CN111777616B - Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof - Google Patents
Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, a preparation method and application thereof, wherein the preparation method of the porphyrin derivative is completed by the following steps: the porphyrin parent is firstly reacted with dibromoalkane, and the obtained product is then reacted with triethylamine to obtain the porphyrin derivative. The porphyrin derivative and the hyaluronic acid chain with negative charges form a novel fluorescent nano sensor by a static self-assembly method. Compared with the existing detection technology, the fluorescence nano-sensor obtained by the invention has good biocompatibility, simple preparation method and high selectivity, can effectively deduct biological background fluorescence when the emission wavelength is in the near infrared region, has good industrial development prospect, and has huge application prospect in the technical fields of analytical chemistry, life science and the like.
Description
Technical Field
The invention belongs to the field of chemical materials and analysis and detection, and particularly relates to a preparation method and application of a porphyrin derivative for detecting hyaluronidase.
Background
Hyaluronic Acid (HA) contains multiple repeating glucuronic acid and N-acetylglucosamine disaccharide units, is negatively charged, HAs good water solubility, and is a main component constituting extracellular matrix (ECM) and intercellular matrix (ICM). Hyaluronidase (HAase) is a specific hydrolase of HA, is an endoglycosidase, can directly hydrolyze beta-1, 4 glycosidic bond of hyaluronic acid, and the final product is mainly a metabolite of tetrasaccharide, and is used as an auxiliary drug for chemotherapy for many years to enhance the permeability of drugs. HAase has been reported to be involved in many physiological and pathological processes, and is highly expressed in malignant tumors such as bladder cancer, prostate cancer, brain cancer, and rectal cancer. Therefore, HAase has attracted extensive attention as a novel tumor marker, and the design and synthesis of a sensor capable of detecting HAase is of great significance for clinical diagnosis and treatment of early cancers.
The detection method of HAase in the prior art mainly comprises a turbidity method, a viscosity method, an zymogram method, an immunoassay method, a colorimetric method, a chemiluminescence auxiliary method and a fluorescence sensing method. Among them, the fluorescence sensing method is widely used because of its advantages such as simple operation, less loss, and high sensitivity. The fluorescence sensing method mainly depends on the mark of a fluorescence sensor to detect HAase, and the traditional fluorescence sensor is mainly divided into an organic small-molecule fluorescence sensor and a fluorescence nano sensor, wherein most of the organic small-molecule fluorescence sensors have poor water solubility, and a large amount of organic solvent is required to be used as a cosolvent in the using process, so that the organic small-molecule fluorescence sensor has strong toxicity and poor light stability; the fluorescence nanosensor has the defects of poor interference resistance although the fluorescence nanosensor has better water dispersibility, biocompatibility and light stability. Most fluorescence sensors in the prior art tend to cause higher biological background fluorescence interference due to absorption and emission in the uv-visible range, resulting in lower signal-to-noise ratio. Therefore, the development of organic molecules and corresponding fluorescence sensors which can effectively overcome the defects of the traditional fluorescence sensing method has important practical significance and application prospect.
Disclosure of Invention
In order to solve the above defects in the prior art, the invention aims to provide a porphyrin derivative based on self-assembly and capable of detecting hyaluronidase and a preparation method thereof, wherein the porphyrin derivative is used as a fluorescent chromophore of a fluorescent sensor to solve the problem of biological background fluorescence interference; the invention also relates to an application of the porphyrin derivative based on self-assembly and capable of detecting hyaluronidase, and the porphyrin derivative and the negatively charged hyaluronidase chain are prepared into a fluorescent nano sensor so as to achieve the purpose of quickly and effectively identifying hyaluronidase.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention relates to a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, which has a general formula as follows:
wherein R1, R2 and R3 are respectively H or O (CH) 2 ) n N(CH 2 CH 3 ) 3 + (ii) a The O (CH) 2 ) n N(CH 2 CH 3 ) 3 + N =2 to 24.
A preparation method of a porphyrin derivative based on self-assembly and capable of detecting hyaluronidase comprises the following steps in sequence:
1. adding SM1 into dry N, N-dimethylformamide, starting stirring, adding anhydrous potassium carbonate, stirring at room temperature, stirring uniformly, adding SM2, refluxing and stirring for 24-48h under the protection of inactive gas, detecting by thin layer chromatography, cooling to room temperature after the reaction is finished, washing with water to remove the N, N-dimethylformamide, and purifying the obtained solid material by column chromatography to obtain SM3, wherein the chemical reaction equation is as follows:
wherein, X 1 ，X 2 ，X 3 Are respectively H or OH; at X 1 =X 2 =X 3 When = H, A 1 =A 2 =A 3 = H; at X 1 =X 2 =H，X 3 When = OH, A 1 =A 2 =H，A 3 =O（CH 2 ） n Br; at X 1 =H，X 2 =X 3 When OH is not zero, A 1 =H，A 2 =A 3 =O（CH 2 ） n Br at X 1 =X 2 =X 3 When OH is not zero, A 1 =A 2 =A 3 = O（CH 2 ） n Br; the O (CH) 2 ） n N =2 to 24 in Br;
2. adding SM3 into dry N, N-dimethylformamide, adding excessive triethylamine, refluxing and stirring for 10 to 20h under the protection of inactive gas, monitoring the reaction by thin-layer chromatography, cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4 which is a porphyrin derivative, wherein the reaction equation is as follows:
wherein A is 1 ，A 2 ，A 3 Are each H or O (CH) 2 ） n Br; in A 1 =A 2 =A 3 When = H, R 1 =R 2 =R 3 = H; in A 1 =A 2 =H，A 3 = O（CH 2 ） n Br is, R 1 =R 2 =H，R 3 = O(CH 2 ) n N(CH 2 CH 3 ) 3 + (ii) a In A 1 =H，A 2 =A 3 =O（CH 2 ） n When Br, R 1 =H，R 2 =R 3 = O(CH 2 ) n N(CH 2 CH 3 ) 3 + (ii) a In A 1 =A 2 =A 3 = O（CH 2 ） n When Br, R 1 =R 2 =R 3 = O(CH 2 ) n N(CH 2 CH 3 ) 3 + (ii) a The O (CH) 2 ） n Br and O (CH) 2 ) n N(CH 2 CH 3 ) 3 + N =2 to 24.
As a limitation to the preparation method of the invention, the reflux temperature is 110 to 140 ℃.
As a further limitation to the above preparation method of the present invention, the SM1: SM2: the molar ratio of the anhydrous potassium carbonate is 1 to 15 to 25.
As a further limitation of the invention, the thin layer chromatography liquid is a mixed liquid of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1 to 10; the eluent for column chromatography is a mixed solution of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1 to 5.
As another limitation to the preparation method, the molar ratio of the SM3 to the triethylamine is 1.
The invention also provides an application of the porphyrin derivative based on self-assembly and capable of detecting hyaluronidase, and the porphyrin derivative and the hyaluronan chain are mixed to prepare the fluorescent nano-device capable of detecting hyaluronidase.
As a limitation of the invention relating to the use of porphyrin derivatives, the hyaluronic acid chain is
Wherein m =100 to 1000.
As another limitation of the present invention relating to the use of the porphyrin derivative, the mass ratio of the porphyrin derivative to the hyaluronic acid chain is 2 to 6.
Due to the adoption of the technical scheme, compared with the prior art, the porphyrin derivative and the corresponding fluorescence sensor have the following beneficial effects:
(1) According to the invention, a functional hydroxyl porphyrin matrix is synthesized by triethyl quaternary ammonium salt to obtain a porphyrin derivative (Mito TPP) with positive charge as a near infrared fluorescence chromophore, so that a sensor is targeted by mitochondria;
(2) The invention is a fluorescence nano-sensor which is formed by self-assembling Mito TPP with positive charges and hyaluronic acid chain with negative charges, the hyaluronic acid further increases the biocompatibility of the nano-sensor and reduces the adverse reaction generated after the fluorescence nano-sensor is injected into the body;
(3) Compared with the traditional organic small molecule fluorescence sensor, the fluorescence nano sensor prepared by the porphyrin derivative provided by the invention has the advantages that the water solubility of the sensor is improved, so that the use of organic solvents is reduced, and the toxicity of the sensor is reduced;
(4) The emission spectrum of the porphyrin derivative is near 650nm, and compared with the traditional fluorescence nanosensor, the emission spectrum of the porphyrin derivative effectively solves the problem of interference generated in the detection process of biological background fluorescence on hyaluronidase dissolved in water.
In conclusion, the invention provides a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, and a preparation method and application thereof. The synthetic route of the porphyrin derivative is simple, the whole process is easy to operate, the steps are short, and the cost investment is low; the prepared fluorescent nano sensor has excellent water solubility and biocompatibility, weak biological background fluorescence and low cytotoxicity, and can directly realize specific recognition on hyaluronidase.
The method is suitable for detecting hyaluronidase and is used for specifically targeting the hyaluronic acid receptor overexpressed on the surface of the cancer cell.
Drawings
The invention is described in further detail below with reference to the figures and the embodiments.
FIG. 1 is a schematic diagram of hyaluronidase recognition by the prepared fluorescent nanosensor;
FIG. 2 is a nuclear magnetic data plot of 5- (4- (triethylamine) -butyloxyphenyl) -10,15, 20-triphenylporphyrin;
FIG. 3 is a particle size distribution diagram of a fluorescent nanosensor;
FIG. 4 is a graph showing the change of fluorescence emission spectrum of the fluorescent nanosensor with the addition time of hyaluronic acid (excitation wavelength: 425 nm);
FIG. 5 is a graph showing the change of fluorescence emission spectra of fluorescence nanosensors when HAase was added at different concentrations;
FIG. 6 is a graph of a fitted curve corresponding to the change in the fluorescence intensity value with the change in the hyaluronidase concentration and a function of the curve;
FIG. 7 is a graph of interference contrast data for fluorescence recovery of various ion pairs for a fluorescent nanosensor;
FIG. 8 is a graph of interference contrast data for various ions on the fluorescence intensity of a fluorescent nanosensor;
FIG. 9 is a graph of HeLa cell viability data of fluorescent nanoprobes of different concentrations.
Detailed Description
Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the description of the preferred embodiment is only for purposes of illustration and understanding, and is not intended to limit the invention.
EXAMPLE 1 porphyrin derivative and preparation method thereof
A porphyrin derivative has a molecular formula of
The preparation method comprises
1. Adding 0.317 mmol of SM1-1 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 4.76 mmol of anhydrous potassium carbonate, stirring at room temperature, adding 4.76 mmol of SM2-1 after stirring and mixing uniformly, stirring at 140 ℃ under reflux for 24h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM = 10), cooling to room temperature after the reaction is finished, washing with water to remove DMF, and purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM = 5) to obtain SM3-1, wherein the chemical reaction equation is:
2. adding 0.131 mmol of SM3-1 into 4ml of dry DMF, adding 5.24mmol of triethylamine, refluxing and stirring at 110 ℃ for 20h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM = 1), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-1, wherein the reaction equation is as follows:
examples 2 to 7 porphyrin derivatives and preparation methods thereof
The experimental procedures and experimental conditions in the preparation methods of examples 2 to 7 were the same as those in example 1, except that the raw materials used were the same
Wherein n =2,5,9,11,17,24, the final porphyrin derivative has the formula
Example 8A porphyrin derivative and a method for preparing the same
A porphyrin derivative has a molecular formula of
The preparation method comprises
1. Adding 0.317 mmol of SM1-2 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 6.02 mmol of anhydrous potassium carbonate, stirring at room temperature, adding 5.39 mmol of SM2-2 after stirring and mixing uniformly, stirring at 130 ℃ under reflux for 30h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM = 8), cooling to room temperature after the reaction is finished, washing with water to remove DMF, and purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM = 4) to obtain SM3-2, wherein the chemical reaction equation is:
2. adding 0.131 mmol of SM3-2 into 4ml of dry DMF, adding 4.72mmol of triethylamine, refluxing and stirring at 125 ℃ for 17h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM = 2), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-2, wherein the reaction equation is as follows:
examples 9 to 14 preparation methods of porphyrin derivatives
The experimental procedures and experimental conditions in the preparation methods of examples 9 to 14 were the same as those in example 1, except that the raw materials used were the same
Wherein n =5, 9,11,17, 22, 24, the final porphyrin derivative has the formula
Example 15A porphyrin derivative and a method for preparing the same
A porphyrin derivative has a molecular formula of
The preparation method comprises
1. Adding 0.317 mmol of SM1-3 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 7.29mmol of anhydrous potassium carbonate, stirring at room temperature, adding 6.34 mmol of SM2-3 after stirring and mixing uniformly, refluxing and stirring at 120 ℃ for 36h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM = 6), cooling to room temperature after the reaction is finished, washing with water to remove DMF, and purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM = 3) to obtain SM3-3, wherein the chemical reaction equation is:
2. adding 0.131 mmol of SM3-3 into 4ml of dry DMF, adding 4.32mmol of triethylamine, stirring at the temperature of 135 ℃ for 14h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM = 3), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-3, wherein the reaction equation is as follows:
examples 16 to 21 porphyrin derivatives and preparation methods thereof
The experimental procedures and experimental conditions in the preparation methods of examples 16 to 21 were the same as those in example 1, except that the raw materials were used
Wherein n =2,5, 11,17, 22, 24, the final porphyrin derivative has the formula
Example 22A porphyrin derivative and a process for the preparation thereof
A porphyrin derivative has a molecular formula of
The preparation method comprises
1. Adding 0.317 mmol of SM4-1 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 8.24mmol of anhydrous potassium carbonate, stirring at room temperature, adding 6.97mmol of SM4-2 after stirring and mixing uniformly, refluxing and stirring at 110 ℃ for 40h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM = 4):
2. adding 0.131 mmol of SM4-3 into 4ml of dry DMF, adding 3.14mmol of triethylamine, refluxing and stirring at 115 ℃ for 12h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM = 7), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-4, wherein the reaction equation is as follows:
examples 23 to 28 porphyrin derivatives and preparation methods thereof
The experimental procedures and experimental conditions in the preparation methods of examples 23 to 28 were the same as those in example 1, except that the raw materials used were the same
Wherein n =2,5, 13, 17, 22, 24, the final porphyrin derivative has the formula
Example 29A porphyrin derivative and preparation method thereof
A porphyrin derivative has a molecular formula of
The preparation method comprises
1. Adding 0.317 mmol of SM1-5 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 9.51 mmol of anhydrous potassium carbonate, stirring at room temperature, adding 7.93 mmol of SM2-5 after stirring and mixing uniformly, refluxing and stirring at 140 ℃ for 48h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM = 10), cooling to room temperature after the reaction is finished, washing with water to remove DMF, and purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM = 5) to obtain SM3-5, wherein the chemical reaction equation is as follows:
2. adding 0.131 mmol of SM3-5 into 4ml of dry DMF, adding 2.62mmol of triethylamine, refluxing and stirring at 110 ℃ for 10h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM = 1), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-5, wherein the reaction equation is as follows:
example 30A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-1 to prepare 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 100) is prepared into 0.05 mg/mL aqueous solution, then 40 μ L of hyaluronic acid is put into a 1.5 mL centrifuge tube, then a pipette is used for taking 16 μ L of SM4-1 solution to be evenly mixed with the hyaluronic acid, 100 μ L of 1 mol/L HEPES buffer solution with pH of 7.0 is added, 844 μ L of water is added for ultrasonic treatment for 1min, and the reaction is continued for 5h, so that the fluorescent nano-sensor can be obtained.
Example 31A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-2 to prepare into 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 300) is prepared into 0.05 mg/mL aqueous solution, then 40 μ L of hyaluronic acid is put into a 1.5 mL centrifuge tube, then a pipetting gun is used for taking 12 μ L of SM4-2 solution to be uniformly mixed with the hyaluronic acid, 100 μ L of 1 mol/L HEPES buffer solution with pH of 7.2 is added, 844 μ L of water is added for ultrasonic treatment for 1.2 min, and the reaction is continued for 6h, so that the fluorescent nano-sensor can be obtained.
Example 32A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-3 to prepare 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 500) preparing 0.05 mg/mL aqueous solution, then putting 40 μ L hyaluronic acid into a 1.5 mL centrifuge tube, then using a pipette to take 8 μ L SM4-3 solution to be uniformly mixed with the hyaluronic acid, adding 100 μ L1 mol/L HEPES buffer solution with pH of 7.4, then adding 844 μ L water, performing ultrasonic treatment for 1.5 min, and continuing to react for 7h to obtain the fluorescent nano-sensor.
Example 33A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-4 to prepare into 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 700) preparing 0.05 mg/mL aqueous solution, then putting 40 μ L hyaluronic acid into a 1.5 mL centrifuge tube, then using a pipette to take 20 μ L SM4-4 solution to be uniformly mixed with the hyaluronic acid, adding 100 μ L1 mol/L HEPES buffer solution with pH of 7.6, then adding 844 μ L water, performing ultrasonic treatment for 1.8 min, and continuing to react for 8h to obtain the fluorescent nano-sensor. />
Example 34A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-5 to prepare into 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 1000) preparationThe concentration of the hyaluronic acid is 0.05 mg/mL of aqueous solution, then 40 mu L of hyaluronic acid is put into a 1.5 mL centrifuge tube, then a pipette is used for taking 24 mu L of SM4-5 solution to be uniformly mixed with the hyaluronic acid, 100 mu L of 1 mol/L HEPES buffer solution with pH of 8.0 is added, 844 mu L of water is added for 2 min, and the reaction is continued for 5h, so that the fluorescent nano-sensor can be obtained.
Example 35 identification of fluorescent nanosensors
The fluorescence nanosensor obtained in example 30 was selected, the particle size distribution of the nanosensor was 148 nm, as shown in fig. 3, the fluorescence intensity after self-assembly rapidly decreased with the increase of response time, and when the response time reached 6h, the fluorescence intensity was almost only 1/6 of that before hyaluronic acid was added, and the fluorescence intensity at this time tended to a stable value, as shown in fig. 4, the fluorescence nanosensor obtained based on the above procedure, i.e., a fluorescence nanosensor capable of detecting hyaluronidase.
Example 36 fluorescence titration experiment of fluorescence nanosensor for detection of Hyaluronidase
10 1.5 mL sample bottles were taken, the fluorescence sensor solutions obtained in example 6 were added, and the fluorescence emission spectra of 12 samples were obtained by incubating 12 sample bottles in the order of [ HAase ] =0 (a), 10U/mL (b), 20U/mL (c), 30U/mL (d), 50U/mL (e), 60U/mL (f), 70U/mL (g), 80U/mL (h), 90U/mL (i), 100U/mL (j), 150U/mL (k), 200U/mL (l) at 37 ℃ for 1 h, and measuring the fluorescence emission spectra of each sample at 425 nm as the excitation wavelength, as shown in FIG. 5. The measurement result shows that: the fluorescence intensity of the polymeric fluorescence sensor at 656 nm gradually increased with increasing hyaluronidase concentration. The corresponding fitted relatively ideal functional graph and the corresponding functional graph (y = a + b x, a =0.9427, b =0.01701, R2= 0.9967) can be made from the 656 nm fluorescence intensity variation versus HA ase concentration variation in fig. 4, see fig. 6.
EXAMPLE 37 comparative assays for the detection of the Effect of other molecules, ions, on hyaluronidase
15 centrifuge tubes of 1.5 mL are taken and respectively filled with the fluorescent nano-sensor solution obtained in the example 30,then HAase with the concentration of 100U/mL, naCl with the concentration of 0.1 mol/L, KCl with the concentration of 0.1 mol/L and CaCl with the concentration of 0.1 mol/L are respectively added 2 、0.1 mol/L MgCl 2 10 mmol/L alanine, 10 mmol/L threonine, 10 mmol/L glycine, 10 mmol/L histidine, 10 mmol/L phenylalanine, 10 mmol/L glucose, 5 mmol/L GSH (glutathione), 1 mmol/L Cys (cysteine) and 5 mmol/L Hcy (homocysteine) solutions are respectively added into sample bottles No. 2 to No. 15, and the sample No. 1 is a blank sample. The fluorescence spectrum data of 15 samples under excitation at a wavelength of 425 nm were then measured, respectively, to obtain the change in fluorescence at emission at a wavelength of 656 nm, and the results are shown in FIG. 7. The measurement result shows that: the above-mentioned various substances, except hyaluronidase, had no significant effect on the fluorescence intensity of the prepared fluorescent nanosensor.
Example 38 comparative assay for Effect of other Small molecules, ions and Hyaluronidase Co-existence
15 1.5 mL centrifuge tubes are respectively filled with the fluorescent nano-sensor solution obtained in example 30, the No. 1 sample is a blank sample, and then HAase with the concentration of 100U/mL, naCl with the concentration of 0.1 mol/L, KCl with the concentration of 0.1 mol/L and CaCl with the concentration of 0.1 mol/L are respectively added 2 、0.1 mol/L MgCl 2 10 mmol/L alanine, 10 mmol/L threonine, 10 mmol/L glycine, 10 mmol/L histidine, 10 mmol/L phenylalanine, 10 mmol/L glucose, 5 mmol/L GSH (glutathione), 1 mmol/L Cys (cysteine) and 5 mmol/L Hcy (homocysteine) solutions were added to 2-14 sample vials. The fluorescence emission spectrum data of 15 samples under 425 nm wavelength excitation were then measured to obtain the fluorescence change at 656 nm wavelength emission, and the results are shown in FIG. 8. The measurement result shows that: in addition to hyaluronidase, the various coexisting ions and molecules described above do not interfere with the response of the fluorescent nanosensor to hyaluronidase.
Example 39 Hela cell viability assay with fluorescent nanoprobes at different concentrations
First, heLa cells were placed in DMEM medium containing 10% fetal bovine serum, then cultured in a constant temperature (37 ℃) and humidity incubator containing 5% CO2 for 24 hours, removed of the medium, washed 3 times with PBS buffer solution, cultured by adding DMEM medium containing fluorescent nanoprobes at different concentrations (0. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 15. Mu.g/mL, 20. Mu.g/mL) for 24 hours, and tested for cytotoxicity by MTT assay according to ISO10993-5 standard, the test results are shown in FIG. 9.
Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The application of the porphyrin derivative capable of detecting hyaluronidase based on self-assembly is characterized in that the porphyrin derivative and the hyaluronidase chain are mixed to prepare the fluorescent nano-device capable of detecting hyaluronidase, wherein the structural formula of the porphyrin derivative is as follows:
2. the application of the porphyrin derivative based on self-assembly detectable hyaluronidase as described in claim 1, wherein the tetrahydrofuran solution of the porphyrin derivative and the aqueous solution of the hyaluronan chain are placed in a centrifuge tube, and added with HEPES buffer solution with pH =7 for dilution, and after 1min of ultrasound, the mixed solution is reacted for 5h to obtain the fluorescent nanosensor capable of detecting hyaluronidase.
3. The use of a self-assembly based porphyrin derivative detectable hyaluronidase according to claim 1, characterized in that the hyaluronan chain has the formula:
wherein m =100.
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