Electrochemical immunosensor for detecting sibutramine content and sibutramine detection kit

Abstract

The invention provides an electrochemical immunosensor for detecting sibutramine content and a preparation method and application thereof, and relates to the technical field of detection. The invention discloses an electrochemical immunosensor for detecting sibutramine content. The electrochemical immunosensor comprises an electrode, a sibutramine artificial antigen and a sibutramine polyclonal antibody, the electrode is a modified working electrode; the sibutramine artificial antigen is obtained by coupling 1-(4-phenol) cyclobutylamine, 5-hydroxytryptamine and a carrier protein; the sibutramine polyclonal antibody is obtained by sibutramine artificial antigen immune animals. The electrochemical immunosensor has high detection sensitivity on sibutramine content.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to an electrochemical immunosensor for detecting sibutramine content and a sibutramine detection kit. Background Art

[0002] With the accelerated pace of modern life and changes in dietary patterns, obesity is becoming increasingly serious worldwide and has become a major public health issue. Sibutramine, a non-amphetamine appetite suppressant, was once widely used in the treatment of obesity. However, recent studies have found that sibutramine may pose serious cardiovascular risks during use. my country has mandated a halt to the production, sale, and use of sibutramine preparations and APIs. However, some unscrupulous vendors continue to sell weight-loss preparations containing sibutramine through illegal markets or informal channels. Therefore, accurate and rapid testing for sibutramine is particularly important.

[0003] Traditional sibutramine detection methods include thin-layer chromatography, high-performance liquid chromatography, mass spectrometry, and high-performance liquid chromatography-mass spectrometry. While these methods achieve accurate detection of the analyte, they suffer from low sensitivity. Immunosensor analysis techniques, represented by electrochemical immunoassays, offer advantages such as high sensitivity and precision, rapid analysis, and strong anti-interference capabilities. Therefore, how to sensitively detect sibutramine using electrochemical immunoassays has become a pressing issue for those skilled in the art. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In view of the shortcomings of the existing technology, the present invention provides an electrochemical immunosensor for detecting sibutramine content and a sibutramine detection kit, which solves the technical problem of low sensitivity of existing sibutramine content detection methods.

[0006] (2) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides an electrochemical immunosensor for detecting the content of sibutramine, wherein the electrochemical immunosensor comprises an electrode, an artificial sibutramine antigen, and a sibutramine polyclonal antibody;

[0009] The electrode is a modified working electrode; the sibutramine artificial antigen is obtained by coupling 1-(4-phenol)cyclobutylamine, 5-hydroxytryptamine and a carrier protein; and the sibutramine polyclonal antibody is obtained by immunizing an animal with the sibutramine artificial antigen.

[0010] Preferably, the modified working electrode is a glassy carbon electrode modified with a nanocomposite, and the nanocomposite is prepared from carboxylated carbon nanotubes, Ketjen black, and chitosan according to a mass ratio of 1:1-1.5:4-5; the carrier protein includes bovine serum albumin and chicken ovalbumin.

[0011] Preferably, the 1-(4-phenol) cyclobutanamine is coupled with the carrier protein by the active ester method.

[0012] Preferably, the preparation method of the sibutramine artificial antigen is as follows:

[0013] S1. Dissolve 1-(4-phenol) cyclobutanamine, 5-hydroxytryptamine, N-carbonylsuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in N,N-dimethylformamide respectively, mix them, and stir at room temperature in the dark for 12-14 h to obtain a first solution;

[0014] S2. Add bovine serum albumin and chicken ovalbumin to the coating buffer respectively to obtain a bovine serum albumin / chicken ovalbumin mixture;

[0015] S3. Slowly drop the first solution into the bovine serum albumin / chicken ovalbumin mixture, and stir and react at room temperature in the dark for 2-4 h to obtain a second solution;

[0016] S4. Dialyze the second solution to obtain the sibutramine artificial antigen.

[0017] Preferably, S1 satisfies at least one of the following conditions:

[0018] The mass-volume ratio of 1-(4-phenol) cyclobutanamine, 5-hydroxytryptamine to N,N-dimethylformamide is (0.2-0.5) mg:(0.1-0.3) mg:(0.1-0.2) mL;

[0019] The mass-volume ratio of N-carbonylsuccinimide to N,N-dimethylformamide is (4-6) mg:(0.2-0.3) mL;

[0020] The mass-volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N,N-dimethylformamide is (4-6) mg:(0.4-0.6) mL;

[0021] Preferably, S2 satisfies at least one of the following conditions:

[0022] The coating buffer includes sodium carbonate (1-3) g / L and sodium bicarbonate (2-4) g / L;

[0023] The mass-volume ratio of bovine serum albumin added to the coating buffer is (1-4) mg:(1-3) mL;

[0024] The mass-to-volume ratio of ovalbumin added to the coating buffer is (1-3) mg:(0.5-1.5) mL.

[0025] Preferably, at least one of the following conditions is satisfied for S4:

[0026] The dialysis solution is 0.5%-1.0% sodium chloride solution;

[0027] The dialysis time is three days, four times a day.

[0028] Preferably, the method for preparing the sibutramine polyclonal antibody is as follows:

[0029] Animals are immunized with the sibutramine artificial antigen solution. The immunization includes one primary immunization and three booster immunizations. The sibutramine artificial antigen solution is mixed and emulsified with Freund's complete adjuvant for primary immunization, and the sibutramine artificial antigen solution is emulsified with Freund's incomplete adjuvant for booster immunization. The time interval between immunization inoculations is 3 weeks; blood is collected on the tenth day after the third booster immunization, and the sibutramine polyclonal antibody is purified by the caprylic acid-ammonium sulfate method.

[0030] Preferably, the animal is a rabbit, and the immunization dose per time is 700-750 μL per rabbit.

[0031] In a second aspect, the present invention provides a sibutramine detection kit, and the sibutramine detection kit includes the sibutramine polyclonal antibody described in the first aspect.

[0032] (III) Beneficial effects

[0033] The present invention provides an electrochemical immunosensor for detecting the content of sibutramine and a sibutramine detection kit. Compared with the prior art, the following beneficial effects are achieved:

[0034] The present invention obtains a sibutramine artificial antigen, which is more similar to serotonin in spatial structure than the commonly used 1-(4-phenol) cyclobutylamine antigen. The sibutramine antibody is bound to the surface of an electrode that enhances the peak current value using a nanocomposite material. The sibutramine polyclonal antibody prepared in this way has high sensitivity and good specificity, providing the core raw material for establishing an electrochemical immunosensor for highly sensitive detection of sibutramine, and has broad development prospects. Description of the drawings

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 Schematic diagram of the process for preparing an electrochemical immunosensor for detecting the content of sibutramine in Example 1.

[0037] Figure 2 Graph of the properties of the nanocomposite material.

[0038] Figure 3 CV characterization graph of the constructed electrochemical immunosensor.

[0039] Figure 4 CV determination graph of a series of gradient sibutramine standard solutions prepared using PBST solution.

[0040] Figure 5 Standard curve corresponding to the CV determination of a series of gradient sibutramine standard solutions prepared using PBST solution. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0042] The embodiments of the present application provide an electrochemical immunosensor for detecting the content of sibutramine and a sibutramine detection kit, thereby solving the technical problem of the low sensitivity of the existing detection methods for the content of sibutramine.

[0043] The general idea of the technical solutions in the embodiments of the present application for solving the above technical problems is as follows:

[0044] The present invention discloses an electrochemical immunosensor for detecting the content of sibutramine and a sibutramine detection kit. The prepared nanocomposite material is immobilized on the electrode surface using chitosan, and then the electrode is activated with an activation solution (2 mM NHS, 1 mM EDC dissolved in 10 mL DMF) for 30 min. The sibutramine antibody is immobilized on the electrode surface modified with the nanocomposite material, and then a standard curve is established. The detection limit of this method for detecting sibutramine is 0.34 pg / mL, and the detection range is 0.01 - 100 pg / mL, showing a good linear relationship within 0.01 - 100 pg / mL. The electrochemical immunosensor obtained by the present invention can quickly, accurately and conveniently detect sibutramine, with a detection limit of 0.34 pg / mL and high sensitivity.

[0045] To better understand the above technical solutions, the above technical solutions will be described in detail below in conjunction with the accompanying drawings of the specification and specific implementation manners.

[0046] Example 1

[0047] This example provides a method for preparing an electrochemical immunosensor for detecting the content of sibutramine, as Figure 1 , including the synthesis of sibutramine artificial antigen, the preparation of sibutramine polyclonal antibody, and the preparation of the working electrode, specifically including the following steps:

[0048] S1. Synthesize sibutramine artificial antigen, including the following steps:

[0049] S11. Take 0.36 mg of 1-(4-phenol)cyclobutanamine and 5-hydroxytryptamine and dissolve them in 0.125 mL of DMF respectively. Take 5 mg of N-carbonyldiimidazole (NHS) and dissolve it in 0.25 mL of DMF. Take 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and dissolve it in 0.5 mL of DMF. After mixing, stir overnight in the dark at 16 °C, centrifuge at 4 °C and 6000 rpm / min for 8 min, and take the supernatant, denoted as the first solution, for standby.

[0050] S12. Take 3 mg of bovine serum albumin (BSA) and dissolve it in 2 mL of coating buffer. Take 2 mg of chicken ovalbumin (OVA) and dissolve it in 1 mL of coating buffer. Mix the two to obtain a BSA / OVA mixture.

[0051] S13. Slowly add 0.15 mL and 0.1 mL of the supernatant to the BSA / OVA mixture respectively, and stir and react at room temperature for 3 h. The resulting solution is denoted as the second solution.

[0052] S14. Use 0.85% sodium chloride solution as the dialysis solution to dialyze the second solution for 3 days (change the dialysis solution every 12 h), and make up the volume to 3 mL with the coating solution to obtain the immunogen 1-(4-phenol)cyclobutanamine-BSA, and make up the volume to 2 mL with the coating solution to obtain the coating antigens 1-(4-phenol)cyclobutanamine-OVA and 5-hydroxytryptamine-OVA.

[0053] Among them, the coating buffer formula is 1.69 g of sodium carbonate and 2.95 g of sodium bicarbonate, and the volume is made up to 1 L with distilled water.

[0054] S2. Prepare sibutramine polyclonal antibody, including the following steps:

[0055] S21. Immunize New Zealand white rabbits with the immunogen 1-(4-phenol)cyclobutanamine-BSA. At the first immunization, emulsify the immunogen with Freund's complete adjuvant at a volume ratio of 1:1 and immunize the rabbits. After that, emulsify the immunogen with Freund's incomplete adjuvant at a volume ratio of 1:1, and perform booster immunizations every three weeks for a total of three times. Take the rabbit serum on the seventh day after the third booster immunization.

[0056] S22. The rabbit serum is centrifuged at 12,000 r / min for 15 min at 4 °C to remove the precipitate and obtain the supernatant.

[0057] S23. 1 volume of the supernatant is mixed with 2 volumes of acetate buffer, the pH is adjusted to 4.8, and n-octanoic acid is added dropwise with stirring at room temperature, with a usage amount of 75 μL of n-octanoic acid / mL of rabbit serum.

[0058] S24. Stir and mix at room temperature for 30 min and let stand at 4 °C for 2 h.

[0059] S25. Centrifuge at 12,000 r / min for 15 min at 4 °C, and filter the supernatant through a sintered glass funnel or a 125-μm nylon mesh.

[0060] S26. Add 1 / 10 volume of PBS buffer (0.1 M, pH 7.4), adjust the pH to 7.4 with 2 M sodium hydroxide, and calculate the total solution volume.

[0061] S27. Add ammonium sulfate at 0.28 g / mL within 30 min under ice bath to make it a 45% saturated solution.

[0062] S28. Let stand at 4 °C for 1 h, centrifuge at 12,000 r / min for 15 min at 4 °C, and discard the supernatant to obtain the precipitate.

[0063] S29. Dialyze the precipitate with PBS buffer (0.1 M, pH 7.4) for three days to obtain sibutramine polyclonal antibody.

[0064] S3. Prepare a modified working electrode, including the following steps:

[0065] S31. Polish the glassy carbon electrode with 1-μm alumina powder, and repeatedly rinse the surface and the outer wall of the electrode around with a large amount of pure water.

[0066] S32. Polish the glassy carbon electrode with 0.3-μm alumina powder and repeatedly rinse with a large amount of pure water.

[0067] S33. Polish the glassy carbon electrode with 0.05-μm alumina powder and repeatedly rinse with a large amount of pure water.

[0068] S34. Ultrasonically clean with ultrapure water and ethanol for 10 s respectively.

[0069] S35. Completely immerse the electrode surface in 10 mL of 0.5 M sulfuric acid solution, and activate the electrode surface by cyclic voltammetry (CV) (scan rate: 0.1 V / s, scan range: -1.00 to +1.00 V).

[0070] S36. Replace the electrolyte with a 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution prepared using 0.1 M KCl. If the peak difference between the oxidation peak and the reduction peak is between 60 and 90, the polishing is successful;

[0071] S37. Weigh 4.8 mg of carboxylated carbon nanotubes, 4.8 mg of Ketjen black, and 19.6 mg of chitosan, and ultrasonically disperse them in 4 mL of 2% acetic acid solution. Then, ultrasonically disperse them in an ice water environment for 1 h to make them uniformly dispersed, obtaining a nanocomposite mixed solution. Measure the properties of the obtained nanocomposite. The results are shown in Figure 2 , from Figure 2 it can be seen that the nanocomposite is successfully prepared and the electrical signal is improved.

[0072] Drop 10 μl of the nanocomposite mixed solution onto the glassy carbon electrode to obtain a modified working electrode. The specific process includes:

[0073] Using the glassy carbon electrode as the working electrode, the silver chloride electrode as the reference electrode, and the platinum wire electrode as the counter electrode to form a three-electrode structure. Drop 10 μl of the nanocomposite mixed solution onto the surface of the working electrode, i.e., the glassy carbon electrode, and then dry it with a nitrogen stream. Use CV electrochemical technology to detect the state of the working electrode. If the electrical signal is amplified, it indicates that the modified working electrode is successfully prepared.

[0074] Use carboxylated carbon nanotubes and Ketjen black to increase the peak current value. The carboxyl groups of the carbon nanotubes are connected to the antibody, simplifying the steps of antibody immobilization.

[0075] S4. Construct an electrochemical immunosensor. The CV characterization diagram of the constructed electrochemical immunosensor is shown in Figure 3 , from Figure 3 it can be seen that the electrochemical immunosensor is successfully constructed. And detect the content of sibutramine, including the following steps:

[0076] S41. Activate the modified working electrode with an activation solution (2 mM NHS, 1 mM EDC dissolved in 10 mL of DMF) for 30 min;

[0077] S42. Drop 3 μl of the sibutramine polyclonal antibody onto the modified working electrode and incubate it at 37 °C for 80 min;

[0078] S43. Drop 3 μl of the blocking solution (1% BSA solution), incubate it at 37 °C for 60 min, and detect the peak current value;

[0079] S44. Drop 3 μl of the standard product, incubate it at 37 °C for 60 min, and detect the peak current value.

[0080] The detection results are as follows:

[0081] CV determination: The CV determination results of the obtained sibutramine electrochemical immunosensor for a series of gradient sibutramine standard solutions are shown in Figure 4 , and it can be seen from Figure 4 that the electrode has good detection ability for sibutramine.

[0082] Standard curve: The direct method standard curve of the obtained sibutramine electrochemical immunosensor is as shown in Figure 5 .

[0083] Sensitivity: The detection limit of the electrochemical immunosensor prepared in this example for detecting sibutramine is 0.34 pg / mL, the detection range is 0.01 - 100 pg / mL, and there is a good linear relationship within 0.01 - 100 pg / mL.

[0084] Comparative example 1:

[0085] The difference between this comparative example and Example 1 is only that the electrode surface does not include the nanocomposite material, and the others are the same as in Example 1.

[0086] The detection limit of the electrochemical immunosensor prepared in this comparative example for detecting sibutramine is 0.02 ng / mL, the detection range is 0.1 - 10 ng / mL, and there is a linear relationship within 0.1 - 10 ng / mL.

[0087] In the present invention, the sibutramine antibody is combined on the surface of the electrode enhanced by the nanocomposite material for peak current value. The prepared polyclonal antibody of sibutramine has high sensitivity. The obtained sibutramine electrochemical immunosensor can meet the detection requirements, is convenient and fast to use, is an important tool for rapid detection of sibutramine, and has broad application prospects.

[0088] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a..." does not exclude the existence of additional identical elements in the process, method, article or device comprising the element.

[0089] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0090] The present invention uses the above embodiments to illustrate the detailed process flow of the present invention, but the present invention is not limited to the above detailed process flow, that is, it does not mean that the present invention must rely on the above detailed process flow to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of the raw materials of the products of the present invention, the addition of auxiliary components, the selection of specific methods, etc. are all within the protection scope and disclosure scope of the present invention.

Claims

1. An electrochemical immunosensor for detecting the content of sibutramine, characterized in that, The electrochemical immunosensor includes an electrode, sibutramine artificial antigen, and sibutramine polyclonal antibody; The electrode is a modified working electrode; the sibutramine artificial antigen is obtained by coupling 1-(4-phenol)cyclobutanamine, 5-hydroxytryptamine with a carrier protein; the sibutramine polyclonal antibody is obtained by immunizing an animal with the sibutramine artificial antigen.

2. The electrochemical immunosensor according to claim 1, characterized in that, The modified working electrode is a glassy carbon electrode modified with a nanocomposite, and the nanocomposite is prepared from carboxylated carbon nanotubes, Ketjen black, and chitosan according to a mass ratio of 1:1 - 1.5:4 - 5; The carrier protein includes bovine serum albumin and chicken ovalbumin.

3. The electrochemical immunosensor according to claim 1, wherein The coupling of 1-(4-phenol)cyclobutanamine and the carrier protein is carried out by the active ester method.

4. The electrochemical immunosensor according to claim 1, wherein The preparation method of the sibutramine artificial antigen is as follows: S1. Dissolve 1-(4-phenol)cyclobutanamine, 5-hydroxytryptamine, N-carbonyldiimidazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in N,N-dimethylformamide respectively, mix them, and stir in the dark at room temperature for 12 - 14 h to obtain a first solution; S2. Add bovine serum albumin and chicken ovalbumin to the coating buffer respectively to obtain a bovine serum albumin / chicken ovalbumin mixture; S3. Slowly drip the first solution into the bovine serum albumin / chicken ovalbumin mixture, and stir and react in the dark at room temperature for 2 - 4 h to obtain a second solution; S4. Dialyze the second solution to obtain the sibutramine artificial antigen.

5. The electrochemical immunosensor according to claim 4, characterized in that, S1 satisfies at least one of the following conditions: The mass-to-volume ratio of 1-(4-phenol)cyclobutanamine, 5-hydroxytryptamine to N,N-dimethylformamide is (0.2 - 0.5)mg:(0.1 - 0.3)mg:(0.1 - 0.2)mL; The mass-to-volume ratio of N-carbonyldiimidazole to N,N-dimethylformamide is (4 - 6)mg:(0.2 - 0.3)mL; The mass-to-volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N,N-dimethylformamide is (4 - 6)mg:(0.4 - 0.6)mL.

6. The electrochemical immunosensor according to claim 4, characterized in that, S2 satisfies at least one of the following conditions: The coating buffer includes sodium carbonate (1 - 3)g / L and sodium bicarbonate (2 - 4)g / L; The mass-to-volume ratio of bovine serum albumin added to the coating buffer is (1 - 4)mg:(1 - 3)mL; The mass-to-volume ratio of chicken ovalbumin added to the coating buffer is (1 - 3)mg:(0.5 - 1.5)mL.

7. The electrochemical immunosensor according to claim 4, characterized in that, S4 satisfies at least one of the following conditions: The dialysis solution is 0.5% - 1.0% sodium chloride solution; The dialysis time is three days.

8. The electrochemical immunosensor according to claim 1, wherein, The preparation method of the sibutramine polyclonal antibody is: Immunize an animal with the sibutramine artificial antigen solution. The immunization includes one primary immunization and three booster immunizations. The sibutramine artificial antigen solution is mixed and emulsified with Freund's complete adjuvant for primary immunization, and the sibutramine artificial antigen solution is emulsified with Freund's incomplete adjuvant for booster immunization. The interval between immunization inoculations is three weeks; blood is collected on the seventh day after the third booster immunization, and the sibutramine polyclonal antibody is purified by the caprylic acid-ammonium sulfate method; The animal is a rabbit, and the immunization dose per time is 700 - 750 μL per rabbit.

9. The electrochemical immunosensor according to claim 1, wherein The preparation method of the nanocomposite material is as follows: Weigh 4 - 6 mg of carboxylated carbon nanotubes, 4 - 6 mg of Ketjen black, and 18 - 21 mg of chitosan, ultrasonically disperse them in 3 - 5 mL of 1 - 3% acetic acid solution, and then ultrasonically treat them in an ice water environment for 0.5 - 1.5 h to obtain the nanocomposite material.

10. A sibutramine detection kit, characterized in that, The sibutramine detection kit includes the sibutramine polyclonal antibody according to any one of claims 1 - 9.

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