96-hole preassembled plate bile acid spectrum pretreatment kit and preparation method thereof

Abstract

The invention discloses a 96-hole preassembled plate bile acid spectrum pretreatment kit and a preparation method thereof, and belongs to the technical field of in-vitro diagnostic reagents. The kit comprises magnetic bead suspension liquid, equilibrium liquid, leacheate 1, leacheate 2 and eluent, wherein the magnetic bead suspension liquid comprises absolute ethyl alcohol and magnetic beads; the equilibrium liquid comprises methanol, formic acid and water; the leacheate 1 comprises formic acid and water; the leacheate 2 comprises methanol and water; the eluent comprises methanol and water; all the reagents are loaded in holes of the preassembled plate; wherein the magnetic bead takes FeO as an inner core, the surface of the magnetic bead is modified with a carboxyl functional group and a porous polymer, the magnetic bead can be specifically combined with a bile acid spectrum in human serum or plasma, and efficient separation and release of an object to be detected can be realized by combining a magnetic separation technology; the kit 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 the bile acid spectrum.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically relating to a 96-well pre-packed bile acid spectrum pretreatment kit and its preparation method. Background Technology

[0002] Bile acids are important products of cholesterol metabolism, and abnormal serum concentrations are closely related to hepatobiliary diseases, intestinal diseases, and metabolic syndromes (such as cirrhosis, cholestasis, and obesity). In clinical testing, bile acids often bind to serum proteins to form complexes, requiring pretreatment steps to release and purify them from their bound state for accurate quantitative analysis.

[0003] Traditional pretreatment methods (such as liquid-liquid extraction and solid-phase extraction columns) suffer from problems such as cumbersome operation, long processing time, low extraction efficiency, and significant matrix interference. For example, liquid-liquid extraction requires multiple shaking and centrifugation cycles, which can easily lead to analyte loss; traditional solid-phase extraction columns lack specificity for the adsorption of hydrophobic bile acids and are easily affected by other lipid-soluble substances. Therefore, developing an efficient, stable, and easy-to-operate pretreatment tool is of great significance for improving the accuracy and efficiency of bile acid spectroscopy detection. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a 96-well pre-packed bile acid spectrometry pretreatment kit and its preparation method, thereby solving the problems in the prior art.

[0005] The objective of this invention can be achieved through the following technical solutions: A magnetic bead with Fe3O4 as its core and its surface modified with carboxyl functional groups and a porous polymer, wherein the porous polymer is polymerized by pyrrolidone polymerization.

[0006] Furthermore, the preparation process of the magnetic beads includes: After washing the carboxyl magnetic beads, they were dispersed in a buffer solution; then EDC and NHS were added to activate the carboxyl groups; nitrochlorobenzene was then added to carry out a coupling reaction, and the reaction was terminated using Tris-HCl buffer. The coupling reaction product was washed and dispersed in deionized water, and pyrrolidone, ammonium persulfate and porogen were added to carry out a polymerization reaction to form a porous polymer; finally, magnetic beads were obtained after washing and drying.

[0007] Furthermore, the pore-forming agent is a mixed solution of anhydrous ethanol and n-hexane.

[0008] Furthermore, the polymerization reaction is carried out at a temperature of 60°C and in a nitrogen atmosphere.

[0009] The above-mentioned magnetic beads are used in the preparation of bile acid spectrum pretreatment kits.

[0010] A bile acid pretreatment kit, comprising: Magnetic bead suspension: comprising anhydrous ethanol and the aforementioned magnetic beads; Equilibrium solution: includes methanol, formic acid, and water; Rinse solution 1: consists of formic acid and water; Eluent 2: consists of methanol and water; Eluent: consists of methanol and water; And a porous pre-assembled plate for carrying 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 vt and the concentration of formic acid is 0.05 vt; in the eluent 1, the concentration of formic acid is 0.05 vt; in the eluent 2, the concentration of methanol is 40 vt; and in the elution solution, the concentration of methanol is 20 vt.

[0013] Furthermore, the porous pre-packed plate includes 96 wells arranged in 8 rows and 12 columns, and the reagent is configured in the wells of the pre-packed plate as follows: Columns 1 and 7 are sample loading wells with a volume of 210 μL; columns 2 and 8 are magnetic bead suspension wells with a volume of 200 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 200 μL; and columns 6 and 12 are elution buffer wells with a volume of 150 μL.

[0014] The above-mentioned kit is used in the separation of bile acid profiles from serum or plasma.

[0015] The beneficial effects of this invention are: 1. In the kit of the present invention, the adsorption capacity for bile acids is significantly improved by the porous structure and special functional groups on the surface of the magnetic beads, and the extraction time is shortened to less than 30 minutes.

[0016] 2. The magnetic bead suspension in the kit of this invention has been optimized and its performance remains stable after one year of storage at room temperature, with an extraction efficiency decrease of ≤5.0% (see Table 4), indicating good stability.

[0017] 3. The pre-loaded plate design of this invention is compatible with automated instruments, reducing human error and making it suitable for high-throughput detection; moreover, the eluent is compatible with LC-MS / MS systems and can be directly injected after simple nitrogen blowing and reconstitution. Attached Figure Description

[0018] 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.

[0019] 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 This is a physical image of the 96-hole pre-mounted plate and magnetic sleeve of the present invention; Figure 4 This is a schematic diagram of the 96-well pre-packed bile acid spectrometry pretreatment kit of the present invention. Detailed Implementation

[0020] 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.

[0021] In this embodiment, some of the raw materials are sourced as follows: 1) 5 μm carboxyl functionalized magnetic beads: Nanjing Dongna Biotechnology Co., Ltd., brand name: MagBeads® 5 μm carboxyl magnetic beads.

[0022] 2) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), CAS: 25952-53-8; 3) N-hydroxysuccinimide (NHS), CAS: 6066-82-6; 4) p-Nitrochlorobenzene, CAS: 100-00-5; 5) N-vinylpyrrolidone (NVP), CAS: 88-12-0; 6) Ammonium persulfate (APS), CAS: 7727-54-0; 7) N,N-dimethylformamide (DMF), CAS: 68-12-2; 8) Dimethyl sulfoxide (DMSO), CAS: 67-68-5; 9) Anhydrous ethanol, CAS: 64-17-5; 10) n-Hexane, CAS: 110-54-3; 11) Acetone, CAS: 67-64-1; 12) Formic acid, CAS: 64-18-6; 13) Methanol, CAS: 67-56-1.

[0023] Example 1 The preparation process of a 96-well pre-packed plate bile acid spectrometry pretreatment kit is as follows: 1. Magnetic bead synthesis (1) Take 5 mL of 5 μm carboxyl magnetic beads (0.8 μmol / mg), wash 3 times with deionized water (centrifuge at 4000 rpm for 5 minutes), and wash once with 0.1M MES buffer (pH 6.0); (2) Add 4 mL of MES (2-morpholinoethanesulfonic acid) buffer, sonicate for 1 minute, then add 38.4 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 23 mg of NHS (N-hydroxysuccinimide), and stir at room temperature for 30 minutes (protected from light). (3) Add 2 mL of DMF (N,N-dimethylformamide) solution containing 12.6 mg of p-nitrochlorobenzene, and shake at 200 rpm for 12 hours (protected from light); (4) Add 1 mL of 1M Tris-HCl buffer (pH 7.4) to terminate the reaction, wash twice with DMF, three times with anhydrous ethanol, and twice with deionized water (centrifuge at 4000 rpm for 5 minutes each time). (5) Disperse in 5 mL of deionized water, add 240 μL of pyrrolidone, 11.5 mg of APS (ammonium persulfate) and pore-forming agent (1.5 mL of anhydrous ethanol + 1.5 mL of n-hexane), purge with nitrogen for 10 minutes, and then polymerize in an oil bath at 60°C for 5 hours. (6) After washing with anhydrous ethanol 3 times, deionized water 3 times, and acetone 1 time, the porous magnetic beads (zdyyjyk05k-2) were obtained by vacuum drying at 60°C for 8 hours.

[0024] like Figure 1 As shown, the synthesized target magnetic beads have a particle size of approximately 5 μm, exhibit regular morphology and uniform dispersion, and show no significant agglomeration or particle size shift due to multi-step surface modification; Figure 2As shown, in the Fourier transform infrared (FTIR) spectrum of the target magnetic beads, the Fourier transform infrared spectrum exhibits characteristic absorption peaks corresponding to each modification step: a characteristic stretching vibration peak of carboxyl group (-COOH) appears around 1710 cm⁻¹, confirming the structural integrity of the initial carboxyl magnetic beads and the reaction basis of the activated NHS active ester; asymmetric and symmetric stretching vibration peaks of nitro group (-NO₂) appear near 1530 cm⁻¹ and 1350 cm⁻¹, confirming that p-nitrochlorobenzene has been successfully coupled to the surface of the magnetic beads; a characteristic absorption peak of amide bond (-CONH-) in pyrrolidone polymer appears near 1650 cm⁻¹, and a stretching vibration peak of CN bond appears around 1270 cm⁻¹, indicating that pyrrolidone has polymerized to form a porous polymer network; the above characteristic peaks synergistically confirm that the surface of the magnetic beads has completed carboxyl functionalization modification and porous polymer layer construction, providing a dual basis of hydrophobic interaction and structural support for the specific adsorption of 15 bile acids in serum or plasma.

[0025] 2. Reagent preparation (1) Magnetic bead suspension: Weigh 30 mg of magnetic beads (zdyyjyk05k-2) and dissolve them in 3 mL of anhydrous ethanol to obtain a concentration of 10 mg / mL; (2) Equilibrium solution: 3 mL methanol + 5 μL formic acid + ultrapure water to a final volume of 10 mL (30 vt% methanol, containing 0.05 vt% formic acid); (3) Eluent 1: 5 μL formic acid + ultrapure water to a final volume of 10 mL (0.05 vt% formic acid solution); (4) Eluent 2: 4 mL methanol + 6 mL ultrapure water (40 wt% methanol solution); (5) Eluent: 2 mL methanol + 8 mL ultrapure water (20 vt% methanol solution).

[0026] 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 get the pre-packed plate kit. Figure 3 ).

[0027] 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.

[0028] 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 96-well pre-packaged plate and add 200 μL of serum sample and 10 μL of internal standard to the sample well (1 / 7); (2) Place the pre-loaded plate in a magnetic rod nucleic acid extractor (such as the ZDYYJYK-48 model), insert the magnetic rod sleeve, and run the "bile acid spectrum 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).

[0029] 2. Testing After the procedure, the eluent from 6 / 12 wells was collected, dried under nitrogen at 60°C, reconstituted, and injected into a high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) system to detect bile acid concentration.

[0030] Example 3 In this embodiment, the performance of the reagent kit in Example 1 is verified; 1. Extraction efficiency Pretreatment of serum samples (containing 15 bile acid subtypes) of known concentrations showed that, compared with traditional solid-phase extraction (SPE) methods, the extraction efficiency of the kit in Example 1 was ≥80% (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 this invention decreased by ≤5.0% (see Table 4), indicating good stability; Table 4 Stability Study 3. Precision 3.1 Intraday Precision 3.1.1 Specimens: Precision verification was performed using quality control materials. The samples contained two concentration levels: high and low.

[0031] 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).

[0032] 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.

[0033] 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).

[0034] 3.3 Results The intra-day and inter-day precision of this invention are shown in Table 5. It can be seen that the intra-day precision CV value ranges from 4.69% to 13.31%, and the inter-day precision CV value ranges from 3.92% to 13.89%. The overall precision meets the reliability requirements of clinical testing.

[0035] Table 5. Intra-day and Inter-day Precision Assessment 4. Recovery rate 4.1 Sample Preparation: Clinical samples were divided into three equal-volume aliquots. Two of these aliquots were then treated with a low concentration (CA: 15.14 nmol / L, CDCA: 45.17 nmol / L, DCA: 30.63 nmol / L, LCA: 9.02 nmol / L, UDCA: 14.69 nmol / L, GCA: 119.20 nmol / L, GCDCA: 184.09 nmol / L, GDCA: 182.24 nmol / L, GLCA: 14.78 nmol / L, GUDCA: 76.33 nmol / L, TCA: 15.21 nmol / L, TCDCA: 60.82 nmol / L, TDCA: 30.04 nmol / L, TLCA: 6.14 nmol / L, TUDCA: 6.24 nmol / L) and a high concentration (CA: 337.00 nmol / L). nmol / L, CDCA: 1001.60 nmol / L, DCA: 662.20 nmol / L, LCA: 207.06 nmol / L, UDCA: 331.23 nmol / L, GCA: 266687nmol / L, GCDCA: 3988.59 nmol / L, GDCA: 3994.75 nmol / L, GLCA: 334.33 nmol / L, GUDCA: 1698.88 nmol / L, TCA: 341.23 nmol / L, TCDCA: 1359.38 nmol / L, TDCA: 673.24 nmol / L, TLCA: 141.10 nmol / L, TUDCA: 140.71 Prepare recovered samples by adding equal volumes of the analyte standard solution (LStd and HStd, nmol / L) to the third sample; add the same volume of blank reagent without the analyte to the third sample as the baseline sample. Repeat the determination three times each for the baseline sample and the low and high concentration recovered samples.

[0036] 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.

[0037] 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.

[0038] 4.3 Results The average recovery rate of bile acid profiles at low concentrations was 94.7%-100.7%, and the average recovery rate at high concentrations was 96.0%-100.8%. The overall recovery rate was within the ideal range of 85%-110%, indicating that the analyte loss during the kit pretreatment process was minimal and the accuracy was high.

[0039] Table 6 Recovery rate study of the present invention The above examples demonstrate that the kit of the present invention can efficiently and stably perform pretreatment of catecholamines and their metabolites, making it suitable for large-scale clinical testing applications. 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.

[0040] 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 magnetic bead, characterized in that, The core is Fe3O4, and the surface is modified with carboxyl functional groups and porous polymers, which are polymerized by pyrrolidone.

2. The magnetic bead according to claim 1, characterized in that, The preparation process of the magnetic beads includes: After washing the carboxyl magnetic beads, they were dispersed in a buffer solution; then EDC and NHS were added to activate the carboxyl groups; nitrochlorobenzene was then added to carry out a coupling reaction, and the reaction was terminated using Tris-HCl buffer. The coupling reaction product was washed and dispersed in deionized water, and pyrrolidone, ammonium persulfate and porogen were added to carry out a polymerization reaction to form a porous polymer; finally, magnetic beads were obtained after washing and drying.

3. The magnetic bead according to claim 2, characterized in that, The pore-forming agent is a mixed solution of anhydrous ethanol and n-hexane.

4. The magnetic bead according to claim 2, characterized in that, The polymerization reaction was carried out at a temperature of 60°C under a nitrogen atmosphere.

5. The use of the magnetic beads according to any one of claims 1-4 in the preparation of a bile acid spectrometry pretreatment kit.

6. A bile acid pretreatment kit, characterized in that, include: Magnetic bead suspension: comprising anhydrous ethanol and magnetic beads as described in any one of claims 1-4; Equilibrium solution: includes methanol, formic acid, and water; Rinse solution 1: consists of formic acid and water; Eluent 2: consists of methanol and water; Eluent: consists of methanol and water; And a porous pre-assembled plate for carrying the magnetic bead suspension, balancing liquid, rinsing liquid 1, rinsing liquid 2 and elution liquid.

7. The bile acid spectrum 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 bile acid spectrum pretreatment kit according to claim 6, characterized in that, The equilibrium solution contains 30 wt% methanol and 0.05 wt% formic acid; the eluent 1 contains 0.05 wt% formic acid; the eluent 2 contains 40 wt% methanol; and the elution solution contains 20 wt% methanol.

9. A bile acid spectrum pretreatment kit according to claim 6, characterized in that, The multi-well pre-packed plate includes 96 wells arranged in 8 rows and 12 columns. The reagent is configured in the wells of the pre-packed plate as follows: Columns 1 and 7 are sample loading wells with a volume of 210 μL; columns 2 and 8 are magnetic bead suspension wells with a volume of 200 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 200 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 separation of bile acid profiles in serum or plasma.

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