Fentanyl artificial hapten, artificial antigen, and preparation method and application thereof

Abstract

The application discloses a fentanyl artificial hapten, a fentanyl artificial antigen and a preparation method and application thereof. The fentanyl artificial hapten of the application retains the characteristic structure of fentanyl to the maximum extent, and has an active group which can be coupled with a carrier protein and can serve as an antigenic determinant; the fentanyl artificial antigen prepared further can be used for immunization to obtain an anti-fentanyl antibody with high affinity, high sensitivity and high specificity, and the titer of the immune serum obtained by immunizing a New Zealand white rabbit is as high as 1:85000. The antigen and antibody can be used for enzyme-linked immunoassay, immunochromatography and the like, have the characteristics of high sensitivity, high specificity, strong operability and the like, and can be used for screening and detection of fentanyl components in various complex samples.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to a fentanyl artificial hapten, an artificial antigen, its preparation method, and its application. Background Technology

[0002] Fentanyl is a potent opioid analgesic. For regulatory purposes, there is a need to develop a rapid method for detecting fentanyl.

[0003] Previously, fentanyl detection mainly relied on high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), and mass spectrometry (MS). However, these methods had drawbacks such as expensive instruments, time-consuming testing, and the need for professional technicians to operate them, failing to meet the requirements of modern detection for speed and accuracy. In recent years, with the development of immunoassay technology, the determination of fentanyl using colloidal gold method, immunofluorescence method, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence method has become the mainstream in the current primary screening market.

[0004] Immunoassay technology can overcome all the above shortcomings. It is an analytical method that uses the specific binding reaction of antigen and antibody to detect various substances (drugs, hormones, proteins, microorganisms, etc.). The key to establishing immunoassay methods for small molecule compounds is the ability to manufacture antibodies with high affinity and high specificity for small molecule compounds. However, since most small molecule compounds (molecular weight less than 1000), including fentanyl, are not immunogenic, that is, they lack T cell epitopes and cannot directly induce the production of specific antibodies in animals, small molecule substances are called haptens. Through appropriate chemical modification, a linker arm with an active group at the end is added to a certain position of the hapten molecule structure, and then it is combined with a macromolecular carrier to generate a hapten-carrier conjugate (i.e., artificial antigen). Artificial antigens can indirectly induce the proliferation and differentiation of B cells through T cell epitopes, thereby producing specific antibodies. Therefore, the efficient synthesis of artificial antigens is a prerequisite and key to ensuring immunoassay. At the same time, efficient artificial antigens and highly specific antibodies can be used to prepare colloidal gold immunochromatographic test strips to meet the requirements of rapid on-site detection. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of the prior art by providing a fentanyl artificial hapten that retains the characteristic structure of fentanyl to the greatest extent and has an active group that can couple with a carrier protein, thus serving as an antigenic determinant.

[0006] A fentanyl artificial hapten, the molecular structure of which is shown in formula (I):

[0007]

[0008] (I).

[0009] A second objective of this invention is to provide a method for preparing the above-mentioned fentanyl artificial hapten, comprising the following steps:

[0010] Step (1): Dissolve β-hydroxyfentanyl in N,N-dimethylformamide, add sodium hydride, and stir at room temperature for 0.5 hours; then add ethyl 5-bromopentanoate, reflux at 65°C and stir for 17 hours. After the reaction is complete, remove the solvent and extract with ethyl acetate, filter, dry and purify to obtain oil A; wherein the molar ratio of β-hydroxyfentanyl to sodium hydride is (1~1.5):3, and the molar ratio of β-hydroxyfentanyl to ethyl 5-bromopentanoate is 1:(1.5~2);

[0011] Under these reaction conditions, β-hydroxyfentanyl is readily soluble in N,N-dimethylformamide, resulting in a high yield and purity of oily compound A, and a relatively simple post-processing procedure.

[0012] Preferably, after filtration and drying, the oily substance A is purified by thin-layer chromatography. The chromatographic solution is ethyl acetate:petroleum ether in a volume ratio of 1:4, and the product Rf = 0.2. Thin-layer chromatography can remove byproducts or impurities in the reaction system to achieve the purpose of general purification.

[0013] Step (2): Dissolve oily substance A in tetrahydrofuran and anhydrous methanol, add 1-1.5N sodium hydroxide aqueous solution, stir rapidly at room temperature for 5 hours, adjust pH to 4-5 with 1N hydrochloric acid solution, extract with dichloromethane multiple times, combine organic phases, dry, filter, remove dichloromethane, and purify to obtain the fentanyl artificial hapten; wherein the mass-volume ratio of oily substance A, tetrahydrofuran, anhydrous methanol and sodium hydroxide aqueous solution is 100mg: (1.5-2)ml: (1.8-2)ml: 10ml.

[0014] Preferably, under these reaction conditions, oily substance A is more easily hydrolyzed, and subsequent processing procedures are simpler and easier to purify.

[0015] Preferably, extraction with dichloromethane three times can maximize the extraction of the target product.

[0016] Preferably, the purification is performed using thin-layer chromatography; thin-layer chromatography can remove byproducts or impurities in the reaction system to achieve approximate purification; the chromatography solvent is 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia water in a volume ratio of 8:1:10:1, and the product Rf = 0.6.

[0017] The reaction formula for the method is shown below:

[0018]

[0019] By using the above method, a linker arm is introduced onto the hydroxyl group of β-hydroxyfentanyl. Introducing a linker arm at this modification site can preserve the characteristic structure of fentanyl to the greatest extent, and the modification site is as far away from the characteristic functional groups of fentanyl as possible, so as to expose its characteristic parts to the outside to avoid interference with specific antigenic determinants and maximize recognition by the immune body.

[0020] Compared with the use of a ring-shaped connecting arm, the connecting arm used in this invention is chain-shaped. The connecting arm is of appropriate length, which ensures that the fentanyl hapten small molecule can be fully exposed on the surface of the artificial antigen, and minimizes the recognition of the connecting arm by T cells during immunization. In this way, the antibodies obtained by immunization have stronger specificity and affinity for fentanyl.

[0021] The third objective of this invention is to provide a fentanyl artificial antigen, obtained by conjugating the above-mentioned fentanyl artificial hapten with a carrier protein; its molecular structure is shown in formula (II):

[0022]

[0023] (II);

[0024] In formula (II), BSA is bovine serum albumin.

[0025] A fourth objective of this invention is to provide a method for preparing the above-mentioned fentanyl artificial antigen, comprising: binding the fentanyl artificial hapten to bovine serum albumin by a mixed anhydride method to obtain the fentanyl artificial antigen.

[0026] Specifically, the preparation of fentanyl artificial antigen using the mixed anhydride method includes the following steps:

[0027] Step (1): Dissolve the fentanyl artificial hapten in a mixture of DMF and triethylamine and stir in an ice bath. Add isobutyl chloroformate during stirring and continue stirring in an ice bath for 2 to 2.5 hours. After the reaction is completed, centrifuge to obtain the supernatant.

[0028] Step (2): Add the supernatant obtained in step (1) to the bovine serum albumin solution, let the resulting mixture stand overnight at 3-5°C, and obtain the supernatant by dialysis and centrifugation to obtain the fentanyl artificial antigen.

[0029] Preferably, unless otherwise specified, the bovine serum albumin solution described in this invention is prepared by dissolving bovine serum albumin in 0.01M PBS buffer (pH 7.2~7.4).

[0030] Preferably, in step (2), the concentration of the bovine serum albumin solution is 5 mg / mL, and the volume ratio of the supernatant to the bovine serum albumin solution is 1:5~6.

[0031] This invention selects bovine serum albumin (BSA) as a macromolecular carrier, which has the following advantages compared with bovine gamma protein (BGG): ① Bovine serum albumin is the most commonly used carrier protein, has stronger immunogenicity, possesses numerous antigenic determinants, and easily induces a stronger immune response; ② Bovine serum albumin molecules contain a large number of lysine residues, have more free amino acids, and can maintain greater solubility under different pH values ​​and ionic strengths. When dissolved in organic solvents (such as N,N-dimethylformamide), its active groups remain soluble, thus preventing protein precipitation; ③ Bovine serum albumin has many lysine residues and contains hundreds of primary amine and carboxyl groups, which can serve as targets for cross-linking with cross-linking reagents such as glutaraldehyde, N-hydroxysuccinimide ester, and EDC. It is easy to couple with haptens, has high coupling efficiency, and is less likely to produce polyclonal antibodies, thus improving antibody specificity. The fentanyl artificial antigen formed by binding bovine serum albumin with fentanyl artificial hapten, after animal immunization, has better specificity for anti-fentanyl antibodies.

[0032] The fifth objective of this invention is to provide an anti-fentanyl antibody, obtained by animal immunization with the fentanyl artificial antigen, which can produce a specific immune response to fentanyl.

[0033] Experiments showed that immunizing New Zealand white rabbits with the fentanyl artificial antigen yielded an immune serum titer of 1:85000. This indicates that the fentanyl artificial antigen of the present invention can produce anti-fentanyl antibodies with high affinity, high sensitivity, and strong specificity, which can be used for the immunodetection and analysis of fentanyl.

[0034] A sixth objective of this invention is to provide the application of the above-mentioned anti-fentanyl antibody in the immunoassay of fentanyl in a sample.

[0035] Preferably, the sample includes a body fluid sample, a tissue sample, or an environmental sample. A body fluid sample refers to a sample of bodily fluids from mammals or humans, including urine, saliva, sweat, blood, etc.; a tissue sample refers to a sample of tissues from mammals or humans, including hair, nails, liver, etc.; and an environmental sample includes powder, residues on object surfaces, water samples, etc.

[0036] Preferably, the immune detection is an immune detection or analysis method that utilizes the principle of antigen-antibody binding, including enzyme-linked immunosorbent assay (ELISA), competitive enzyme-linked immunosorbent assay, or immunochromatographic detection.

[0037] Specifically, the anti-fentanyl antibody of this invention can be used to prepare a fentanyl competitive inhibition ELISA detection kit, comprising an anti-fentanyl antibody, fentanyl standards, and an enzyme-labeled secondary antibody. Due to the limitations of the molecular structure of fentanyl itself, the detection kit of this invention is prepared based on the principle of competitive inhibition ELISA. In use, the fentanyl standards are first coated, then the sample to be tested is added, followed by the anti-fentanyl antibody and the enzyme-labeled secondary antibody in sequence. The reaction is then carried out, the OD450 value is detected, and the competitive inhibition rate is calculated. The coating amount of the fentanyl standards is preferably based on the detection threshold of the anti-fentanyl antibody against fentanyl. The enzyme-labeled secondary antibody can be goat anti-mouse IgG-HRP.

[0038] Specifically, the anti-fentanyl antibody of this invention can be used to prepare a competitive enzyme-linked immunosorbent assay (ELISA) kit, comprising an ELISA plate coated with anti-fentanyl antibody, fentanyl standards, enzyme-labeled antigen, substrate, and stop solution. In use, the sample to be tested and the enzyme-labeled fentanyl antigen are added together to the ELISA plate for reaction. If fentanyl remains in the sample, it will compete with the enzyme-labeled fentanyl antigen for binding to the antibody on the plate. The substrate is then added, and the reaction is terminated. The colorimetric intensity is inversely proportional to the amount of fentanyl remaining in the sample. The fentanyl content in the sample is calculated based on a standard curve generated using the fentanyl standards.

[0039] Specifically, the anti-fentanyl antibody of the present invention can be used to prepare a fentanyl immunoassay test strip, comprising a sample pad, a labeling pad, a reaction membrane, and a sample absorption pad; the labeling pad is coated with the anti-fentanyl antibody labeled with colored substances such as latex, colloidal gold, or colored microspheres, and with colorless substances such as fluorescence; the detection line (T line) of the reaction membrane is coated with a fentanyl-bovine serum albumin complex, and the control line (C line) of the reaction membrane is coated with a secondary antibody. The test strip operates on a competitive principle, and the amount of fentanyl-bovine serum albumin complex coating is preferably based on the detection threshold of the anti-fentanyl antibody for fentanyl. During detection, if only the C line shows color, it indicates that the sample contains fentanyl and the detected fentanyl content is higher than the detection limit; if both the C and T lines show color, it indicates that the detected fentanyl content in the sample is lower than the detection threshold, or that the sample does not contain fentanyl; if neither the C nor the T line shows color, it indicates that the test strip has expired.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] The fentanyl artificial hapten of this invention features a novel modification site selection, and no precedent for artificial haptens with the same site has been found to date. The fentanyl artificial hapten of this invention retains the characteristic structure of fentanyl to the greatest extent possible and possesses an active group that can couple with carrier proteins, serving as an antigenic determinant. Further preparation of the fentanyl artificial antigen yields anti-fentanyl antibodies with high affinity, high sensitivity, and strong specificity. The titer of the immune serum obtained from immunizing New Zealand white rabbits is as high as 1:85000, which can be used for rapid and accurate immunodetection and immunoanalysis of fentanyl. Attached Figure Description

[0042] Figure 1 This is a flowchart illustrating the preparation process of fentanyl artificial antigen II according to the present invention.

[0043] Wherein, DMF represents N,N-dimethylformamide, NaH represents sodium hydride, THF represents tetrahydrofuran, MeOH represents anhydrous methanol, RT represents room temperature, Et3N represents triethylamine, and BSA represents bovine serum albumin, the same below;

[0044] Figure 2 This is the liquid chromatogram of fentanyl artificial hapten I of the present invention;

[0045] In the spectrum, the horizontal axis represents time, in minutes; the vertical axis represents the response value, in mAU.

[0046] Figure 3 This is the mass spectrum of the fentanyl artificial hapten I of this invention;

[0047] Wherein, Relative Abundance represents relative abundance; m / z represents mass-to-charge ratio;

[0048] Figure 4 Ultraviolet scans of fentanyl artificial hapten I, fentanyl artificial antigen II, and bovine serum albumin;

[0049] Where Abs represents the ultraviolet-visible absorption spectrum, and WL (nm) represents the wavelength (nm);

[0050] Figure 5 The flowchart for the preparation of fentanyl artificial antigen IV in Comparative Example 1 is shown.

[0051] Wherein, Pyridine represents pyridine, and DCC represents N,N'-dicyclohexylcarbodiimide, the same below;

[0052] Figure 6 The flowchart for the preparation of comparative example 2, fentanyl artificial antigen V;

[0053] BGG stands for bovine gamma protein, and the same applies below.

[0054] Figure 7 The flowchart for the preparation of comparative example 3, fentanyl artificial antigen VI;

[0055] Figure 8 The flowchart for the preparation of comparative example 4, fentanyl artificial antigen VII;

[0056] Figure 9 The flowchart for the preparation of comparative example 5, fentanyl artificial antigen VIII;

[0057] Figure 10 The flowchart for the preparation of comparative example 6, fentanyl artificial antigen IX;

[0058] Figure 11 This is a flowchart of the preparation process of comparative example 7, fentanyl artificial antigen X. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0060] Example 1

[0061] This embodiment provides a method for preparing fentanyl artificial antigen II (reaction process as follows) Figure 1 ), including the following steps:

[0062] (1) Preparation of artificial hapten I:

[0063] ① Dissolve 181 mg (0.514 mmol) of β-hydroxyfentanyl in 5 ml of N,N-dimethylformamide and place in a 50 ml round-bottom flask. Add 37 mg (1.542 mmol) of sodium hydride and stir at room temperature for 0.5 hours. Add 122 μL (0.771 mmol) of ethyl 5-bromopentanoate and reflux at 65 °C for 17 hours. After the reaction is complete, directly transfer to dryness to obtain 298 mg of yellow oil. Extract twice with 20 ml × 2 ethyl acetate, collect the organic phase, directly transfer to dryness to obtain 268 mg of yellow oil. Purify by thin-layer chromatography to obtain 196 mg of pale yellow oil A. The solvent and eluent are anhydrous ethanol.

[0064] The pale yellow oily substance A was analyzed by TLC with ethyl acetate:petroleum ether = 4:1 (volume ratio) as the chromatographic solvent, and the product Rf = 0.2.

[0065] The chromatographic solvent used in the thin-layer chromatography method was ethyl acetate:petroleum ether = 4:1 (volume ratio), and the product Rf = 0.2.

[0066] ② Dissolve 196 mg (0.408 mmol) of a pale yellow oily substance A in 2.94 ml of tetrahydrofuran and 3.53 ml of anhydrous methanol. Add 19.6 ml of 1N sodium hydroxide aqueous solution. The solution becomes turbid. Stir rapidly at room temperature for 5 hours. Adjust the pH to 4-5 with 1N hydrochloric acid solution. Extract three times with 30 ml of dichloromethane. Combine the organic phases, dry, filter, transfer to dryness, and purify by thin-layer chromatography to obtain 170 mg of fentanyl artificial hapten I. The solvent and eluent are anhydrous ethanol.

[0067] The fentanyl artificial hapten was detected by TLC with a chromatography buffer of 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia = 8:1:10:1, and the product Rf = 0.6.

[0068] The thin-layer chromatography method used a chromatographic solution of 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia water = 8:1:10:1, with anhydrous ethanol as the solvent and eluent, and the product Rf = 0.6.

[0069] The liquid chromatogram of fentanyl artificial hapten I is shown below. Figure 2 (Ultraviolet detector, wavelength 288 nm), the mass spectrum of fentanyl artificial hapten I is shown below. Figure 3 .

[0070] from Figure 2 It can be seen that the purity of the purified fentanyl artificial hapten reaches over 99.9%. Figure 3 It can be seen that the mass-to-charge ratio (m / z) of the M+H peak of the fentanyl artificial hapten obtained in this embodiment is 453.27, which is consistent with its theoretical relative molecular weight of 452. The mass-to-charge ratios (m / z) of its other two main fragment ion peaks are 336.27 and 376.12, respectively, which are consistent with the theoretical molecular weights of its two main fragments of 335 and 375. Based on the above data, it can be preliminarily determined that the final compound obtained in step ② is the fentanyl artificial hapten I designed in this invention.

[0071] (2) Preparation of fentanyl artificial antigen II:

[0072] ③ Place 170 mg (0.376 mmol) of fentanyl artificial hapten I in a 50 ml round-bottom flask, add 8.5 ml of N,N-dimethylformamide (DMF), then add 52 μL (0.376 mmol) of triethylamine, stir in an ice bath for 30 min, then add 97 μL (0.752 mmol) of isobutyl chloroformate, stir in an ice bath for 2 h, centrifuge after the reaction is complete, and collect the supernatant for later use.

[0073] ④ Weigh 14.5g (0.0405mol) disodium hydrogen phosphate dodecahydrate, 43.875g (0.75mol) sodium chloride, and 1.495g (0.00958mol) sodium dihydrogen phosphate dihydrate, dissolve them in double-distilled water, and bring the volume to 5.0L to obtain 0.01M PBS buffer with pH=7.4.

[0074] ⑤ Weigh 0.215g of bovine serum albumin and dissolve it in 43ml of PBS buffer from step ④ to obtain a bovine serum albumin solution with a concentration of 5mg / ml.

[0075] ⑥ Under rapid stirring, the supernatant from step ③ is slowly added dropwise to the bovine serum albumin solution. The volume ratio of the supernatant to the bovine serum albumin solution is 1:5. The resulting mixture is stored at 4°C overnight to obtain the artificial antigen mixture.

[0076] ⑦ Transfer the artificial antigen mixture into a dialysis bag and dialyze nine times with the PBS buffer from step ④. After dialysis, centrifuge and collect the supernatant to obtain artificial antigen II: fentanyl-bovine serum albumin conjugate. See the UV scan images before and after preparation of fentanyl artificial antigen II. Figure 4 .

[0077] Figure 4 In the diagram, curve a represents the UV scan of fentanyl artificial hapten I, curve b represents the UV scan of fentanyl artificial antigen II, and curve c represents the UV scan of bovine serum albumin (BSA). The maximum absorption wavelength of fentanyl artificial hapten I is 288 nm, that of BSA is 268 nm, and that of fentanyl artificial antigen II is 240 nm. Compared with fentanyl hapten I and BSA, the maximum absorption wavelength of fentanyl artificial antigen II shows a significant change, indicating that fentanyl hapten I and BSA were successfully conjugated.

[0078] Comparative Example 1

[0079] This embodiment describes a method for preparing fentanyl artificial antigen IV (reaction process as follows). Figure 5 ), including the following steps:

[0080] (1) Preparation of fentanyl artificial hapten III:

[0081] ① Dissolve 181 mg (0.514 mmol) of β-hydroxyfentanyl in 20 ml of pyridine, place in a 50 ml single-necked round-bottom flask, add 103 mg (1.03 mmol) of succinic anhydride, reflux at 100 °C with stirring for 17 hours. After the reaction is complete, transfer directly to dryness to obtain 256 mg of yellow oily substance, which is purified by thin-layer chromatography to obtain 180 mg of fentanyl artificial hapten III. The solvent and eluent are anhydrous ethanol;

[0082] TLC detection of fentanyl artificial hapten III was performed using a chromatography buffer of 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia water = 8:1:10:1 (volume ratio), and the product Rf = 0.5.

[0083] The chromatographic solution used in the thin-layer chromatography method was 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia water = 8:1:10:1 (volume ratio), and the product Rf = 0.5.

[0084] (2) Preparation of fentanyl artificial antigen IV:

[0085] ② Place 180 mg (0.398 mmol) of fentanyl artificial hapten III in a 50 ml round-bottom flask, add 9 ml of N,N-dimethylformamide (DMF), then add 69 mg (0.6 mmol) of N-hydroxysuccinimide (NHS) and 124 mg (0.602 mmol) of cyclohexylcarbodiimide (DCC), stir at room temperature and react overnight. After the reaction is complete, centrifuge and collect the supernatant for later use.

[0086] ③ Weigh 14.5g (0.0405mol) disodium hydrogen phosphate dodecahydrate, 43.875g (0.75mol) sodium chloride, and 1.495g (0.00958mol) sodium dihydrogen phosphate dihydrate, dissolve them in double-distilled water, and bring the volume to 5.0L to obtain 0.01M PBS buffer with pH=7.4.

[0087] ④ Weigh 0.225g of bovine serum albumin and dissolve it in 45ml of PBS buffer from step ③ to obtain a bovine serum albumin solution with a concentration of 5mg / ml.

[0088] ⑤ Under rapid stirring, the supernatant from step ② is slowly added dropwise to the bovine serum albumin solution. The volume ratio of the supernatant to the bovine serum albumin solution is 1:5. The resulting mixture is stored at 4°C overnight to obtain the artificial antigen mixture.

[0089] ⑥ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 9 times with the PBS buffer from step ③. After dialysis, centrifuge and collect the supernatant to obtain artificial antigen IV: fentanyl-bovine serum albumin conjugate.

[0090] Comparative Example 2

[0091] This embodiment describes a method for preparing fentanyl artificial antigen V (reaction process as follows). Figure 6 ), including the following steps:

[0092] (1) Preparation of fentanyl artificial hapten III:

[0093] ① Same as Comparative Example 1.

[0094] (2) Preparation of fentanyl artificial antigen V:

[0095] Bovine gamma protein was used as a carrier and conjugated with fentanyl artificial hapten III. The conjugation steps ②-⑥ were the same as those in Comparative Example 1 to obtain fentanyl artificial antigen V.

[0096] Comparative Example 3

[0097] This embodiment describes a method for preparing fentanyl artificial antigen VI (reaction process as follows). Figure 7 ), including the following steps:

[0098] (1) Preparation of fentanyl artificial hapten III:

[0099] ① Same as Comparative Example 1.

[0100] (2) Preparation of fentanyl artificial antigen VI:

[0101] ② Weigh 180 mg (0.398 mmol) of fentanyl artificial hapten III into a 50 ml round-bottom flask, add 9 ml of N,N-dimethylformamide (DMF), then add 55 μL (0.398 mmol) of triethylamine, stir in an ice bath for 30 min, then add 103 μL (0.796 mmol) of isobutyl chloroformate, continue stirring in an ice bath for 2 h, centrifuge after the reaction is complete, and collect the supernatant for later use.

[0102] ③-⑥ Same as Comparative Example 1, fentanyl artificial antigen VI was obtained.

[0103] Comparative Example 4

[0104] This embodiment describes a method for preparing fentanyl artificial antigen VII (reaction process as follows). Figure 8 ), including the following steps:

[0105] (1) Preparation of fentanyl artificial hapten III:

[0106] ① Same as Comparative Example 1.

[0107] (2) Preparation of fentanyl artificial antigen:

[0108] Bovine gamma protein was used as a carrier and conjugated with fentanyl artificial hapten III. The conjugation steps ②-⑥ were the same as those in Comparative Example 3 to obtain fentanyl artificial antigen VII.

[0109] Comparative Example 5

[0110] This embodiment describes a method for preparing fentanyl artificial antigen VIII (reaction process as follows). Figure 9 ), including the following steps:

[0111] (1) Preparation of fentanyl artificial hapten I:

[0112] ①-② are the same as in Example 1.

[0113] (2) Preparation of fentanyl artificial antigen VIII:

[0114] ③ Weigh 170 mg (0.376 mmol) of fentanyl artificial hapten I and place it in a 50 ml round-bottom flask. Add 8.5 ml of N,N-dimethylformamide (DMF), then add 65 mg (0.564 mmol) of N-hydroxysuccinimide (NHS) and 116 mg (0.564 mmol) of cyclohexylcarbodiimide (DCC). Stir the mixture at room temperature overnight. After the reaction is complete, centrifuge and collect the supernatant for later use.

[0115] ④-⑦ are the same as in Example 1, and fentanyl artificial antigen VIII is obtained.

[0116] Comparative Example 6

[0117] This embodiment describes a method for preparing fentanyl artificial antigen IX (reaction process as follows). Figure 10 ), including the following steps:

[0118] (1) Preparation of fentanyl artificial hapten I:

[0119] ①-② are the same as in Example 1.

[0120] (2) Preparation of fentanyl artificial antigen IX:

[0121] Bovine gamma protein was used as a carrier and conjugated with fentanyl artificial hapten I. The conjugation steps ③-⑦ were the same as those in Comparative Example 5 to obtain fentanyl artificial antigen IX.

[0122] Comparative Example 7

[0123] This embodiment describes a method for preparing fentanyl artificial antigen X (reaction process as follows). Figure 11 ), including the following steps:

[0124] (1) Preparation of fentanyl artificial hapten I:

[0125] ①-② are the same as in Example 1.

[0126] (2) Preparation of fentanyl artificial antigen X:

[0127] Bovine gamma protein was used as a carrier and conjugated with fentanyl artificial hapten I. The conjugation steps ③-⑦ were the same as in Example 1 to obtain fentanyl artificial antigen X.

[0128] Application Example 1: Fentanyl Colloidal Gold Immunochromatographic Detection Reagent Strip

[0129] (1) Preparation of colloidal gold

[0130] Colloidal gold particles were prepared using the simple trisodium citrate reduction method. A conical flask containing 100 ml of 0.01% HAuCl4 solution was placed on a magnetic stirrer and heated to boiling. A certain amount of 1% trisodium citrate was quickly added while adjusting the stirring speed. Initially, the solution turned slightly blue, then light blue, then blue, and finally red. After boiling for 7–10 minutes, a transparent orange-red color was achieved, at which point heating was stopped, and the solution was stored for later use.

[0131] (2) Colloidal gold-labeled fentanyl antibody

[0132] Take 10 ml of colloidal gold solution into a 50 ml centrifuge tube, stir well, and adjust the pH to the optimal value using 0.1 M K2CO3. While stirring slowly, gradually add an appropriate amount of (mouse IgG) FYL-Ab, then mix well and let stand for 30 min. Afterward, continue stirring slowly and slowly add PEG 20,000 (final concentration 0.05%), mix well and let stand for 30 min. Centrifuge at 8000 rpm for 30 min at 4°C. After centrifugation, carefully aspirate the supernatant, reconstitute it with 15 mM Tris buffer containing 0.05% PEG 20,000, mix well and let stand for 30 min. Add 0.05% sodium azide for preservation; repeat the centrifugation at 8000 rpm for 30 min at 4°C. After centrifugation, discard the supernatant, measure its OD value using a UV spectrophotometer, and store for later use.

[0133] (3) Treatment of polyester fiber membrane

[0134] reagents Dosage Purified water 90% <![CDATA[Na2HPO4 . 12H2O]]> 5.74g / L <![CDATA[K2HPO4]]> 1.46 g / L Tween-20 0.1%

[0135] The treatment solution for the polyester fiber membrane was prepared according to the above formula. The final volume was adjusted using purified water, and the pH was adjusted to 7.4 ± 0.1 using 6M HCl and 6M NaOH. An 84mm × 301mm polyester membrane was rolled up and placed in a 50ml centrifuge tube. An appropriate amount of the prepared solution was added, and the mixture was placed on a rotary mixer and mixed at a constant speed for 5 hours. After mixing, the membrane was removed, laid flat on a grid rack, and dried at 37℃.

[0136] (4) Sample pad treatment

[0137] reagents Dosage Purified water 90% Borax 19.05g / L S-17 5g / L Sodium cholate 5g / L PVP (Polyvinylpyrrolidone) 10g / L Sodium azide 0.02%

[0138] Prepare the treatment solution for the glass fiber membrane according to the above formula, and finally make up the volume with purified water. Adjust the pH to 8.5±0.1 with 6M HCl and 6M NaOH. Take the untreated glass fiber membrane, spread it flat on the grid rack, and spray the above-prepared solution evenly with a disposable syringe. Spread the solution evenly with the cap of a 50ml disposable centrifuge tube. Place the grid rack in a 37℃ oven to dry.

[0139] (5) Assembly of immunochromatographic assay strips

[0140] The following components are attached to the plastic plate from bottom to top: a sample pad, a gold-labeled pad (a polyester fiber membrane with dried colloidal gold-labeled mouse IgG and FYL-Ab), a nitrocellulose membrane (coated with goat anti-mouse IgG and FYL-Ag), and an absorbent pad. Each component overlaps by approximately 1.5 mm. When the test sample is added to the sample plate, the sample rises to the surface via capillary action, completing the reaction.

[0141] (6) Result interpretation

[0142] Positive result: A red line appears on the C line (control line) of the test strip display area; Negative result: A red line appears on both the C line (control line) and the T line (test line) of the test strip display area; Invalid result: No red lines appear on either the C line (control line) or the T line (test line) of the test strip display area, or only a red line appears on the T line.

[0143] The above application examples are not intended to limit the present invention. The present invention is not limited to the above application examples. Any application example that meets the requirements of the present invention is within the protection scope of the present invention.

[0144] Example 1: Performance determination of fentanyl artificial antigen

[0145] (1) Identification of fentanyl artificial antigen:

[0146] Molar absorptivity ε: Fentanyl artificial hapten solutions with concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, and 40 μg / ml were prepared using PBS buffer. UV scanning revealed that the maximum absorption wavelength of the fentanyl artificial hapten was 240 nm. Absorbance values ​​were measured at 240 nm, with each concentration tested in parallel. The molar absorptivity (i.e., molar absorption coefficient) is calculated using the formula: ε = absorbance value / molar concentration.

[0147] Determination of conjugate protein concentration: Prepare 1 ml of bovine serum albumin solutions with concentrations of 0 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, and 120 μg / ml using PBS buffer. Add 3 ml of Coomassie brilliant blue staining solution, mix immediately, and incubate at 30°C for 5 minutes. Perform parallel tests for each concentration. Measure the absorbance at 655 nm and plot the relationship between protein concentration and absorbance. Dilute the fentanyl artificial antigen solution (prepared with PBS buffer) at a certain ratio, measure the absorbance of the fentanyl artificial antigen at 655 nm, and read the corresponding protein concentration values ​​of the fentanyl artificial antigen solution from the curve.

[0148] Conjugation ratio determination: Prepare a 100 μg / ml bovine serum albumin PBS solution. Dilute the conjugate (i.e., fentanyl artificial antigen) to 100 μg / ml with PBS. Measure the absorbance A1 at 240 nm. Measure the absorbance A2 using PBS as a blank. The conjugation ratio γ is then calculated as: γ = [(A1 - A2) / ε] / (100 × 10⁻⁶). -3 / 66000).

[0149] Where ε is the molar absorptivity (L / mol), 66000 is the molecular weight of bovine serum albumin, and 100 × 10⁻⁶ is the molecular weight of bovine serum albumin. -3 This refers to bovine serum albumin concentration (g / L).

[0150] When bovine gamma protein is used as a carrier, the coupling ratio is calculated as follows: γ = [(A1 - A2) / ε] / (100 × 10⁻⁶) -3 / 43000); where 43000 is the molecular weight of bovine gamma protein.

[0151] Table 1. Conjugation ratio and molar absorption coefficient of each fentanyl artificial antigen

[0152] serial number Artificial antigen Coupling ratio Coupling protein concentration molar absorption coefficient Example 1 Ⅱ 28 3.568mg / ml 5928.88 Comparative Example 1 Ⅳ 20 3.124mg / ml 6018.38 Comparative Example 2 Ⅴ 10 1.685mg / ml 6018.38 Comparative Example 3 Ⅵ 16 2.687 mg / ml 6018.38 Comparative Example 4 Ⅶ 6 0.354 mg / ml 6018.38 Comparative Example 5 Ⅷ 24 3.356 mg / ml 5928.88 Comparative Example 6 Ⅸ 8 0.896mg / ml 5928.88 Comparative Example 7 Ⅹ 12 2.088mg / ml 5928.88

[0153] As shown in Table 1, the structure of the artificial hapten, the activation method of the artificial hapten, and the structure of the carrier protein all affect the binding ratio when the artificial hapten crosslinks with the carrier protein.

[0154] (2) Animal immunization

[0155] New Zealand white rabbits were immunized with the prepared fentanyl artificial antigens, and the titers of the resulting immune serum were detected by ELISA. The results are shown in Table 2.

[0156] Table 2. Results of titer tests for various immune sera.

[0157] serial number Fentanyl Artificial Antigen Immune serum titer Example 1 Ⅱ 1:85000 Comparative Example 1 Ⅳ 1:12000 Comparative Example 2 Ⅴ 1:6400 Comparative Example 3 Ⅵ 1:18000 Comparative Example 4 Ⅶ / Comparative Example 5 Ⅷ 1:18000 Comparative Example 6 Ⅸ / Comparative Example 7 Ⅹ 1:14000

[0158] As shown in Table 2, compared with Example 1, the immune sera obtained by immunizing animals using fentanyl artificial antigens in each comparative example had lower titers and could not be used in immunoassays. Specifically, fentanyl artificial antigens VII and IX obtained in Comparative Examples 4 and 6 showed significant precipitation immediately during dialysis, while fentanyl artificial antigen V obtained in Comparative Example 2 was relatively turbid and showed significant precipitation after freezing, indicating poor stability; therefore, it could not be used as an immunoassay antigen. However, the immune sera obtained by immunizing animals using fentanyl artificial antigen II had a titer of 1:85000, making it perfectly suitable for immunoassays and providing a more convenient, rapid, and accurate method for fentanyl detection.

[0159] Example 2: Performance Determination of Fentanyl Urine Colloidal Gold Immunochromatographic Detection Strip

[0160] This test example performs a functional test on the fentanyl colloidal gold immunochromatographic assay strip prepared according to Example 1. The specific operation is as follows:

[0161] (1) Sample configuration

[0162] Fresh clinically negative urine samples were collected and fentanyl standard was added to prepare samples at the following concentrations: 0 ng / ml, 0.25 ng / ml, 0.5 ng / ml, 0.75 ng / ml, 1 ng / ml, 1.25 ng / ml, 1.5 ng / ml, 1.75 ng / ml, 2 ng / ml, and 3 ng / ml.

[0163] Cross-interference substance solution: Different drugs and common medicines are added to negative urine samples to prepare a 100 μg / ml concentration solution sample.

[0164] (2) Detection and Result Analysis

[0165] When the above sample is added to the sample application area of ​​the reagent card, if both the test line and the control line of the reagent strip show color, the test result is negative (-); if the control line shows color but the test line does not, the test result is positive (+); if the control line does not show color but the test line shows color, or if neither the control line nor the test line shows color (X), the test result is invalid.

[0166] The sensitivity test results for this test are shown in Table 3. The limit of detection for fentanyl in urine colloidal gold immunochromatographic test strips is 1 ng / ml. Detections above 1 ng / ml are all positive, and those below 1 ng / ml are all negative.

[0167] The drug cross-interference experiment is shown in Table 4. Samples of 100 μg / ml concentration of 88 common drugs were prepared using negative urine, and the results were all negative.

[0168] Table 3. Sensitivity test results of fentanyl urine colloidal gold immunochromatographic test strips

[0169] Concentration (ng / ml) result 0 -,-,-,-,- 0.25 -,-,-,-,- 0.5 -,-,-,-,- 0.75 -,-,-,-,- 1 -,+,+,-,+ 1.25 +,+,+,+,+ 1.5 +,+,+,+,+ 1.75 +,+,+,+,+ 2 +,+,+,+,+ 3 +,+,+,+,+

[0170] Table 4. Results of drug cross-interference test for fentanyl colloidal gold immunochromatographic test strips

[0171] reagents result reagents result reagents result reagents result Acetaminophen - Nifedipine - Oxyquinacrine - Propranolol - Cyclobenzalin - Ampicillin - Tryptophan - Prednisone - Methoxynamine - Erythromycin - bilirubin - Lidocaine - Sulinic acid - Norethindrone - Tyrosine - pseudoephedrine - Phenacetin - Venlafaxine Hydrochloride - papaverine hydrochloride - 5-hydroxytryptamine - desipramine - nortriptyline - Bupropion hydrochloride - Clomipramine - Acetylcarni - Verapamil - Ibuprofen - Loperamide - Vitamin B1 - Vitamin C - penicillin - Quinine - Thioridazine - Fenoprofen - caffeine - sulfadiazine - Naphthylidine - That's for sure. - Trimipramine - Cola Ding - Aminopterin - Aspartame - Fen Nai Jing - Maprotiline hydrochloride - Salbutamol - Furosemide - Carbamazepine - ranitidine - Naloxone - Gentian acid - Benzylpiperidine - Tetrahydrocortisone - Trifluralin - Hydroxyphenylethanolamine - Hydroxytyramine - Hydrocortisone acetate - Digoxin - Diphenylethanol - Chloramphenicol - Tyramine - naltrexone - Bud methyl ester - ketamine - L-carnitine - Trimethoprim - benzoic acid - phenprobarthol - Methionine - Amitriptyline - Bovine hemoglobin - Niacinamide - salicylic acid - diphenhydramine hydrochloride - Osasipa - Chlorthiazide - Creatine - Naproxen - hydrated trichloroacetaldehyde - Ketoprofen - Hypnosone - Dosepin - Benzoyl Iconine - Chlorpromazine - Scobarbital - amobarbital - Hydralazine - La Bellor - Dexapyrine -

Claims

1. A fentanyl artificial hapten, characterized in that, Its molecular structure is shown in formula (Ⅰ): ； （Ⅰ）。 2. The method for preparing the fentanyl artificial hapten according to claim 1, characterized in that, The preparation method includes the following steps: Step (1): Dissolve β-hydroxyfentanyl in N,N-dimethylformamide, add sodium hydride, and stir at room temperature for 0.5-1 h; then add ethyl 5-bromopentanoate, and stir under reflux for 17-20 h; after the reaction is complete, remove the solvent and extract with ethyl acetate, filter, dry and purify to obtain oil A; wherein the molar ratio of β-hydroxyfentanyl to sodium hydride is (1-1.5):3, and the molar ratio of β-hydroxyfentanyl to ethyl 5-bromopentanoate is 1:(1.5-2); Step (2): Dissolve oily substance A in a mixture of tetrahydrofuran and anhydrous methanol, add sodium hydroxide aqueous solution, stir at room temperature for 5-6 hours, adjust pH to 4-5, extract with dichloromethane multiple times, combine organic phases, dry, filter, remove dichloromethane and purify to obtain the fentanyl artificial hapten; wherein the mass-volume ratio of oily substance A, tetrahydrofuran, anhydrous methanol and sodium hydroxide aqueous solution is 100mg: (1.5-2)ml: (1.8-2)ml: 10ml.

3. The preparation method according to claim 2, characterized in that, In step (2), the equivalent concentration of the sodium hydroxide aqueous solution is 1 to 1.5 N.

4. A fentanyl artificial antigen, obtained by conjugating the fentanyl artificial hapten of claim 1 with bovine serum albumin; characterized in that, The molecular structure of the fentanyl artificial antigen is shown in formula (II): ； （Ⅱ） In formula (II), BSA is bovine serum albumin.

5. The method for preparing the fentanyl artificial antigen according to claim 4, characterized in that, The preparation method includes the following steps: Step (1): Dissolve the fentanyl artificial hapten in a mixture of DMF and triethylamine and stir in an ice bath. Add isobutyl chloroformate during stirring and continue stirring in an ice bath for 2 to 2.5 hours. After the reaction is completed, centrifuge to obtain the supernatant. Step (2): Add the supernatant obtained in step (1) to the bovine serum albumin solution, let the resulting mixture stand overnight at 3-5°C, and obtain the supernatant by dialysis and centrifugation to obtain the fentanyl artificial antigen.

6. The preparation method according to claim 5, characterized in that, The concentration of the bovine serum albumin solution in step (2) is 5 mg / mL, and the volume ratio of the supernatant to the bovine serum albumin solution is 1: (5-6).

Images