CN116375606B - Rapid detection device for phenformin in hypoglycemic drugs and health-care products, and preparation and application thereof - Google Patents

Rapid detection device for phenformin in hypoglycemic drugs and health-care products, and preparation and application thereof Download PDF

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CN116375606B
CN116375606B CN202310362034.4A CN202310362034A CN116375606B CN 116375606 B CN116375606 B CN 116375606B CN 202310362034 A CN202310362034 A CN 202310362034A CN 116375606 B CN116375606 B CN 116375606B
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phenformin
halogenated
hapten
stirring
reaction
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CN116375606A (en
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吴民富
李莎
詹清敏
林立栋
吴民华
贺劲锋
高柔敏
林颖泓
罗林
徐振林
周彩琴
刘艳灿
张少敏
吴晖琳
刘考钰
周欢欣
饶芳芳
方育芳
卢玉萍
林木健
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Foshan Polytechnic
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Abstract

The invention discloses a rapid detection device for phenformin in hypoglycemic drugs and health-care products, and preparation and application thereof, and relates to phenformin hapten, antigen, antibody, rapid detection device, and preparation and application thereof in the detection of phenformin in hypoglycemic drugs and health-care products. The invention provides a preparation method of a phenformin hapten, an artificial antigen prepared by the phenformin hapten coupling carrier protein, and an antibody which is specifically directed against the phenformin and is produced by a body of an immunized experimental animal, wherein the antibody has high titer, the half inhibition concentration of the phenformin is 16.5ng/mL, and the linear range (IC is quantitatively detected 20 ～IC 80 ) The detection method has the advantages of high sensitivity, strong specificity, low cost, short detection time and the like, and can be used for rapidly detecting the phenformin in the hypoglycemic drugs and health care products, wherein the minimum detection limit is 2.2 ng/mL, and the detection method is 4.8-180.5 ng/mL.

Description

Rapid detection device for phenformin in hypoglycemic drugs and health-care products, and preparation and application thereof
Technical Field
The invention relates to the technical field of food safety detection, in particular to a rapid detection device for phenformin in hypoglycemic medicines and health-care products, and preparation and application thereof.
Background
Diabetes belongs to common and frequently-occurring diseases, and the incidence rate of the diabetes is in an annual rising trend in China along with the improvement of the living standard of people, so that the diabetes threatens the public health. Some illegal merchants illegally add sugar-reducing western medicines to health-care foods to falsely advertise and misguide consumers to obtain violence. Patients can cause adverse reactions such as serious damage to liver and kidney functions, hypoglycemia reaction, cytopenia and the like after long-term use under unknowing conditions. Along with the improvement of the living standard of people, the requirements on quality of diets, medicines, health care products and the like are higher and higher, and the countries and the society correspondingly supervise the quality of diets, medicines, health care products and the like by law and moral supervision. In this regard, many efforts have been made by corresponding scientific research staff to develop more effective detection means for detecting illegal additives prohibited by statutes to suppress the occurrence of such events. The detection method which is mature at present mainly comprises the following steps: high Performance Liquid Chromatography (HPLC), mass spectrometry, liquid chromatography, fluorescence spectrometry, electrochemical methods, etc., while all of which have their advantages, most require complicated processing and long preparation times.
The immunological detection analysis method is an analysis method based on specific recognition and reversible binding reaction of antigen and antibody, and has the advantages of high sensitivity, high specificity, low requirement on instruments, rapidness, simple and convenient operation, low cost and the like. However, an immunological detection analysis technology is established and applied to detecting the phenformin in hypoglycemic medicines and health products, and the key technology is that antibodies with strong specificity and high sensitivity can be obtained, and the aim is to be achieved if proper phenformin hapten is designed and synthesized. However, there are no reports on the synthesis of phenformin hapten.
Therefore, there is an urgent need to design and develop a suitable phenformin hapten, and thus establish a corresponding phenformin rapid detection method, so as to realize rapid detection of phenformin in hypoglycemic drugs and health care products through an immunological method.
Disclosure of Invention
The invention aims to provide a rapid detection device for phenformin in hypoglycemic drugs and health-care products, and preparation and application thereof, and aims to detect phenformin in hypoglycemic drugs or health-care products.
According to one aspect of the present invention, there is provided a phenformin hapten having the structure shown in formula (I):
wherein n is 1,2,3,4 or 5.
According to another aspect of the present invention, there is provided a method for preparing a phenformin hapten, comprising the steps of:
s1, weighing p-hydroxyphenylethylamine, dissolving in tetrahydrofuran, adding triethylamine, and slowly adding (Boc) 2 O, stirring at room temperature, evaporating to dryness after the reaction is completed, and performing column chromatography to obtain an intermediate I;
s2, dissolving the intermediate I in DMF, adding potassium carbonate and halogenated ester, reacting overnight at 85 ℃, cooling after the reaction, adding water for dilution, EA extraction, water washing, distilling to dryness, and performing column chromatography to obtain an intermediate II;
s3, dissolving the intermediate II in dichloromethane, stirring at room temperature, introducing HCl gas for 30min, stirring in an ice bath, filtering, and airing to obtain an intermediate III;
s4, adding the intermediate III and dicyandiamide into a single-mouth bottle, stirring at room temperature, raising the reaction bath temperature to 145 ℃, stirring for 2 hours, cooling to room temperature, stirring, directly carrying out column chromatography on the reaction liquid, separating the target liquid, and evaporating to dryness to obtain an intermediate IV;
and S5, sequentially adding the intermediate IV and methanol into a single-port bottle, stirring at room temperature until the mixture is clear, adding lithium hydroxide and purified water, continuing stirring at room temperature, monitoring the complete reaction of materials by TLC, regulating the pH value of a reaction system to 5-6 by using hydrochloric acid, performing reduced pressure distillation until the mixture is dry, performing column chromatography on the remainder, separating out target liquid, and performing distillation drying to obtain the phenformin hapten.
In some embodiments, the p-hydroxyphenylethylamine and (Boc) of step S1 2 The molar ratio of O is 1 (1-5), and the molar ratio of the intermediate I to the halogenated ester in the step S2 is1-5, wherein the halogen atom in the halogenated ester is F, cl, br or I, and the halogenated ester is one of methyl 6-halohexanoate, ethyl 6-halohexanoate, propyl 6-halohexanoate, tert-butyl 6-halohexanoate, methyl 5-halovalerate, ethyl 5-halovalerate, propyl 5-halovalerate, tert-butyl 5-halovalerate, methyl 4-halobutyrate, ethyl 4-halobutyrate, propyl 4-halobutyrate, tert-butyl 4-halobutyrate, methyl 3-halopropionate, ethyl 3-halopropionate, propyl 3-halopropionate, tert-butyl 3-halopropionate, methyl 2-haloacetate, ethyl 2-haloacetate, propyl 2-haloacetate or tert-butyl 2-haloacetate.
In some embodiments, the molar ratio of intermediate III to dicyandiamide in step S4 is 1 (1-2.5), and the molar ratio of intermediate IV to lithium hydroxide in step S5 is 1 (3-7).
According to yet another aspect of the invention, the phenformin antigen is a conjugate of a phenformin hapten and a carrier protein, the carrier protein being one of bovine serum albumin, human serum albumin, chicken egg albumin or hemocyanin.
According to a fourth aspect of the present invention there is provided the use of a phenformin hapten, a phenformin antigen in the immunological detection of phenformin.
According to a fifth aspect of the present invention, the phenformin antibody is prepared from phenformin antigen by animal immunization, and the phenformin antibody is an anti-phenformin monoclonal antibody.
According to a sixth aspect of the present invention there is provided the use of a phenformin antibody in the immunological detection of phenformin.
According to a seventh aspect of the present invention, there is provided a phenformin colloidal gold immunochromatography detection apparatus, the detection apparatus comprising a test strip and a reaction cup, the test strip comprising a reaction membrane, the reaction membrane being provided with a detection zone and a quality control zone, the detection zone being coated with phenformin antigen, the reaction cup containing a phenformin antibody labeled with colloidal gold.
According to an eighth aspect of the present invention, there is provided a method for detecting phenformin in hypoglycemic drugs and health care products, wherein the method comprises detecting phenformin in a sample, which is a drug or a health care product, by using a phenformin colloidal gold immunochromatography detection device.
The invention has the beneficial effects that: the invention provides a preparation method of a phenformin hapten, an artificial antigen prepared by the phenformin hapten coupling carrier protein, and an antibody which is specifically directed against the phenformin and is produced by a body of an immunized experimental animal, wherein the antibody has high titer, the half inhibition concentration of the phenformin is 16.5ng/mL, and the linear range (IC is quantitatively detected 20 ～IC 80 ) The detection method has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time and the like, and can meet the rapid detection of the phenformin in the hypoglycemic drugs and health care products, wherein the minimum detection limit is 2.2 ng/mL, and the detection method is 4.8-180.5 ng/mL.
Drawings
Fig. 1 is a mass spectrum of a phenformin hapten according to one embodiment of the present invention.
FIG. 2 is a synthetic route pattern of a phenformin hapten according to one embodiment of the present invention.
Figure 3 is a graph of an indirect competition ELISA standard established based on a phenformin monoclonal antibody according to one embodiment of the invention.
Detailed Description
The invention will be described in further detail with reference to specific embodiments thereof, it being understood that these embodiments are for purposes of illustration only and not for purposes of limiting the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the invention, and are defined in the claims appended hereto. Unless otherwise specified, all materials and reagents of the invention are those commercially available in the conventional market.
Example 1 a method for preparing a phenformin hapten comprising the steps of:
s1. Weighing 6.5g of p-hydroxyphenylethylamine, dissolving in tetrahydrofuran, adding 5mL of triethylamine, and slowly adding (Boc) 2 O, stirring at room temperature, evaporating to dryness after the reaction is completed, and performing column chromatography to obtain an intermediate I, p-hydroxyphenylethylamine and (Boc) 2 The molar ratio of O is 1:1.2;
s2, dissolving 8.9g of the obtained intermediate I in 30mLDMF, adding 5.0g of potassium carbonate and halogenated ester, reacting overnight at 85 ℃, cooling, diluting with water, extracting by EA, washing with water, distilling to dryness, and performing column chromatography to obtain intermediate II, wherein the molar ratio of the intermediate I to the halogenated ester is 1:1.5;
s3, taking 960mg of the intermediate II, dissolving in 20mL of dichloromethane, stirring at room temperature, introducing HCl gas for 30min, stirring in an ice bath, filtering, and airing to obtain an intermediate III;
s4, adding 640mg of intermediate III and 200mg of dicyandiamide into a 50mL clean single-mouth bottle, stirring at room temperature, raising the reaction bath temperature to 145 ℃, stirring for 2 hours, cooling to room temperature, stirring, directly carrying out column chromatography on the reaction liquid, taking eluent as MeOH/DCM, separating target liquid, and evaporating to dryness to obtain an intermediate IV;
s5, sequentially adding 600mg of intermediate IV and 8.0mL of methanol into a 50mL clean single-port bottle, stirring at room temperature until the mixture is dissolved, adding 200mg of lithium hydroxide and 6.0mL of purified water, continuing stirring at room temperature, monitoring the complete reaction of materials by TLC, regulating the pH value of a reaction system to 5-6 by using 6N hydrochloric acid, carrying out reduced pressure distillation until the mixture is dry, carrying out column chromatography on residues, separating out target solution by using MeOH/DCM as eluent, and carrying out distillation until the mixture is dry to obtain 300mg of white solid, namely the phenformin hapten.
The hapten of the phenformin is identified by mass spectrometry, and the obtained mass spectrum is shown in figure 1 of the specification. As can be seen from the mass spectrum, when n=3, the molecular ion peak of the phenformin hapten is EI-MS (negative) m/z:342.5[ M+Cl] - Which is consistent with the molecular weight 307.35 of the phenformin hapten, shows that the phenformin hapten shown in the formula (I) is successfully synthesized. In the embodiment, the synthesis flow chart of the phenformin hapten is shown in the attached figure 2 of the specification.
EXAMPLE 2 preparation of antigen for phenformin immunization and antigen for coating
2.1 Preparation of antigen for phenformin immunization:
weighing 50mg of phenformin hapten, dissolving in 2.5mL of DMF, adding 25mg of NHS and 30mg of EDC.HCl, and reacting for 6 hours at room temperature to prepare an activated liquid; 45mg bovine Lactoferrin (LF) is dissolved in 3mL of 0.1M boric acid buffer solution with the pH value of 9.0, 1mL of DMF and 0.6mL of the activating solution are added, the mixture is dialyzed by PBS (0.01 mol/L phosphate buffer solution with the pH value of 7.4) after being reacted for 4 hours at room temperature, each 4 times of h is changed for 1 time, the solution is changed for 7 to 8 times, the solution is centrifuged for 5 minutes at 4000 revolutions/min after dialysis, and the supernatant is taken to prepare the antigen for the phenformin immunity, namely the phenformin hapten-LF conjugate, and the antigen is stored at the temperature of minus 20 ℃.
2.2 preparation of the antigen for phenformin coating:
3.8 g mg g of phenformin hapten is weighed and dissolved in 200 mu l of anhydrous DMF, then 3 mu l of tri-n-butylamine and 1.6 mu l of isobutyl chloroformate are sequentially added, and stirring reaction is carried out at room temperature for 1 h, thus obtaining a reaction liquid; 22 mg of BSA is dissolved in 2mL of carbonate buffer solution, 100 mul of the reaction solution is slowly added dropwise under stirring, the reaction is stirred at room temperature for 3h, then dialysis and centrifugation are carried out, thus obtaining the antigen for coating the phenformin, namely the phenformin hapten-BSA conjugate, and the conjugate is preserved at the temperature of minus 20 ℃ for standby.
EXAMPLE 3 preparation and purification of phenformin monoclonal antibodies
3.1 Immunization of animals
Healthy BALB/c mice of 6 to 8 weeks of age were selected for immunization, and after the antigen for phenformin immunization obtained in example 2 was mixed and emulsified with an equivalent amount of Freund's adjuvant, BALB/c mice were subjected to subcutaneous multipoint injection of the back of the neck (except for sprint immunization). The first immunization is carried out by using complete Freund's adjuvant, and the dosage is 180 mug/dose; boosting is carried out after 4 weeks at a dose of 90 mug/dose, the mixture is mixed and emulsified by incomplete Freund's adjuvant, and then the boosting is carried out for a plurality of times for 3 weeks; the dose is halved again during the sprint immunization, 45 mug/mouse, and the complete antigen is diluted by normal saline for intraperitoneal injection. The tail-breaking blood sampling detection can be carried out after the third immunization of the mice, and the titer and IC of the serum of the mice can be detected by an indirect competition enzyme-linked immunosorbent assay (IC-ELISA) 50 High selective potency, IC 50 Low mice were fused;
3.2 Cell fusion and cloning
Spleen cells of immunized BALB/c mice were taken at a ratio of 10:1 are fused with SP2/0 myeloma cells, and a phenformin monoclonal hybridoma cell strain which stably secretes the phenformin monoclonal antibody is obtained by screening;
3.3 Cell cryopreservation and resuscitation
Preparation of phenformin monoclonal hybridoma cell into 5×10 frozen stock solution 6 Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. Taking out the freezing tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen solution, and transferring into a culture flask for culture;
3.4 Preparation and purification of monoclonal antibodies
Incremental culture method: placing the phenformin monoclonal hybridoma cells in a cell culture medium, culturing at 37 ℃, purifying the obtained culture solution by an octanoic acid-saturated ammonium sulfate method to obtain the phenformin monoclonal antibody, and preserving at-20 ℃. Wherein, the cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI-1640 culture medium, wherein the weight percentage of the calf serum in the cell culture medium is 20%, the weight percentage of the sodium bicarbonate in the cell culture medium is 0.2%, and the pH of the cell culture medium is 7.4.
EXAMPLE 4 Standard Curve of phenformin monoclonal antibody and detection of its specificity
4.1 An indirect competition ELISA method for detecting phenformin antibodies comprising the steps of:
(1) The phenformin hapten-BSA conjugate prepared in the example 2 is taken as a coating antigen, diluted to 50 mug/L by coating liquid, a 96-well ELISA plate is coated, 100 mug/well is added, and the mixture is incubated overnight at 37 ℃;
(2) Removing the coating liquid, washing for 2 times, and beating to dry;
(3) 120 mu L of blocking solution (namely 1wt% of fish skin collagen) is added into each hole, and the mixture is blocked for 3 hours at 37 ℃;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, and taking out;
(5) The phenformin monoclonal antibody prepared in example 3 was diluted 20000-fold with PBST, and phenformin standard was diluted in gradient;
(6) Adding 50 mu L of phenformin standard diluent (three groups are arranged in parallel) into each row, adding 50 mu L/hole of 16000 times diluted phenformin monoclonal antibody diluent, incubating for 40min at 37 ℃, washing for 5 times, and beating to dryness;
(7) Adding goat anti-mouse secondary antibody-HRP (6000-fold dilution) 100 mu L/hole, incubating for 30min at 37 ℃, washing for five times, and beating to dryness;
(8) Adding a developing solution, and developing for 10min at 100 mu L per hole;
(9) 50. Mu.L of 2mol/L H are added 2 SO 4 The reaction was stopped and the OD was read at 450 nm.
The standard curve of the indirect competition ELISA for detecting the antibody against phenformin is shown in FIG. 3, and it is understood from FIG. 3 that the half Inhibitory Concentration (IC) of the phenformin monoclonal antibody against phenformin prepared in example 3 50 ) The quantitative detection linear range (IC 20-IC 80) is 4.8-180.5 ng/mL, and the lowest detection limit is 2.2 ng/mL; the antibody for detecting the phenformin, which is prepared by the invention, can meet the detection requirement, and has strong recognition capability on the phenformin.
4.2 The specificity of the antibody of the phenformin for detecting the phenformin is determined by carrying out a cross-reaction experiment on the phenformin and analogues thereof, wherein the specificity of the antibody is expressed by a cross-reaction rate (CR), and the smaller the cross-reaction rate is, the stronger the specificity is. The phenformin and its analogues were diluted by fold ratio and measured by indirect competition ELISA, and the IC of each phenformin analogue was obtained by the same procedure as in example 4, sensitivity verification method of 4.1 50 Values, phenformin cross-reactivity (CR) was calculated according to the following formula:
CR（％）＝IC 50 (phenformin)/IC 50 (analog) ×100%
The drug phenformin in 4.1 was changed to metformin, glibenclamide, glimepiride, repaglinide, and the above test was performed at the same dilution factor to determine the cross-reactivity of the monoclonal antibody prepared in example 3 to other structural analogues, as shown in table 1. It can be seen from Table 1 that the monoclonal antibodies do not significantly cross-react with the rest of the similar drugs. It is shown that the antibody can be used for detecting phenformin.
Table 1 table of the cross-reactivity of monoclonal antibodies with phenformin analogs
Example 5 preparation of phenformin colloidal gold chromatography detection apparatus
5.1 Preparation of colloidal gold solution
Diluting 1% chloroauric acid solution into 0.01% (mass fraction) by using double distilled deionized water, placing 100mL of 0.01% chloroauric acid solution into a conical flask, heating to boil by using a constant-temperature electromagnetic stirrer, adding 2.0 mL of 1% trisodium citrate solution under continuous high temperature and continuous stirring, continuing to stir and heat at a constant speed until the solution is transparent red, stopping cooling to room temperature, recovering to original volume by using deionized water, obtaining colloidal gold solution, and preserving at 4 ℃. The prepared colloidal gold solution has pure appearance, is transparent and has no sediment or floaters;
5.2 Preparation of phenformin monoclonal antibody-colloidal gold marker
Under the magnetic stirring, regulating the pH value of the colloidal gold solution to 7.2 by using 0.2mol/L potassium carbonate, adding 20-60 mug of the phenformin monoclonal antibody into the colloidal gold solution according to the standard of adding the phenformin monoclonal antibody into each milliliter of the colloidal gold solution, continuously stirring and uniformly mixing for 30min, standing for 10min, adding 10% Bovine Serum Albumin (BSA) solution, and standing for 10min, wherein the volume percentage of the phenformin monoclonal antibody in the colloidal gold solution is 1%. Centrifuging at 12000rpm at 4deg.C for 40min, discarding supernatant, and re-suspending the precipitate with 1/10 of the original colloidal gold solution volume of re-dissolving buffer to obtain phenformin monoclonal antibody-colloidal gold marker, and storing at 4deg.C;
reconstitution buffer: 0.02mol/L phosphate buffer solution containing 0.3-0.5% of bovine serum albumin, 0.1-0.3% of tween-20, 3-6% of trehalose and pH=7.2;
5.3 preparation of microporous reaction cup
Adding 100 mu l of phenformin monoclonal antibody-colloidal gold marker into a micropore reaction cup, putting into a freeze dryer, pre-freezing for 3 hours at the cold trap temperature of-50 ℃, and then drying for 6 hours in vacuum, thus obtaining the micropore reaction cup freeze-dried with the phenformin monoclonal antibody-colloidal gold marker, and sealing and preserving, wherein the freeze-drying amount of the phenformin monoclonal antibody-colloidal gold marker is 0.20-0.40 mu g/mL;
5.4 Preparation of sample absorbent pad
The sample absorption pad is placed in phosphate buffer solution containing 0.02mol/L of bovine serum albumin to be soaked for 2 hours, and is dried for 2 hours at 50 ℃ for standby. The pH of the phosphate buffer solution of 0.02mol/L is 7.2, wherein the volume percentage of the bovine serum albumin is 1.0%;
5.5 Preparation of reaction film
The coating process comprises the following steps: respectively diluting the phenformin coating with antigen to the concentration of 10mg/mL by using a phosphate buffer solution, and coating the phenformin coating with the antigen on a detection area (T line) on a nitrocellulose membrane by using a gold-labeled gold-spraying spot film instrument, wherein the coating concentration is 0.5 mg/mL; the goat anti-mouse antibody (purchased commercially) was diluted to 10mg/mL with phosphate buffer at a concentration of 0.01 mol/L and pH=7.4, and coated on the quality control region (line C) of the nitrocellulose membrane with a gold-labeled gold-dot membrane applicator at a concentration of 1.0. 1.0 mg/mL. And (5) drying the coated reaction film for 6 hours at 50 ℃ to prepare for later use.
5.6 preparation of phenformin colloidal gold chromatography detection device
5.6.1 Assembly of test strips
Sequentially adhering a sample absorption pad, a reaction membrane and a water absorption pad on a bottom plate, wherein the bottom plate is a PVC bottom plate, the sample absorption pad is a piece of filter paper, the water absorption pad is a piece of filter paper, and the reaction membrane is a nitrocellulose membrane. The end of the sample absorbing pad is connected with the initial end of the reaction membrane, the end of the reaction membrane is connected with the initial end of the water absorbing pad, the initial end of the sample absorbing pad is aligned with the initial end of the bottom plate, and the end of the water absorbing pad is aligned with the end of the bottom plate.
5.6.2 Assembling of phenformin colloidal gold chromatography detection paper box
And (3) assembling the test strip obtained in the step 5.6.1 and the microporous reaction cup obtained in the step 5.3 into a test paper box, storing in an environment of 2-8 ℃ and storing for 12 months in the effective period.
Example 6 method for detecting phenformin in hypoglycemic drugs and health products
Taking 1mL of liquid hypoglycemic medicine as a sample, diluting by 10 times with Tris-PBS buffer solution, and shaking uniformly to obtain a sample diluent for later use. The sample diluent is dripped into a reaction cup of the phenformin colloidal gold chromatography detection device prepared in the example 5, and is evenly mixed, then a test strip is inserted, and the reaction is carried out for 6min at 20-40 ℃. And taking out the test strip, and judging the result. If the T line and the C line simultaneously show purple red strips, the result is negative, namely the liquid hypoglycemic medicine does not contain phenformin; if the color of the T line is lighter than that of the C line or the color of the C line is developed but the T line is not developed, the result is positive, namely the liquid health care product contains the phenformin; if neither the C line nor the T line is colored, the detection device is failed.
Example 7A method for detecting phenformin in hypoglycemic drugs and health products
About 0.1g of the solid health product is taken, and 10mL of water is added for full stirring. Standing for 2min to obtain sample solution for standby. Taking 1mL of the sample solution, diluting by 10 times with Tris-PBS buffer solution, and shaking uniformly to obtain a sample diluent for later use. The diluted sample solution is dripped into a reaction cup of the phenformin colloidal gold chromatography detection device prepared in the example 5, and is uniformly mixed, then a test strip is inserted, and the reaction is carried out for 6min at 20-40 ℃. And taking out the test strip, and judging the result. If the T line and the C line simultaneously show purple red strips, the result is negative, namely the solid health care product contains the phenformin; if the color of the T line is lighter than that of the C line or the color of the C line is developed but the T line is not developed, the result is positive, namely the solid health care product has no phenformin; if neither the C line nor the T line is colored, the detection device is failed.
The foregoing is merely illustrative of some embodiments of the invention, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept.

Claims (6)

1. The phenformin hapten is characterized in that the structure is shown as a formula (I):
wherein n is 1,2,3,4 or 5.
2. A method of preparing the phenformin hapten of claim 1, comprising the steps of:
s1, weighing p-hydroxyphenylethylamine, dissolving in tetrahydrofuran, adding triethylamine, and slowly adding (Boc) 2 O, stirring at room temperature, evaporating to dryness after the reaction is completed, performing column chromatography to obtain an intermediate I,
s2, dissolving the intermediate I in DMF, adding potassium carbonate and halogenated ester, reacting overnight at 85 ℃, cooling after the reaction is finished, adding water for dilution, EA extraction, washing with water, distilling to dryness, and then performing column chromatography to obtain an intermediate II, wherein halogenated atoms in the halogenated ester are F, cl, br or I, the halogenated ester is one of 6-halogenated methyl caproate, 6-halogenated ethyl caproate, 6-halogenated propyl caproate, 6-halogenated butyl caproate, 5-halogenated methyl valerate, 5-halogenated ethyl valerate, 5-halogenated propyl valerate, 5-halogenated butyl valerate, 4-halogenated methyl butyrate, 4-halogenated ethyl butyrate, 4-halogenated propyl butyrate, 4-halogenated butyl butyrate, 3-halogenated methyl propionate, 3-halogenated ethyl propionate, 3-halogenated propyl propionate, 2-halogenated methyl acetate, 2-halogenated ethyl acetate, 2-halogenated propyl acetate or 2-halogenated butyl acetate,
s3, dissolving the intermediate II in dichloromethane, stirring at room temperature, introducing HCl gas for 30min, stirring in an ice bath, filtering, airing to obtain an intermediate III,
s4, adding the intermediate III and dicyandiamide into a single-mouth bottle, stirring at room temperature, raising the reaction bath temperature to 145 ℃, stirring for 2 hours, cooling to room temperature, stirring, directly carrying out column chromatography on the reaction liquid, separating the target liquid, evaporating to dryness to obtain an intermediate IV,
s5, sequentially adding the intermediate IV and methanol into a single-mouth bottle, stirring at room temperature until the mixture is dissolved, adding lithium hydroxide and purified water, continuing stirring at room temperature, monitoring the complete reaction of materials by TLC, regulating the pH value of a reaction system to 5-6 by using hydrochloric acid, carrying out reduced pressure distillation until the mixture is dry, carrying out column chromatography on the residues, separating out target liquid, carrying out distillation drying to obtain the phenformin hapten,
3. the method for preparing a phenformin hapten according to claim 2, wherein p-hydroxyphenylethylamine and (Boc) are used in the step S1 2 The molar ratio of O is 1 (1-5), and the molar ratio of the intermediate I to the halogenated ester in the step S2 is 1 (1-5).
4. The method for preparing a phenformin hapten according to claim 2, wherein the molar ratio of the intermediate III to dicyandiamide in the step S4 is 1 (1-2.5), and the molar ratio of the intermediate IV to lithium hydroxide in the step S5 is 1 (3-7).
5. The phenformin antigen is characterized in that the phenformin antigen is the conjugate of the phenformin hapten and carrier protein of claim 1, and the carrier protein is one of bovine serum albumin, human serum albumin, chicken egg albumin or hemocyanin.
6. Use of the phenformin hapten of claim 1 and the phenformin antigen of claim 5 in a method of non-disease diagnosis and treatment in immunological detection of phenformin.
CN202310362034.4A 2023-04-07 2023-04-07 Rapid detection device for phenformin in hypoglycemic drugs and health-care products, and preparation and application thereof Active CN116375606B (en)

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