Fluorescence sensing kit for detecting carbonic anhydrase activity and application

Abstract

The invention discloses a fluorescent sensing kit for detecting carbonic anhydrase activity and application, and belongs to the technical field of biology. Based on the principle that carbonic anhydrase can catalyze a substrate to generate p-nitrophenol and can quench fluorescence of synthesized carbon quantum dots, a carbonic anhydrase activity detection method is established, and the activity of carbonic anhydrase is determined by detecting fluorescence intensity; based on the principle that a carbonic anhydrase inhibitor can restore fluorescence of carbon quantum dots by inhibiting the activity of carbonic anhydrase, a screening method of the carbonic anhydrase activity inhibitor is established. According to the method, carbonic anhydrase activity detection and inhibitor screening can be efficiently and accurately achieved, the screening efficiency is high, the false positive rate is low, a new method is provided for rapid detection of carbonic anhydrase activity, and a new strategy is provided for screening and evaluation of carbonic anhydrase activity inhibitors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a fluorescence sensing kit for detecting carbonic anhydrase activity and application. BACKGROUND

[0002] Carbonic anhydrase (CA) is a zinc-dependent metalloenzyme that exists in organisms and is widely distributed in animals, plants, algae and bacteria. Carbonic anhydrase plays an important role in various physiological processes, including pH regulation, gluconeogenesis, urea production, gastric acid secretion and CO2 transport in tissues, and is considered as a potential therapeutic target for diseases such as glaucoma, obesity, epilepsy, osteoporosis, high altitude reaction and edema. So far, the commercially available small molecule inhibitors such as acetazolamide and methazolamide still lack selectivity and can cause a series of side effects after taking, affecting human health. Fortunately, natural drugs have rich structural diversity, which can provide more choices for the development of new carbonic anhydrase inhibitors. Many natural drugs have been proven to be safe and effective in traditional applications, with fewer side effects and more easily accepted by patients. Therefore, it is necessary to develop a method that can comprehensively, objectively, efficiently and quickly discover and screen carbonic anhydrase inhibitors, and screening carbonic anhydrase inhibitors from natural drugs will bring a new breakthrough to overcome the shortcomings of current synthetic drugs. SUMMARY

[0003] The purpose of the present application is to provide a fluorescence sensing kit for detecting carbonic anhydrase activity and application to solve the problems existing in the prior art. The present application is based on the principle that carbonic anhydrase can catalyze the generation of p-nitrophenol from the substrate, and the fluorescence of the synthesized carbon quantum dots can be quenched. A carbonic anhydrase activity detection method is established, and the activity of carbonic anhydrase is determined by detecting the fluorescence intensity. Based on the principle that carbonic anhydrase inhibitors can inhibit the activity of carbonic anhydrase and restore the fluorescence of carbon quantum dots, a carbonic anhydrase activity inhibitor screening method is established. The method of the present application can efficiently and accurately realize the detection of carbonic anhydrase activity and the screening of inhibitors, with high screening efficiency and low false positive rate, providing a new method for rapid detection of carbonic anhydrase activity and a new strategy for screening and evaluation of carbonic anhydrase activity inhibitors.

[0004] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0005] The present application provides a fluorescence sensing kit for detecting carbonic anhydrase activity, which comprises p-nitrophenyl acetate and carbon quantum dots.

[0006] Further, the preparation method of the carbon quantum dots is as follows: p-aminophenol and ethylenediamine are mixed according to a molar ratio of 1:1, and reacted at 45-55℃ for 4-6h.

[0007] The application also provides application of the fluorescent sensing kit in detection of carbonic anhydrase activity for non-diagnostic purposes.

[0008] The application also provides a method for using the fluorescent sensing kit, wherein after mixing and reacting the to-be-detected carbonic anhydrase solution and the p-nitrophenyl acetate solution, the mixed solution is mixed and incubated with the carbon quantum dot solution, the fluorescence intensity is detected, and the standard curve prepared by the standard carbonic anhydrase is substituted to calculate the activity of the to-be-detected carbonic anhydrase solution.

[0009] The volume ratio of the to-be-detected carbonic anhydrase solution to the p-nitrophenyl acetate solution is 1:1, and the volume ratio of the mixed solution to the carbon quantum dot solution is 1-2:1.

[0010] The concentration of the p-nitrophenyl acetate solution is 400-600 μg / mL, and the concentration of the carbon quantum dot solution is 0.1-0.3 mg / mL.

[0011] The temperature of the mixing reaction is 35-38 ℃, and the time is 80-100 min; and the temperature of the mixing incubation is 20-25 ℃, and the time is 25-35 min.

[0012] The application also provides a kit for evaluating the inhibition effect of a carbonic anhydrase activity inhibitor, wherein the kit comprises carbonic anhydrase, p-nitrophenyl acetate and the carbon quantum dot.

[0013] The application also provides application of the kit in evaluation of the inhibition effect of a carbonic anhydrase activity inhibitor.

[0014] The application also provides a method for evaluating the inhibition effect of a carbonic anhydrase activity inhibitor by using the kit, wherein after mixing and reacting the to-be-detected carbonic anhydrase activity inhibitor, the carbonic anhydrase solution and the p-nitrophenyl acetate solution, the mixed solution is mixed and incubated with the carbon quantum dot solution, the fluorescence intensity is detected, and the fluorescence intensity of the initial carbonic anhydrase solution is compared to analyze the inhibition effect of the carbonic anhydrase activity inhibitor.

[0015] The volume ratio of the to-be-detected carbonic anhydrase activity inhibitor to the carbonic anhydrase solution and the p-nitrophenyl acetate solution is 1:1:1, and the volume ratio of the mixed solution to the carbon quantum dot solution is 1-2:1.

[0016] The concentration of the carbonic anhydrase solution is 30-50 μg / mL, the concentration of the p-nitrophenyl acetate solution is 400-600 μg / mL, and the concentration of the carbon quantum dot solution is 0.1-0.3 mg / mL.

[0017] The temperature of the mixing reaction is 35-38 ℃, and the time is 80-100 min; and the temperature of the mixing incubation is 20-25 ℃, and the time is 25-35 min.

[0018] The present application also provides a kit for screening carbonic anhydrase activity inhibitors, wherein the kit comprises a carbonic anhydrase immobilized enzyme;

[0019] The preparation method of the carbonic anhydrase immobilized enzyme is as follows: after reacting Fe3O4 in an ethanol aqueous solution with ammonium hydroxide and tetraethyl orthosilicate for 1-3 h, N-(trimethoxysilylpropyl) ethylenediamine is added for further reaction for 20-35 min, and then the reaction is maintained at 30 DEG C for 2-4 h to obtain Fe3O4@SiO2-TMSED; after reacting the Fe3O4@SiO2-TMSED with glutaraldehyde for 1.5-2.5 h, a carbonic anhydrase solution is incubated for 3-5 h, and finally lysine is added for reaction for 1.5-2.5 h to obtain the carbonic anhydrase immobilized enzyme.

[0020] The present application also provides the use of the above-mentioned kit in screening carbonic anhydrase activity inhibitors.

[0021] The present application also provides a method for screening carbonic anhydrase activity inhibitors by using the above-mentioned kit, wherein the carbonic anhydrase immobilized enzyme is mixed with a to-be-tested active substance and incubated for 1 h, followed by solid-liquid separation, and then the obtained solid component is eluted with an acetonitrile aqueous solution, and the eluate is collected; and the active monomer component in the eluate is identified by using a high performance liquid chromatograph.

[0022] The present application discloses the following technical effects:

[0023] The present application provides a method for detecting carbonic anhydrase activity based on fluorescence sensing technology and a method for screening carbonic anhydrase activity inhibitors based on affinity adsorption technology. The present application establishes a method for detecting carbonic anhydrase activity based on the principle that carbonic anhydrase can catalyze the generation of p-nitrophenol from a substrate, and can quench the fluorescence of synthesized carbon quantum dots; and the activity of carbonic anhydrase is determined by detecting the fluorescence intensity. The present application establishes a method for screening carbonic anhydrase activity inhibitors based on the principle that carbonic anhydrase inhibitors can inhibit the activity of carbonic anhydrase, and can restore the fluorescence of carbon quantum dots. The present application also prepares a carbonic anhydrase immobilized enzyme, which is immobilized on Fe3O4@SiO2-TMSED to form a Fe3O4@SiO2-TMSED-GLU-CA affinity chromatography carrier material. By using the principle of affinity adsorption, carbonic anhydrase activity inhibitors are adsorbed onto the immobilized enzyme, and the components having carbonic anhydrase activity inhibiting effect are determined by using HPLC analysis technology and liquid chromatography-mass spectrometry technology, so that the screening of carbonic anhydrase activity inhibitors is faster and more convenient.

[0024] The method of the present application can efficiently and accurately realize the detection of carbonic anhydrase activity and the screening of inhibitors, has high screening efficiency and low false positive rate, and provides a new method for the rapid detection of carbonic anhydrase activity and a new strategy for the screening and evaluation of carbonic anhydrase activity inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0026] Figure 1 The research process diagram of the present application; wherein, a is the process diagram of the fluorescence sensing technology; b is the process diagram of the affinity adsorption technology; c is the process diagram of the inhibitor screening;

[0027] Figure 2 The structure characterization and fluorescence activity detection results of carbon quantum dots; wherein, A is the synthesis process diagram of carbon quantum dots; B is the TEM image of carbon quantum dots; C is the size distribution diagram of carbon quantum dots; D is the high-resolution XPS spectrum of C 1s ; E is the high-resolution XPS spectrum of N 1s ; F is the high-resolution XPS spectrum of O 1s ; G is the XPS spectrum of carbon quantum dots; H is the FT-IR spectrum of carbon quantum dots; I is the fluorescence spectrum and absorption spectrum of carbon quantum dots;

[0028] Figure 3 The establishment and verification results of the carbonic anhydrase activity detection method; wherein, A is the principle diagram of the carbonic anhydrase activity detection; B is the fluorescence intensity of different concentrations of carbonic anhydrase; C is the standard curve of the carbonic anhydrase activity detection; D is the fluorescence intensity of different enzymes; E is the fluorescence intensity of different ions and biological macromolecules; F is the fluorescence intensity of different amino acids; G is the fluorescence intensity of different concentrations of carbonic anhydrase positive inhibitors; H is the anti-interference experiment results of the carbonic anhydrase activity detection; I is the repeatability investigation results of the carbonic anhydrase activity detection;

[0029] Figure 4 The structure characterization and detection results of the immobilized enzyme Fe3O4@SiO2-TMSED-GLU-CA; wherein, A is the synthesis process diagram of the immobilized enzyme; B is the TEM image of Fe3O4@SiO2-TMSED, the scale is 50nm; C is the TEM image of the immobilized enzyme, the scale is 200nm; D is the XPS spectrum of Fe3O4@SiO2-TMSED and Fe3O4@SiO2-TMSED-GLU-CA; E is the element mapping of Fe3O4@SiO2-TMSED-GLU-CA, the scale is 200nm; F is the FTIR spectrum of Fe3O4@SiO2-TMSED and Fe3O4@SiO2-TMSED-GLU-CA, the scale is 200nm;

[0030] Figure 5 The results of the establishment and verification of the screening method for carbonic anhydrase activity inhibitors; wherein A is a schematic diagram of the screening principle of carbonic anhydrase activity inhibitors; B is the adsorption rate of different carbonic anhydrase activity inhibitors; C is the HPLC result graph before and after the adsorption of acetazolamide; D is the HPLC result graph before and after the adsorption of ciprofloxacin; E is the HPLC result graph before and after the adsorption of florfenicol; F is the HPLC result graph before and after the adsorption of argatroban;

[0031] Figure 6 The screening results of carbonic anhydrase activity inhibitors in traditional Chinese medicine extracts; wherein A is a schematic diagram of the screening process of carbonic anhydrase activity inhibitors; B is the screening results of CA inhibitors in 62 traditional Chinese medicine samples;

[0032] Figure 7 The screening results of carbonic anhydrase activity inhibitors in papaya extract and cassia twig extract; wherein A is the HPLC spectrum of the papaya eluent after Fe3O4@SiO2-TMSED-GLU-CA affinity adsorption; B is the high performance liquid chromatogram of papaya monomer compounds; C is the EIC and MS data of papaya monomer compounds; D is the HPLC spectrum of the cassia twig eluent after Fe3O4@SiO2-TMSED-GLU-CA affinity adsorption; E is the high performance liquid chromatogram of cassia twig monomer compounds; F is the EIC and MS data of cassia twig monomer compounds;

[0033] Figure 8 The inhibition rates of ursolic acid, oleanolic acid, cis-cinnamic acid and trans-cinnamic acid on carbonic anhydrase activity; wherein A is the inhibition rate of ursolic acid of different concentrations on carbonic anhydrase activity; B is the inhibition rate of oleanolic acid of different concentrations on carbonic anhydrase activity; C is the inhibition rate of cis-cinnamic acid of different concentrations on carbonic anhydrase activity; D is the inhibition rate of trans-cinnamic acid of different concentrations on carbonic anhydrase activity. DETAILED DESCRIPTION

[0034] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, features and embodiments of the present application.

[0035] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the range, and any other stated value or intermediate value in the range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0036] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would understand. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification controls.

[0037] Many modifications and variations of the present disclosure described in the detailed description of the specification can be made without departing from the scope or spirit of the present disclosure, which will be apparent to those skilled in the art. Other implementations of the disclosure will be apparent to those skilled in the art from the specification. The specification and examples of the disclosure are merely illustrative.

[0038] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", "provide", "providing", "carry", "carrying" or any other variation thereof, are open-ended terms that are intended to mean including but not limited to.

[0039] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the test materials used in the following examples are all purchased from conventional biochemical reagent stores.

[0040] The research process of the present disclosure is as shown in Figure 1 The technical principle is as follows:

[0041] The present disclosure adopts fluorescence sensor technology to quickly identify samples with carbonic anhydrase activity inhibition, and the principle is that carbonic anhydrase (CA) can catalyze the substrate acetic acid p-nitrophenyl ester (NPA) to generate p-nitrophenol, which can quench the fluorescence of the synthesized carbon quantum dots; when the screened sample has carbonic anhydrase activity inhibition, the fluorescence of the carbon quantum dots is restored. Further, through affinity adsorption technology combined with liquid chromatography-mass spectrometry technology, the specific components in the sample with carbonic anhydrase activity inhibition can be identified. The specific operation is to fix carbonic anhydrase on Fe3O4@SiO2-TMSED to form Fe3O4@SiO2-TMSED-GLU-CA immobilized enzyme; the sample to be screened is incubated with the immobilized enzyme for a certain period of time, and after removing the unadsorbed liquid, a suitable solvent is used to elute to obtain an eluate; HPLC analysis is used to compare the peak area changes of the initial liquid of the traditional Chinese medicine extract, the unadsorbed liquid and the eluate, so as to screen the traditional Chinese medicine components that have adsorption effect on carbonic anhydrase; and then liquid chromatography-mass spectrometry technology is used to identify the components. This method can efficiently and accurately realize the detection of carbonic anhydrase activity and the screening of inhibitors, and has high screening efficiency and low false positive rate.

[0042] Example 1

[0043] This embodiment prepares a carbon quantum dot (CDs), and the preparation process is illustrated in the schematic diagram below. Figure 2 As shown in A. The process is as follows:

[0044] Carbon quantum dots were synthesized by mixing p-aminophenol solution (PAP) and ethylenediamine solution (EDA) at a molar ratio of 1:1 and stirring at 500 rpm for 5 h at 50 °C. The quantum dots were further purified by column chromatography using ethyl acetate, and then the solvent was freeze-dried to obtain black solid powder carbon quantum dots (CDs), which were stored at 4 °C for later use.

[0045] The structure of the carbon quantum dots prepared by the above process was characterized, and the results are shown in the figure. Figure 2 TEM images show that CDs are spherical in shape with a narrow diameter distribution; XPS spectra show C 1s There are two distinct peaks at 284.8 eV and 283.8 eV, corresponding to CC and C=C, N, respectively. 1s Assigned to CN and C=N, the peak values ​​are 400.4 and 396.6 respectively, O 1s The peaks observed at 532.0 and 532.5 eV are attributed to C=O and CO bonds; 3437.12 cm⁻¹ in the FT-IR image. -1 The absorption band at corresponds to the hydroxyl group (-OH), and the peaks at 1604.76 and 1327.01 cm⁻¹ are caused by the stretching vibrations of C=O and CO. These characterization results indicate that CDs were successfully synthesized via the reaction of PAP and EDA.

[0046] Example 2

[0047] This embodiment establishes a fluorescence-based method for detecting carbonic anhydrase (CA) activity and establishes a standard curve for carbonic anhydrase activity detection. The detection principle is described in [link to documentation]. Figure 3 A. The process is as follows:

[0048] A series of CA solutions with concentrations ranging from 0 to 100 μg / mL were prepared. 200 μL of CA solution was mixed with 200 μL of 500 μg / mL p-nitrobenzene acetate (NPA), and incubated at 37 °C for 90 min. Then, 200 μL of the mixture was taken out and mixed with 200 μL of 0.2 mg / mL LCDs solution. The mixture was incubated at room temperature for 30 min. The maximum fluorescence intensity was recorded at an excitation wavelength of 415 nm within the range of 440–600 nm. The results are shown below. Figure 3 B and C. The results showed that as the CA concentration increased, the maximum fluorescence value gradually decreased, that is, the CA concentration in the range of 0-50 μg / mL was negatively proportional to the maximum fluorescence value, and the linear equation was y = -21.466x + 3196.7 (R 2 =0.9915). ThroughFigure 3 As can be seen from C, the fluorescence value is moderate when the carbonic anhydrase concentration is 30-50 μg / mL, which lays the foundation for the subsequent establishment of a screening method for carbonic anhydrase activity inhibitors.

[0049] To verify the specificity of the above method, this embodiment uses equal concentrations of different enzymes, including human hyaluronidase (HAase), acetylcholinesterase (AChE), β-lactamase (β-Lac), neuraminidase (NA), β-galactosidase (β-Gal), glucose oxidase (GOX), thrombin (THR), trypsin (TRY), tyrosinase (TYR), and lysozyme (LYS), to replace the 10 μg / mL CA solution in the above process. Al 3+ Na + Ba 2+ NH4 + Zn 2+ Mg 2+ K + Cu 2+ Different ions and biomolecules, such as hemoglobin (Hb) and bovine serum albumin (BSA), were used to replace the 50 μg / mL CA solution in the above process. Different amino acids, such as valine (Val), glycine (Gly), tryptophan (Try), tyrosine (Tyr), cysteine ​​(Cys), lysine (Lys), glutamic acid (Glu), aspartic acid (Asp), histidine (His), and proline (Pro), were used to replace the 50 μg / mL CA solution in the above process. The maximum fluorescence value was measured; the higher the column, the higher the fluorescence intensity. The detection results are shown in […]. Figure 3 DF.

[0050] The above results show that the detection method of the present invention has good selectivity and can be used for subsequent experiments.

[0051] Example 3

[0052] This embodiment uses a carbonic anhydrase positive inhibitor to perform a carbonic anhydrase activity detection experiment. The procedure is as follows:

[0053] A series of acetazolamide (a carbonic anhydrase-positive inhibitor) solutions with concentrations ranging from 0 to 10 μg / mL were prepared. 200 μL of each solution was added, followed by 200 μL of 50 μg / mL CA solution and 200 μL of 500 μg / mL NPA solution, respectively. All solutions were mixed thoroughly and reacted at 37°C for 90 min. Then, 200 μL of the reaction solution was added to 100 μL of CDs solution and mixed thoroughly. The mixture was incubated at room temperature for 30 min. The maximum fluorescence intensity was recorded at an excitation wavelength of 415 nm within the range of 440–600 nm. Results are shown below. Figure 3The results showed that the maximum fluorescence value gradually increased with increasing acetazolamide concentration. These results indicate that acetazolamide inhibits carbonic anhydrase activity, leading to increased solution fluorescence. Therefore, the carbonic anhydrase activity detection method established in this invention can be used for the detection of carbonic anhydrase activity and subsequent screening of inhibitors.

[0054] Example 4

[0055] This embodiment uses a carbonic anhydrase negative inhibitor to perform an anti-interference experiment for carbonic anhydrase activity detection. The procedure is as follows:

[0056] Take 200 μL each of acetazolamide (a carbonic anhydrase-positive inhibitor), ciprofloxacin (a carbonic anhydrase-negative inhibitor), florfenicol (a carbonic anhydrase-negative inhibitor), and argatroban (a carbonic anhydrase-negative inhibitor), and then add 200 μL of 50 μg / mL CA solution and 200 μL of 500 μg / mL NPA solution, respectively. Mix all solutions thoroughly and react at 37 °C for 90 min. Then, take 200 μL of the reaction solution and add 100 μL of LCDs solution, mix thoroughly, and incubate at room temperature for 30 min. Record the maximum fluorescence intensity at an excitation wavelength of 415 nm in the range of 440–600 nm. The results are shown below. Figure 3 The maximum fluorescence intensity of H, ciprofloxacin, florfenicol, and argatroban in the solution was significantly lower than that in the solution of acetazolamide. Furthermore, the relative standard deviation of fluorescence intensity was calculated for eight CA samples reacted simultaneously; the results are shown below. Figure 3 The relative standard deviation (RSD) of the reaction was 2.49%, indicating that the method has good repeatability and is suitable for subsequent experiments.

[0057] The above results further confirm that the carbonic anhydrase activity detection method established in this invention can be used for the detection of carbonic anhydrase activity and subsequent screening of inhibitors.

[0058] Example 5

[0059] This embodiment establishes a screening method for carbonic anhydrase activity inhibitors. The feasibility of this screening method is verified using extracts of traditional Chinese medicine as raw materials. The process is as follows:

[0060] Take 62 EP tubes and add 200 μL of 50 μg / mL CA solution and 200 μL of 500 μg / mL NPA solution, then add 200 μL of 400 μg / mL herbal extracts, namely: Cinnamomum cassia Presl, Pipermethysticum, Magnolia officinalis Rehd. et Wils, Cinnamomum cassia Presl, Commiphora myrrha Engl, Eucommia ulmoides Oliv, Taxillus chinensis (DC.) Danser, Phellodendron chinense Schneid, Crataegus pinnatifida Bge, Cistanche deserticola YCMa, and Rhodiola crenulata (Hook.f. et al.). Thoms.) H. Ohba), Ligusticum chuanxiong Hort, Eugenia caryophyllata Thunb, Rheum palmatum L, Ginkgo biloba L, Chrysanthemum morifolium Ramat, Lonicera japonica Thunb, Albizia julibrissin Durazz, Morus alba L, Eriobotrya japonica (Thunb.) Lindl, Patrinia arvense Linn, Ligustrum lucidum Ait, Acorus tatarinowii Schott, Forsythia suspensa (Thunb.)Vahl), Licorice (Glycyrrhiza uralensis Fisch), Magnolia biondii Pamp, Foeniculum vulgare Mill, Terminalia chebula Retz, Aconitum carmichaelii Debx, Leonurus japonicus Houtt, Juglansmandshurica Maxim, Paeonia lactiflora Pall, Atractylodes macrocephala Koidz, Coptis chinensis Franch, Crataegus pinnatifida Bge (stir-frying), Salvia miltiorrhiza Bge, Epimedium brevicornu Maxim, Prunella vulgaris L, Dipsacus asper Wall. ex Henry, Xanthium sibiricum Patr), nutmeg (Myristica fragrans Houtt), morinda officinalis How, aucklandia lappa Decne, sophora alopecuroides L, polygonum cuspidatum Sieb. et Zucc, lycium barbarum L, scleromitrion diffusum (Willd.) RJWang, scutellaria baicalensis Georgi, cuscuta australis R.Br, angelica sinensis (Oliv.) Diels, paeonia lactiflora Pall, gentiana macrophylla Pall, polygonum multiflorum Thunb, lycium chinense Mill.Alternatively, Lycium barbarum L, saffron (Crocus sativus L), Achyranthes bidentata Bl, Stellaria dichotoma L. var. lanceolata Bge, Pheretima aspergillum (E. Perrier) , Astragalus membranaceus (Fisch.) , Saposhnikovia divaricata (Turcz.) Schishk, Cyperus rotundus L, and Chaenomelesspeciosa (Sweet) Nakai were used. Acetazolamide was used as a positive control. All solutions were mixed and reacted at 37°C for 90 min. Then, 200 μL of each reaction solution was added to 100 μL of LCDs solution and mixed, then incubated at room temperature for 30 min. The maximum fluorescence intensity was recorded at an excitation wavelength of 415 nm in the range of 440–600 nm. Results are shown below. Figure 6 B. The results showed that the maximum fluorescence value of the system solution with added papaya and cinnamon twig increased significantly, indicating that the activity of carbonic anhydrase decreased after reacting with papaya and cinnamon twig extracts. That is, papaya and cinnamon twig extracts have the effect of inhibiting carbonic anhydrase activity, and there are components in papaya and cinnamon twig extracts that can inhibit carbonic anhydrase activity.

[0061] Example 6

[0062] This embodiment prepares an affinity adsorption support material for screening inhibitors of carbonic anhydrase activity—immobilized enzyme Fe3O4@SiO2-TMSED-GLU-CA. A schematic diagram of its preparation process is shown below. Figure 4 As shown in A. The process is as follows:

[0063] 1. Preparation of Fe3O4 by coprecipitation method: 2.5 g of FeCl2·6H2O and 5 g of FeCl2·4H2O were added separately to 500 mL round-bottom flasks, dissolved in 250 mL of distilled water, and heated to 60 °C. While stirring, 10 mL of ammonium hydroxide solution was slowly added dropwise. The reaction was terminated after 3 h. After cooling, a black substance was obtained, collected using an external magnetic field, and washed three times with distilled water. The washed product was dried at 60 °C for 24 h.

[0064] 2. An improved sol-gel method was adopted. Synthesis of Fe3O4@SiO2-TMSED: 2g of freshly prepared Fe3O4 was dispersed in 100mL of a water-ethanol mixture (volume ratio 1:1), 5mL of ammonium hydroxide and 2mL of tetraethyl orthosilicate (TEOS) were added and stirred for 2h. Then, 1mL of N-(trimethoxysilylpropyl)ethylenediamine (TMSED) was added, and stirring was continued for another 30min. The reaction mixture was then maintained at 30℃ for 3h to promote the synthesis of Fe3O4@SiO2-TMSED. The mixture was washed three times alternately with water and ethanol to remove impurities, and finally dried in an oven at 60℃.

[0065] 3. Synthesis of Fe3O4@SiO2-TMSED-GLU-CA: 50 mg of Fe3O4@SiO2-TMSED was dispersed in 2 mL of 0.1 M phosphate buffer (pH 7.4) and reacted with 1 mL of glutaraldehyde solution at room temperature for 2 h. The mixture was then centrifuged, and the particles were washed three times with purified water to remove excess glutaraldehyde. To activate the covalent bond between CA and GLU, the synthesized Fe3O4@SiO2-TMSED-GLU was dispersed in 4 mL of 0.1 M phosphate buffer containing CA (2 mg / mL). After incubation at room temperature for 4 h, the particles were washed three times with distilled water. The particles were then reacted with 10 mL of lysine (1 mg / mL) for 2 h. The washed particles were dispersed in 4 mL of PBS to obtain Fe3O4@SiO2-TMSED-GLU-CA, which was stored at -20℃ for later use. The surface morphology of Fe3O4@SiO2-TMSED and Fe3O4@SiO2-TMSED-GLU-CA was characterized using transmission electron microscopy (TEM). The chemical composition of Fe3O4@SiO2-TMSED and Fe3O4@SiO2-TMSED-GLU-CA was analyzed by X-ray photoelectron spectroscopy (XPS). Their structural properties were analyzed using Fourier transform infrared spectroscopy (FT-IR).

[0066] See results Figure 4It is evident that the Fe3O4@SiO2-TMSED and Fe3O4@SiO2-TMSED-GLU-CA MNPs exhibit regular shapes, uneven dispersion, and consistent basic morphology, but differ in material distribution density. This distribution density may be related to the coating effect of enzyme molecules on the surface of Fe3O4@SiO2-TMSED MNPs, the high surface-to-volume ratio, and the magnetism of the material, indicating that CA is fixed on the material surface. The higher N content in Fe3O4@SiO2-TMSED-GLU-CA compared to Fe3O4@SiO2-TMSED may be related to the successful fixation of CA. Elemental mapping diagrams show that C, N, O, Si, and Fe elements are uniformly distributed in the synthesized Fe3O4@SiO2-TMSED-GLU-CA. In addition, the FTIR spectra of Fe3O4@SiO2-TMSED and Fe3O4@SiO2-TMSED-GLU-CA showed the following values: 3433.26 (NH), 2927.92 (CH3), 2854.62 (CH2), 1631.76 (CO-NH), 1519.89 (CC), 1400.30 (CN), 1053.12 (Si-O), 798.52 (Si-C), and 582.49 cm⁻¹. -1 Characteristic peaks of (Fe-O). NH, CH, CN, and Si-C are related to the presence of TEOS and TMSED in the prepared MNPs, indicating the formation of an organic-inorganic hybrid structure. In the spectrum of Fe3O4@SiO2-TMSED-GLU-CA, all peaks related to the MNPs generated in the above steps can be observed. However, the peaks of CA cannot be clearly distinguished due to overlap with the peaks of other NPs.

[0067] Example 7

[0068] This embodiment uses carbonic anhydrase positive inhibitors (acetazolamide) and negative inhibitors (ciprofloxacin, florfenicol, and argatroban) as experimental materials. A method for screening carbonic anhydrase activity inhibitors based on affinity adsorption technology was established and validated using the affinity adsorption support material—immobilized enzyme Fe3O4@SiO2-TMSED-GLU-CA—prepared in Example 6. The process is as follows:

[0069] To four EP tubes containing Fe3O4@SiO2-TMSED-GLU-CA, add 200 μL each of acetazolamide (ACE), ciprofloxacin (CIP), florfenicol (FLO), and argatroban (ARG). After mixing, shake in a shaker for 1 hour, allow to stand, and centrifuge to obtain the supernatant, which is the adsorption solution. Then, add 200 μL of an acetonitrile-water mixture (1:1 volume ratio) to the remaining solid, mix well, shake in a shaker at room temperature for 1 hour, and centrifuge to obtain the supernatant, which is the eluent. Analyze the initial solution, unadsorbed solution, and eluent using high-performance liquid chromatography (HPLC). Calculate the adsorption rate using α = (adsorbed amount / initial amount) × 100%. Results are shown below. Figure 5 As shown in the figure, the results indicate that the carbonic anhydrase positive inhibitor acetazolamide exhibits good adsorption with Fe3O4@SiO2-TMSED-GLU-CA, while the three negative inhibitors show poor adsorption with Fe3O4@SiO2-TMSED-GLU-CA. The signal intensity of the unadsorbed acetazolamide solution decreased compared to the initial solution, indicating that the adsorption of acetazolamide by the immobilized enzyme led to the signal intensity decrease. The signal intensity of the unadsorbed solutions of the other three negative inhibitors showed no significant change compared to the initial solution, indicating that the negative inhibitors were not adsorbed by the immobilized enzyme. These results demonstrate that the immobilized enzyme Fe3O4@SiO2-TMSED-GLU-CA can be used for screening carbonic anhydrase inhibitors.

[0070] Example 8

[0071] This embodiment, based on the results of Example 5, uses the affinity adsorption support material prepared in Example 6—immobilized enzyme Fe3O4@SiO2-TMSED-GLU-CA—to screen and capture components in papaya and cinnamon twig extracts that can bind to carbonic anhydrase and inhibit its activity. A schematic diagram of the screening principle is shown below. Figure 6 A. The process is as follows:

[0072] 200 μL of papaya and cinnamon twig extracts were added to an EP tube containing Fe3O4@SiO2-TMSED-GLU-CA. After mixing, the tube was shaken for 1 h and centrifuged to obtain the supernatant (unadsorbed liquid). Then, 200 μL of acetonitrile-water (1:1) was added to the remaining solid, mixed, and shaken at room temperature for 1 h. The supernatant (elution liquid) was then centrifuged to obtain the supernatant. The initial solution, unadsorbed liquid, and eluent of the papaya and cinnamon twig extracts were analyzed by high-performance liquid chromatography (HPLC). The specific HPLC operating parameters for papaya were as follows: XBridge C18 column (4.6 mm × 150 mm, 5 μm), mobile phase 0.3% phosphoric acid (A): acetonitrile (B), gradient elution program 0 min - 70% B; 40 min - 70% B; column temperature: 27℃; flow rate: 1 mL / min. The specific HPLC operating parameters for Cinnamomum cassia were as follows: XBridge C18 column (4.6 mm × 150 mm, 5 μm); mobile phase: 0.1% phosphoric acid (A): acetonitrile (B); gradient elution program: 0 min - 32% B; 30 min - 70% B; 32 min - 32% B; column temperature: 30℃; flow rate: 1 mL / min. Results are shown below. Figure 7 The results of A and D indicate that chromatographic peaks can be clearly seen in the high performance liquid chromatograms of papaya and cinnamon twig eluents, indicating that Fe3O4@SiO2-TMSED-GLU-CA successfully captured four components in papaya and cinnamon twig that can bind to carbonic anhydrase.

[0073] Further liquid chromatography-mass spectrometry (LC-MS) was used to identify components in papaya and cinnamon twig that can bind to carbonic anhydrase. The eluates from papaya and cinnamon twig were analyzed using LC-MS with the following parameters: 0 min - 90% A; 6 min - 10% A; 9 min - 10% A; 12 min - 90% A; 15 min - 90% A (A: 0.1% phosphate water; B: 0.1% phosphate methanol). The ion transfer tube temperature was 350℃, the evaporation temperature was 300℃, the sheath gas pressure was 35 arb, the auxiliary gas pressure was 10 arb, and the spray voltage was 3.5 kV. In both positive and negative ion modes, a primary spectrum of ions in the 100-1000 ion range was acquired using Full MS-ddms2 mode, while a secondary spectrum of the top 5 ions was acquired using a collision energy of HCD30. The results are shown below. Figure 7 Based on the C and F, it was preliminarily determined that the components in papaya and cinnamon twig that can bind with carbonic anhydrase are ursolic acid, oleanolic acid, cis-cinnamic acid, and trans-cinnamic acid.

[0074] Further, high-performance liquid chromatography (HPLC) was used in conjunction with reference standards to determine the components in papaya and cinnamon twig that can bind to CA. 100 μg / m solutions of ursolic acid, oleanolic acid, cis-cinnamic acid, and trans-cinnamic acid reference standards were prepared. The initial, unadsorbed, and eluent solutions of papaya and cinnamon twig extracts, along with the ursolic acid, oleanolic acid, cis-cinnamic acid, and trans-cinnamic acid reference standard solutions, were analyzed by HPLC. Results are shown below. Figure 7 B and E. The results showed that the chromatographic peaks in the eluent were identified as ursolic acid, oleanolic acid, cis-cinnamic acid, and trans-cinnamic acid. These results determined that the components in papaya and cinnamon twig that can bind to CA are ursolic acid, oleanolic acid, cis-cinnamic acid, and trans-cinnamic acid, respectively.

[0075] Example 9

[0076] This embodiment tested the half-maximal inhibitory concentration (IC50) of ursolic acid, oleanolic acid, cis-cinnamic acid, and trans-cinnamic acid—components in papaya and cinnamon twig that inhibit carbonic anhydrase activity. 50 Values ​​were determined. A series of solutions of cinnamic acid, ursolic acid, and oleanolic acid were prepared. 200 μL and 50 μg / mL CA solution, and 200 μL and 500 μg / mL NPA solution were taken respectively, mixed thoroughly, and reacted at 37℃ for 90 min. Then, 200 μL of each reaction solution was added to 100 μL of CDs solution, mixed thoroughly, and incubated at room temperature for 30 min. The maximum fluorescence intensity at an excitation wavelength of 415 nm within the range of 440-600 nm was recorded. Results are shown below. Figure 8 AD. The results showed that ursolic acid, oleanolic acid, cis-cinnamic acid, and trans-cinnamic acid had different IC50 values. 50 The values ​​were 291.29 μM, 446.35 μM, 3.98 mM, and 5.37 mM, respectively. These results further confirm that cinnamic acid, ursolic acid, and oleanolic acid have a good inhibitory effect on carbonic anhydrase activity.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fluorescence sensing kit for detecting carbonic anhydrase activity, characterized in that, The kit contains p-nitrophenyl acetate and carbon quantum dots.

2. The fluorescence sensing reagent kit according to claim 1, characterized in that, The carbon quantum dots are prepared by mixing p-aminophenol and ethylenediamine in a molar ratio of 1:1 and reacting them at 45-55℃ for 4-6 hours.

3. The use of the fluorescence sensing kit according to claim 1 or 2 in the detection of carbonic anhydrase activity for non-diagnostic purposes.

4. A method of using the fluorescence sensing kit according to claim 1 or 2, characterized in that, After mixing and reacting the carbonic anhydrase solution to be tested with p-nitrophenyl acetate solution, the mixed solution was incubated with carbon quantum dot solution, the fluorescence intensity was detected, and the activity of the carbonic anhydrase solution to be tested was calculated by substituting the fluorescence intensity into the standard curve prepared by standard carbonic anhydrase. The volume ratio of the carbonic anhydrase solution to the p-nitrophenyl acetate solution is 1:1; the volume ratio of the mixed solution to the carbon quantum dot solution is 1-2:

1. The concentration of the p-nitrobenzene acetate solution is 400-600 μg / mL; the concentration of the carbon quantum dot solution is 0.1-0.3 mg / mL. The mixing reaction is carried out at a temperature of 35-38℃ for 80-100 min; the mixing incubation is carried out at a temperature of 20-25℃ for 25-35 min.

5. A kit for evaluating the inhibitory effect of carbonic anhydrase activity inhibitors, characterized in that, The kit contains carbonic anhydrase, p-nitrophenyl acetate, and the carbon quantum dots as described in claim 2.

6. The use of the kit according to claim 5 in evaluating the inhibitory effect of carbonic anhydrase activity inhibitors.

7. A method for evaluating the inhibitory effect of carbonic anhydrase activity inhibitors using the kit described in claim 5, characterized in that, The carbonic anhydrase activity inhibitor to be tested was mixed with carbonic anhydrase solution and p-nitrobenzene acetate solution and reacted. The mixed solution was then incubated with carbon quantum dot solution, and the fluorescence intensity was detected and compared with the fluorescence intensity of the initial carbonic anhydrase solution to analyze the inhibitory effect of the carbonic anhydrase activity inhibitor. The volume ratio of the carbonic anhydrase activity inhibitor to the carbonic anhydrase solution and the p-nitrobenzene acetate solution is 1:1:1; the volume ratio of the mixed solution to the carbon quantum dot solution is 1-2:

1. The concentration of the carbonic anhydrase solution is 30-50 μg / mL; the concentration of the p-nitrobenzene acetate solution is 400-600 μg / mL; and the concentration of the carbon quantum dot solution is 0.1-0.3 mg / mL. The mixing reaction is carried out at a temperature of 35-38℃ for 80-100 min; the mixing incubation is carried out at a temperature of 20-25℃ for 25-35 min.

8. A kit for screening inhibitors of carbonic anhydrase activity, characterized in that, The kit contains carbonic anhydrase immobilized enzyme; The method for preparing the carbonic anhydrase immobilized enzyme is as follows: Fe3O4@SiO2-TMSED is obtained by reacting iron(III) oxide with ammonium hydroxide and tetraethyl orthosilicate in an ethanol aqueous solution for 1-3 hours, followed by the addition of N-(trimethoxysilylpropyl)ethylenediamine and further reaction for 20-35 minutes. The reaction is then maintained at 30°C for 2-4 hours to obtain Fe3O4@SiO2-TMSED. The Fe3O4@SiO2-TMSED is then reacted with glutaraldehyde for 1.5-2.5 hours, followed by incubation with carbonic anhydrase solution for 3-5 hours. Finally, lysine is added and the reaction is carried out for 1.5-2.5 hours to obtain the carbonic anhydrase immobilized enzyme.

9. The use of the kit according to claim 8 in screening carbonic anhydrase activity inhibitors.

10. A method for screening carbonic anhydrase activity inhibitors using the kit according to claim 8, characterized in that, After mixing and incubating the carbonic anhydrase immobilized enzyme with the target active substance for 1 hour, solid-liquid separation was performed. The obtained solid component was eluted with acetonitrile aqueous solution, and the eluent was collected. The active monomer components in the eluent were identified by high performance liquid chromatography.

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