetic bead.

[0009] The above-mentioned magnetic beads are used in the preparation of a pretreatment kit for fat-soluble vitamins.

[0010] A fat-soluble vitamin pretreatment kit, comprising: Magnetic bead suspension: comprising anhydrous ethanol and the magnetic beads as described in claim 4; Equilibrium solution: consists of methanol and ultrapure water; Eluent 1: consists of methanol and ultrapure water; Eluent 2: consists of methanol and ultrapure water; Eluent: includes isopropanol and acetonitrile; And a porous pre-assembled plate containing the magnetic bead suspension, balancing liquid, rinsing liquid 1, rinsing liquid 2 and elution liquid.

[0011] Furthermore, the concentration of magnetic beads in the magnetic bead suspension is 10 mg / mL.

[0012] Furthermore, in the equilibrium solution, the concentration of methanol is 30 wt%; in both eluent 1 and eluent 2, the concentration of methanol is 20 wt%; and in the elution solution, the volume ratio of isopropanol to acetonitrile is 1:3.

[0013] Furthermore, the porous pre-packed plate includes 96 wells arranged in 8 rows and 12 columns, and the reagents are configured in the wells of the pre-packed plate as follows: Columns 1 and 7 are sample loading wells with a volume of 510 μL; columns 2 and 8 are magnetic bead suspension wells with a volume of 150 μL; columns 3 and 9 are equilibration buffer wells with a volume of 500 μL; columns 4 and 10 are eluent 1 wells with a volume of 500 μL; columns 5 and 11 are eluent 2 wells with a volume of 500 μL; and columns 6 and 12 are elution buffer wells with a volume of 150 μL.

[0014] 10. The use of the kit according to any one of claims 6-9 in the separation of fat-soluble vitamins from serum or plasma.

[0015] The beneficial effects of this invention are: 1. The bifunctional groups and porous structure on the surface of the magnetic beads of the present invention significantly improve the specific adsorption capacity for fat-soluble vitamins, and shorten the extraction time to less than 30 minutes; 2. The pre-loaded plate design of this invention is compatible with automated instruments, reducing human error and making it suitable for high-throughput detection. Furthermore, the desolvation process is compatible with LC-MS / MS systems, allowing for direct sample injection after simple nitrogen reconstitution. In addition, the kit of this invention features ease of operation, high extraction efficiency, and good stability, significantly shortening pretreatment time and improving detection accuracy, providing an efficient solution for the clinical detection of fat-soluble vitamins. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a transmission electron microscope image of the magnetic beads of the present invention; Figure 2 This is the Fourier transform infrared spectrum of the magnetic beads of the present invention; Figure 3 Here is a picture of the 96-hole pre-mounted plate and magnetic sleeve. Figure 4 This is a schematic diagram of the detection of a 96-well pre-packed plate fat-soluble vitamin pretreatment kit. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] In this embodiment, some of the raw materials are sourced as follows: 10 μm Carboxyl-functionalized magnetic beads: Nanjing Dongna Biotechnology Co., Ltd., Brand: MagBeads® 10 μm Carboxyl-functionalized magnetic beads.

[0020] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), CAS: 25952-53-8; N-hydroxysuccinimide (NHS), CAS: 6066-82-6; p-Nitrochlorobenzene, CAS: 100-00-5; N-vinylpyrrolidone (NVP), CAS: 88-12-0; Ammonium persulfate (APS), CAS: 7727-54-0; N,N-Dimethylformamide (DMF), CAS: 68-12-2; Dimethyl sulfoxide (DMSO), CAS: 67-68-5; Anhydrous ethanol, CAS: 64-17-5; Methanol, CAS: 67-56-1; Isopropanol, CAS: 67-63-0; Ethanolamine, CAS: 141-43-5; Triethylamine, CAS: 121-44-8 Acetone, CAS: 67-64-1.

[0021] Example 1 The preparation process of a 96-well pre-packed plate fat-soluble vitamin pretreatment kit includes the following steps: 1. Magnetic bead synthesis (1) Take 5 mL of 10 μm carboxyl magnetic beads (0.5 μmol / mg), wash 3 times with deionized water, and wash once with 0.1M MES (2-morpholinoethanesulfonic acid) buffer (pH 6.0); (2) Add 5 mL of MES buffer, sonicate for 60 s, then add 60 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 36 mg of NHS (N-hydroxysuccinimide), and stir at room temperature for 30 min to activate. (3) Add 5 mL of DMF solution containing 20 mg of p-nitrochlorobenzene, add 50 μL of triethylamine dropwise, and shake at 200 rpm for 12 h (protected from light); (4) Add 1 mL of 0.1M ethanolamine to terminate the reaction, and wash twice each with DMF, anhydrous ethanol, and deionized water; (5) Disperse in 7.5 mL of deionized water, add 600 μL of pyrrolidone, 28 mg of APS (ammonium persulfate) and porogen (3 mL of ethanol + 1.5 mL of water), purge with nitrogen for 10 min, and then polymerize at 60 °C for 4-6 h; (6) After washing with anhydrous ethanol, deionized water and acetone, the porous magnetic beads (zdyyjyk10k-1) were obtained by vacuum drying at 40℃ for 12 h.

[0022] like Figure 1 As shown, the synthesized porous magnetic beads, with Fe3O4 as the core, were modified by carboxyl activation, p-nitrochlorobenzene coupling, and pyrrolidone polymerization. The particle size remained at approximately 10 μm, exhibiting uniform morphology and good dispersibility. Figure 2 As shown, in its Fourier transform infrared spectrum, the characteristic stretching vibration peak of the carboxyl group (-COOH) appears at around 1710 cm⁻¹, and the characteristic absorption peak of the amide bond in the pyrrolidone polymer appears near 1650 cm⁻¹. At the same time, the absorption peak of the hydroxyl group (-OH) at around 3400 cm⁻¹ is weakened, which confirms that the surface of the magnetic beads has been successfully functionalized with carboxyl groups and modified with porous polymers, providing a structural basis for the specific adsorption of fat-soluble vitamins.

[0023] 2. Reagent preparation Magnetic bead suspension: 30 mg of magnetic beads were dissolved in 3 mL of anhydrous ethanol, with a concentration of 10 mg / mL; Equilibrium solution: 30 wt% methanol (3 mL methanol + 7 mL ultrapure water); Eluent 1: 20 vt% methanol (2 mL methanol + 8 mL ultrapure water); Eluent 2: 20 wt% methanol (2 mL methanol + 8 mL ultrapure water); Eluent: Isopropanol to acetonitrile volume ratio is 1:3 (3 mL isopropanol + 9 mL acetonitrile).

[0024] 3. Pre-assembled panels Add the reagents to the corresponding wells of the 96-well plate according to Table 1, seal with aluminum foil, and you will have a pre-packed reagent kit (e.g., ...). Figure 3 (As shown).

[0025] Table 1. Pre-assembled plate hole configuration Among them, 1 / 7 represents the hole positions in columns 1 and 7; The hole positions indicated by 2 / 8 are: columns 2 and 8; The hole positions indicated by 3 / 9 are: the 3rd and 9th columns; 4 / 10 indicates the hole positions in columns 4 and 10. 5 / 11 indicates the hole positions in columns 5 and 11. The hole positions indicated by 6 / 12 are: column 6 and column 12.

[0026] Example 2 This embodiment describes the usage method of the reagent kit described in Example 1, such as... Figure 4 The steps shown are as follows: 1. Sample pretreatment (1) Tear open the aluminum film of the pre-packaged plate and add 200 μL of serum sample, 200 μL of methanol, 100 μL of pure water and 10 μL of internal standard to the sample well (1 / 7); (2) Place the pre-loaded plate in a commercially available magnetic rod method nucleic acid extractor or magnetic solid phase extractor, insert the magnetic rod sleeve, and run the "fat-soluble vitamin extraction" program (parameters are shown in Table 2). Table 2 Extraction program parameters The speed is 5 Hz (5 times up and down per second).

[0027] 2. Testing After the procedure, the eluent from 6 / 12 wells was collected and directly injected into a high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) system to detect the concentration of fat-soluble vitamins.

[0028] Example 3 In this embodiment, the performance of the reagent kit prepared in Example 1 is verified; 1. Extraction efficiency Pretreatment of serum samples (containing vitamins A, D2, D3, E, and K) with known concentrations showed that, compared with solid-phase extraction methods, the extraction efficiency of the kit prepared in this invention (Example 1) was ≥85% (see Table 3). Table 3 Comparison of Extraction Efficiency 2. Stability: After the kit was stored at room temperature for 12 months, the extraction efficiency of the present invention (the kit prepared in Example 1) decreased by ≤3.0% (see Table 4), indicating that it has good stability; Table 4 Stability Study 3. Precision: 3.1 Intra-day precision 3.1.1 Specimens: Precision verification was performed using quality control materials. The samples contained two concentration levels: high and low.

[0029] 3.1.2 Procedure: Take two quality control samples with high and low concentrations as samples, and repeat the test 3 times with the same batch of reagents. Calculate the mean (AV) of the 3 test results, and calculate the standard deviation (SD) and coefficient of variation (CV).

[0030] 3.2 Daytime precision 3.2.1 Specimens: Precision verification was performed using quality control materials. The samples contained two concentration levels: high and low.

[0031] 3.2.2 Procedure: Take three quality control samples with high and low concentrations as samples, repeat the test three times with the same batch of reagents, and measure continuously for three days. Calculate the mean (AV) of the test results, and calculate the standard deviation (SD) and coefficient of variation (CV).

[0032] 3.3 Results The intra-day and inter-day precision of this invention are shown in Table 5: the intra-day precision CV value range for fat-soluble vitamins (VA, VD2, VD3, VE, VK) is 5.92%-14.24%, and the inter-day precision CV value range is 3.46%-12.82%. The overall precision meets the reliability requirements for clinical testing.

[0033] Table 5. Intra-day and Inter-day Precision Assessment 4. Recovery rate 4.1 Sample preparation: Clinical samples were divided into three equal volumes. Two of these samples were prepared by adding equal volumes of low- and high-concentration analyte standard solutions (LStd and HStd) to each sample. The third sample was prepared as a baseline sample by adding an equal volume of blank reagent without the analyte. The baseline sample and the recovered samples at low concentrations (VA: 149.38 ng / mL, VD2: 3.63 ng / mL, VD3: 7.34 ng / mL, VE: 1550.00 ng / mL, VK: 0.07 ng / mL) and high concentrations (VA: 1465.38 ng / mL, VD2: 36.80 ng / mL, VD3: 71.64 ng / mL, VE: 14778.57 ng / mL, VK: 3.05 ng / mL) were each measured three times.

[0034] 4.2 Experimental Procedure: The basic sample group was added in the following order: 10 μL mixed internal standard + 10 μL methanol + 490 μL blood sample; The order of adding the recovered sample groups was: 10 μL mixed internal standard + 10 μL standard solution + 490 μL blood sample.

[0035] The specific steps are carried out in conjunction with the pretreatment operation instructions of the magnetic bead method reagent kit and the operation procedure of the fully automated extractor. After processing, the samples are tested on the instrument.

[0036] 4.3 Results The average recovery rates of low concentrations of various fat-soluble vitamins were 94.1%-101.5%, and the average recovery rates of high concentrations were 92.7%-100.0%. The overall recovery rates were within the ideal range of 85%-110%, indicating that the analyte loss during the pretreatment of the kit was minimal and the accuracy was high.

[0037] Table 6 Recovery rate study of the present invention The above experiments show that the kit of the present invention can efficiently and stably perform pretreatment of fat-soluble vitamins, and is suitable for large-scale clinical testing applications.

[0038] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0039] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing magnetic beads, characterized in that, Includes the following steps: S1. After washing the carboxyl magnetic beads, disperse them in a buffer solution; then add EDC and NHS to activate the carboxyl groups; then add nitrochlorobenzene and triethylamine for a coupling reaction, and terminate the reaction with ethanolamine. S2, the coupling reaction product is washed and dispersed in deionized water, pyrrolidone, ammonium persulfate and porogen are added, and a polymerization reaction is carried out to form a porous polymer; finally, after washing and drying, magnetic beads are obtained.

2. The method for preparing magnetic beads according to claim 1, characterized in that, The average particle size of the carboxyl magnetic beads is 10 μm.

3. The method for preparing magnetic beads according to claim 1, characterized in that, The pore-forming agent is a mixed solution of ethanol and water in a volume ratio of 2:

1.

4. A magnetic bead, characterized in that, The magnetic beads were prepared using the method described in any one of claims 1-3.

5. The application of the magnetic beads according to claim 4 in the preparation of a pretreatment kit for fat-soluble vitamins.

6. A pretreatment kit for fat-soluble vitamins, characterized in that, include: Magnetic bead suspension: comprising anhydrous ethanol and the magnetic beads as described in claim 4; Equilibrium solution: consists of methanol and ultrapure water; Eluent 1: consists of methanol and ultrapure water; Eluent 2: consists of methanol and ultrapure water; Eluent: includes isopropanol and acetonitrile; And a porous pre-assembled plate containing the magnetic bead suspension, balancing liquid, rinsing liquid 1, rinsing liquid 2 and elution liquid.

7. The fat-soluble vitamin pretreatment kit according to claim 6, characterized in that, The concentration of magnetic beads in the magnetic bead suspension is 10 mg / mL.

8. The fat-soluble vitamin pretreatment kit according to claim 6, characterized in that, In the equilibrium solution, the concentration of methanol is 30 wt%; in both eluent 1 and eluent 2, the concentration of methanol is 20 wt%; and in the elution solution, the volume ratio of isopropanol to acetonitrile is 1:

3.

9. A fat-soluble vitamin pretreatment kit according to claim 6, characterized in that, The multi-well pre-packed plate includes 96 wells in 8 rows and 12 columns. The reagents are configured in the wells of the pre-packed plate as follows: Columns 1 and 7 are sample loading wells with a volume of 510 μL; columns 2 and 8 are magnetic bead suspension wells with a volume of 150 μL; columns 3 and 9 are equilibration buffer wells with a volume of 500 μL; columns 4 and 10 are eluent 1 wells with a volume of 500 μL; columns 5 and 11 are eluent 2 wells with a volume of 500 μL; and columns 6 and 12 are elution buffer wells with a volume of 150 μL.

10. The use of the kit according to any one of claims 6-9 in the isolation of fat-soluble vitamins from serum or plasma.

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