96-hole preassembled plate catecholamine and metabolite pretreatment kit thereof
By modifying Fe3O4 magnetic beads with a silicon-based support, carboxyl-phenylboronic acid bifunctional groups, and porous polymers, combined with a 96-well pre-packed plate design, the cumbersome and inefficient pretreatment methods for catecholamines were solved, achieving efficient and accurate detection of catecholamines and their metabolites.
Patent Information
- Application Number
- CN202511858452.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-02-06
AI Technical Summary
Existing pretreatment methods for catecholamines and their metabolites are cumbersome, time-consuming, have low extraction efficiency, and are easily affected by matrix interference, thus affecting detection accuracy.
The magnetic beads, which are formed by modifying the surface of Fe3O4 magnetic beads with silicon-based carriers, carboxyl-phenylboronic acid bifunctional groups and porous polymers, are used in the design of 96-well pre-packed plates. Combined with specific solution preparation, they achieve efficient adsorption and automated extraction.
It significantly improves the specific adsorption capacity of catecholamines and their metabolites, shortens the extraction time, reduces human operation errors, is suitable for high-throughput detection, and improves detection accuracy and efficiency.
Smart Images
Figure CN121476484A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of in vitro diagnostic reagents, and particularly relates to a 96-well preloaded plate catecholamine and metabolite pretreatment kit. BACKGROUND
[0002] Catecholamine and its metabolites (dopamine (DA), adrenaline (E), norepinephrine (NE), methoxyepinephrine (MN), methoxy-norepinephrine (NMN), 3-methoxytyramine (3-MT)) are important neurotransmitters and hormones in the human body, and their abnormal concentrations are closely related to hypertension, pheochromocytoma, Parkinson's disease and many other diseases. In clinical detection, catecholamine and its metabolites are low in content in samples and are easily interfered by matrix, and thus need to be separated and purified through a pretreatment step to enable accurate quantitative analysis.
[0003] Traditional pretreatment methods (such as liquid-liquid extraction and solid-phase extraction column) have problems such as complicated operation, long time consumption, low extraction efficiency, and significant matrix effect. For example, liquid-liquid extraction needs to use a large amount of organic solvent, which is easy to cause environmental pollution and has poor repeatability; and traditional solid-phase extraction columns have insufficient specificity in adsorbing catecholamine, and are easily interfered by other polar substances. Therefore, it is of great significance to develop a pretreatment tool that is efficient, stable and easy to operate to improve the accuracy and efficiency of catecholamine and its metabolite detection. SUMMARY
[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a 96-well preloaded plate catecholamine and metabolite pretreatment kit to solve the problems in the prior art.
[0005] The purpose of the present application can be achieved by the following technical solutions: A magnetic bead, taking Fe3O4 as a core, and being modified on the surface with a silicon-based carrier, a carboxyl-phenylboronic acid bifunctional group and a porous polymer, wherein the porous polymer is formed by copolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate.
[0006] Further, the preparation process of the magnetic bead comprises: S1, modifying the silicon-based magnetic bead with APTES to obtain an aminated magnetic bead; S2, after dispersing the aminated magnetic bead in an organic solvent, adding succinic anhydride and triethylamine to introduce a carboxyl group through reaction, and obtaining a carboxyl-modified magnetic bead; S3, after dispersing the carboxyl-modified magnetic bead in a buffer solution, adding EDC and NHS to activate the carboxyl group, and then coupling with 4-carboxyphenylboronic acid to obtain a coupling reaction product; S4, the coupling reaction product is dispersed in anhydrous ethanol, glycidyl methacrylate, ethylene glycol dimethacrylate, pore-forming agent and AIBN are added, and a copolymerization reaction is carried out to form a porous polymer layer. After drying, the target magnetic beads are obtained.
[0007] Furthermore, the pore-forming agent is a mixed solution of toluene and n-heptane.
[0008] Furthermore, the polymerization reaction is carried out at a temperature of 65°C and in a nitrogen atmosphere.
[0009] The application of the aforementioned magnetic beads in the pretreatment kit for preparing catecholamines and their metabolites.
[0010] A pretreatment kit for catecholamines and their metabolites includes: Magnetic bead suspension: comprising anhydrous ethanol and the aforementioned magnetic beads; Equilibrium solution: includes acetonitrile, formic acid, and water; Rinse solution 1: consists of acetonitrile and water; Rinse solution 2: consists of acetonitrile and water; Eluent: includes acetonitrile, formic acid 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] Further, in the equilibrium solution, the concentration of acetonitrile is 30 vt and the concentration of formic acid is 0.5 vt; in the eluent 1, the concentration of acetonitrile is 20 vt; in the eluent 2, the concentration of acetonitrile is 20 vt; and in the elution solution, the concentration of acetonitrile is 20 vt and the concentration of methanol is 0.5 vt.
[0013] Furthermore, the porous pre-packed plate includes 96 wells arranged in 8 rows and 12 columns; the reagent positions in the wells of the pre-packed plate are configured as follows: Columns 1 and 7 are sample loading wells with a volume of 240 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 catecholamines and their metabolites 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 catecholamines, and shorten the extraction time to less than 30 minutes; 2. The pre-loaded plate design of this invention is compatible with automated instruments, reduces human error, and is suitable for high-throughput detection; moreover, it is compatible with LC-MS / MS systems after desolvation, and can be directly injected after simple nitrogen blowing and reconstitution.
[0016] 3. The kit of the present invention is characterized by simple operation, high extraction efficiency and good stability. It can significantly shorten the pretreatment time and improve the detection accuracy, providing an efficient solution for the clinical detection of catecholamines and their metabolites. Attached Figure Description
[0017] 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.
[0018] Figure 1 This is a transmission electron microscope image of the magnetic beads of the present invention.
[0019] Figure 2 This is the Fourier transform infrared spectrum of the magnetic bead.
[0020] Figure 3 This is a picture of the 96-hole pre-installed plate and magnetic sleeve.
[0021] Figure 4 This is a schematic diagram of the 96-well pre-packed plate catecholamine and its metabolite pretreatment kit for detection. Detailed Implementation
[0022] 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.
[0023] In this embodiment, some of the raw materials are sourced as follows: 1) 5 μm amino-functionalized magnetic beads: Nanjing Dongna Biotechnology Co., Ltd., brand name: MagBeads® 5 μm amino magnetic beads.
[0024] 2) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), CAS: 25952-53-8; 3) N-hydroxysuccinimide (NHS), CAS: 6066-82-6; 4) Succinic anhydride, CAS: 108-30-5; 5) 4-Carboxyphenylboronic acid, CAS: 14047-29-1; 6) Glycidyl methacrylate (GMA), CAS: 106-91-2; 7) Ethylene glycol dimethacrylate (EGDMA), CAS: 97-90-5; 8) Azobisisobutyronitrile (AIBN), CAS: 78-67-1; 9) Toluene, CAS: 108-88-3; 10) n-Heptane, CAS: 142-82-5; 11) DMF, CAS: 68-12-2; 12) Anhydrous ethanol, CAS: 64-17-5; 13) Triethylamine, CAS: 121-44-8 14) Acetone, CAS: 67-64-1; 15) Acetonitrile, CAS: 75-05-8; 16) Formic acid, CAS: 64-18-6 17) Ammonium acetate, CAS: 631-61-8.
[0025] Example 1 The preparation process of the 96-well pre-packed plate catecholamine and metabolite pretreatment kit includes: 1. Magnetic bead synthesis (1) Carboxyl modification: Amino magnetic beads (specific surface area of about 50 m² / g) were dispersed in 20 mL DMF, 1.5 g succinic anhydride and 0.5 mL triethylamine were added, and the mixture was stirred at room temperature for 24 hours. After magnetic separation, the mixture was washed with DMF (3×20 mL), anhydrous ethanol (3×20 mL) and deionized water (3×20 mL) in sequence, and dried under vacuum at 60 °C for 6 hours to obtain carboxyl modified magnetic beads.
[0026] (2) Coupling with phenylboronic acid: 1 g of carboxyl-modified magnetic beads were dispersed in 20 mL of 0.1 M MES (2-morpholinoethanesulfonic acid) buffer (pH 5.5), 480 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 287 mg of NHS (N-hydroxysuccinimide) were added, and the mixture was activated at room temperature for 30 minutes; 415 mg of 4-carboxyphenylboronic acid was added, and the mixture was stirred at room temperature for 12 hours; after magnetic separation, the mixture was washed with 0.1 M PBS buffer (pH 7.4, 3×20 mL) and deionized water (3×20 mL) to obtain Fe3O4@SiO2-COOH-PBA.
[0027] (3) Construction of porous polymer layer: 1 g of the above magnetic beads (Fe3O4@SiO2-COOH-PBA) was dispersed in 10 mL of anhydrous ethanol, and 2 mL of glycidyl methacrylate, 1 mL of ethylene glycol dimethacrylate, pore-forming agent (2 mL of toluene + 1 mL of n-heptane) and 50 mg of AIBN (azobisisobutyronitrile) were added. After purging with nitrogen for 15 minutes, the mixture was stirred in an oil bath at 65 °C for 6 hours (300 rpm). After magnetic separation, the mixture was washed with acetone (3×20 mL), anhydrous ethanol (3×20 mL), and deionized water (3×20 mL) in sequence, and dried under vacuum at 60 °C for 12 hours to obtain porous magnetic beads (zdyyjyk10k-3).
[0028] 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 2 As shown, in its Fourier transform infrared spectrum, a stretching vibration peak of amino (-NH2) appears in the 3300-3400 cm⁻¹ region, confirming the presence of amino groups; a characteristic absorption peak of carboxyl (-COOH) appears around 1720 cm⁻¹, indicating that the succinic anhydride reaction effectively achieves carboxyl modification; a benzene ring skeleton vibration peak appears in the 1600-1500 cm⁻¹ region, and a characteristic peak of boric acid group (-B(OH)2) appears near 1350 cm⁻¹, confirming the successful coupling of 4-carboxyphenylboronic acid; characteristic stretching vibration peaks of ester bond (-COO-) appear near 1250 cm⁻¹ and 1730 cm⁻¹, confirming that glycidyl methacrylate and ethylene glycol dimethacrylate copolymerize to form a porous polymer layer. The above characteristic peaks together confirm that the magnetic bead surface has completed multilayer modification of silicon-based support, carboxyl-phenylboronic acid bifunctional groups and porous polymer, providing a structural basis for the specific adsorption of catecholamines and their metabolites.
[0029] 2. Reagent preparation (1) Magnetic bead suspension: Weigh 30 mg of magnetic beads (zdyyjyk10k-3) and dissolve them in 3 mL of anhydrous ethanol to obtain a concentration of 10 mg / mL; (2) Equilibrium solution: 3 mL acetonitrile + 50 μL formic acid + ultrapure water to a final volume of 10 mL (30 vt% acetonitrile, containing 0.5 vt% formic acid); (3) Rinse solution 1: 2 mL acetonitrile + 8 mL ultrapure water (20 vt% acetonitrile solution); (4) Rinse solution 2: 2 mL acetonitrile + 8 mL ultrapure water (20 vt% acetonitrile solution); (5) Eluent: 2 mL acetonitrile + 50 μL formic acid + ultrapure water to a final volume of 10 mL (20 vt% acetonitrile, containing 0.5 vt% formic acid).
[0030] 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).
[0031] 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.
[0032] Example 2 This embodiment describes the usage process of the reagent kit, such as... Figure 4 As shown, it includes the following steps: 1. Sample pretreatment (1) Tear open the aluminum film of the pre-packaged plate, add 200 μL of serum sample, 10 μL of internal standard and 30 mM ammonium acetate to the sample well (1 / 7), and mix thoroughly; (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 "Extraction of Catecholamines and Their Metabolites" program (parameters are shown in Table 2). Table 2 Extraction program parameters The speed is 5 Hz (5 times up and down per second).
[0033] 2. Testing After the procedure, the eluent from wells 6 / 12 was collected, dried under nitrogen at 60°C, reconstituted, and injected into an LC-MS / MS system to detect the concentrations of catecholamines and their metabolites.
[0034] Example 3 In this embodiment, the performance of the reagent kit prepared in Example 1 is verified according to the usage method described in Example 2. The verification process includes: 1. Extraction efficiency: Pretreatment of serum samples with known concentrations (containing 6 catecholamines and their metabolites) showed that, compared with traditional solid-phase extraction methods, the extraction efficiency of the kit prepared in this invention (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 the present invention (the kit prepared in Example 1) decreased by ≤5.0% (see Table 4), indicating that it has 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.
[0035] 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).
[0036] 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.
[0037] 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).
[0038] 3.3 Results The intra-day and inter-day precision of this invention are shown in Table 5: the intra-day precision CV value of catecholamines and their metabolites ranges from 4.66% to 12.34%, and the inter-day precision CV value ranges from 3.92% to 13.89%. The overall precision meets the reliability requirements for clinical testing.
[0039] 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 (DA: 158.50 ng / mL, E: 387.17 ng / mL, NE: 509.44 ng / mL, MN: 100.00 ng / mL, NMN: 101.27 ng / mL, 3-MT: 10.85 pg / mL) and high concentrations (DA: 6781.11 ng / mL, E: 16661.11 ng / mL, NE: 21355.55 ng / mL, MN: 4375.00 ng / mL, NMN: 4278.33 ng / mL, 3-MT: 150.50 pg / mL) were each measured three times.
[0040] 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.
[0041] 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.
[0042] 4.3 Results The average recovery rates of catecholamines and their metabolites at low concentrations were 93.6%-104.3%, and the average recovery rates at high concentrations were 91.6%-105.0%. The overall recovery rates were within the ideal range of 85%-110%, indicating that the analyte loss during the kit pretreatment process was minimal and the accuracy was high.
[0043] Table 6 Recovery rate study of the present invention The above experiments 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.
[0044] 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.
[0045] 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, With Fe3O4 as the core, the surface is modified with a silicon-based carrier, carboxyl-phenylboronic acid bifunctional groups, and a porous polymer, which is formed by copolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate.
2. The magnetic bead according to claim 1, characterized in that, The preparation process of the magnetic beads includes: S1, silicon-based magnetic beads are modified with APTES to obtain aminated magnetic beads; S2, after the aminated magnetic beads are dispersed in an organic solvent, succinic anhydride and triethylamine are added, and the reaction introduces carboxyl groups to obtain carboxyl-modified magnetic beads; S3, after the carboxyl-modified magnetic beads were dispersed in buffer solution, EDC and NHS were added to activate the carboxyl groups, and then coupled with 4-carboxyphenylboronic acid to obtain the coupling reaction product; S4, the coupling reaction product is dispersed in anhydrous ethanol, glycidyl methacrylate, ethylene glycol dimethacrylate, pore-forming agent and AIBN are added, and a copolymerization reaction is carried out to form a porous polymer layer. After drying, the target magnetic beads are obtained.
3. The magnetic bead according to claim 1, characterized in that, The pore-forming agent is a mixed solution of toluene and n-heptane.
4. The magnetic bead according to claim 1, characterized in that, The polymerization reaction was carried out at a temperature of 65°C under a nitrogen atmosphere.
5. The use of the magnetic beads according to any one of claims 1-4 in the pretreatment kit for preparing catecholamines and their metabolites.
6. A pretreatment kit for catecholamines and their metabolites, 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 acetonitrile, formic acid, and water; Rinse solution 1: consists of acetonitrile and water; Rinse solution 2: consists of acetonitrile and water; Eluent: includes acetonitrile, formic acid 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 pretreatment kit for catecholamines and their metabolites according to claim 6, characterized in that, The concentration of magnetic beads in the magnetic bead suspension is 10 mg / mL.
8. The pretreatment kit for catecholamines and their metabolites according to claim 6, characterized in that, In the equilibrium solution, the concentration of acetonitrile is 30 vt and the concentration of formic acid is 0.5 vt; in the eluent 1, the concentration of acetonitrile is 20 vt; in the eluent 2, the concentration of acetonitrile is 20 vt; in the elution solution, the concentration of acetonitrile is 20 vt and the concentration of methanol is 0.5 vt.
9. A pretreatment kit for catecholamines and their metabolites according to claim 6, characterized in that, The porous pre-packed plate comprises 96 wells in 8 rows and 12 columns; the reagents are positioned in the wells of the pre-packed plate as follows: Columns 1 and 7 are sample loading wells with a volume of 240 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 catecholamines and their metabolites from serum or plasma.