CN112666143A

CN112666143A - Fluorescence detection method for alkaline phosphatase activity of environment

Info

Publication number: CN112666143A
Application number: CN202011501476.5A
Authority: CN
Inventors: 陈庆国; 刘梅; 杨金生; 竺柏康
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-04-16

Abstract

The invention discloses a fluorescence detection method for alkaline phosphatase activity of environment, and relates to the technical field of chemical analysis and detection. The detection method specifically comprises the following steps: mixing and incubating the fluorescent probe and alkaline phosphatase standard solutions with different known activities in a buffer solution, and performing a fluorescence spectrum test; drawing a standard curve by utilizing the linear relation between the activity of the alkaline phosphatase standard solution and the fluorescence spectrum intensity; mixing and incubating the fluorescent probe and the alkaline phosphatase solution to be detected in a buffer solution, and performing fluorescence spectrum test to obtain the fluorescence spectrum intensity of the alkaline phosphatase solution to be detected; and calculating to obtain the activity of the alkaline phosphatase to be detected by using the standard curve and the measured fluorescence spectrum intensity. The fluorescence detection method can be used for quantitatively detecting the activity of the alkaline phosphatase, has the detection limit of 0.093U/L, and has the advantages of high sensitivity, strong anti-interference capability, excellent linear relation and the like. And can be used for detecting the activity of alkaline phosphatase in marine environment.

Description

Fluorescence detection method for alkaline phosphatase activity of environment

Technical Field

The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a fluorescence detection method for alkaline phosphatase activity of environment.

Background

Phosphate is considered to be the most important source of phosphorus for algae and bacteria, and the lack of phosphorus in seawater can limit the growth of algae, limit the primary productivity of the sea, and further affect the overall ecological cycle system. Early studies have shown that Dissolved Organophosphorus (DOP) compounds in water can be utilized as a nutrient source for phosphorus by algae and bacteria, which contributes to ALP in seawater, when seawater is deficient in inorganic phosphorus. Alkaline phosphatase (ALP) is an extracellular enzyme that can convert monophosphorous lipids into a desired inorganic phosphorus source (Pi), and plays an important role in maintaining marine ecology. During the past decades, the artificial input of pollutants such as heavy metals into marine environments has greatly increased the concentration of heavy metals. These pollutants are persistent, not subject to bacterial decomposition, and have deleterious effects on various animals, plants, and microorganisms in the ocean. In the case of organisms, when the accumulation of heavy metals exceeds the tolerance of the organism, the contaminated organisms exhibit metabolic changes, slow growth, reduced biomass, etc. In the case of seawater, when the heavy metal ion exceeds a certain range, it will cause damage to the primary productivity of the whole sea area. Therefore, the development and design of the high-selectivity alkaline phosphatase fluorescent probe and the application of the high-selectivity alkaline phosphatase fluorescent probe in researching the ALP activity of seawater under the stress of metal ions can provide a new way for researching the influence of heavy metal pollution on the marine ecological environment.

Recently, various detection means for detecting ALP have been developed, including electrochemical, chromatographic, colorimetric techniques and peptide microarray assays, among others. Although these provide effective methods for ALP detection, they do not have good specificity for the substrate, and among them, there is a non-negligible threat from the acid phosphatase in phosphomonoesterase, because the alkaline phosphatase and the acid phosphatase are enzymes without specific hydrolysis function for the substrate, and they have different optimum pH values, about 5 and 8, respectively, in the conventional method for detecting ALP by fluorescence and ultraviolet absorption, including disodium p-nitrophenylphosphate and commercialized probes 4-MUP, ELF-97, etc., the sample is dissolved in Tris-HCl buffer solution (pH 8.0), and the activity of ACP is reduced while the maximum activity of ALP is ensured, so that the interference of ACP is avoided. However, this type of method is disadvantageous for analyzing ALP activity in real environment, since the alkaline phosphatase in the environment is not present in a stable environment at pH 8.0 to a large extent. For this reason, it is very necessary to design a probe capable of singly detecting ALP in various environments for accurate quantification.

Disclosure of Invention

The present invention aims to provide a fluorescent detection method for environmental alkaline phosphatase activity, which can quantitatively detect ALP activity, has a wide linear detection range and a low detection limit, and can specifically respond to ALP through pH without being interfered by ACP.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a preparation method of the fluorescent probe comprises the following steps:

s1: adding 4-hydroxy-benzo [ b ] thiophene-7-carboxaldehyde and ethyl benzindole into a mixed solution of n-butanol/toluene, stirring for dissolving, and heating for reflux reaction;

s2: removing the solvent after the reaction is finished, standing and layering the mixture in a mixed system of water and dichloromethane, and further purifying an organic phase to obtain a fluorescent compound Cy 5-M;

s3: dissolving the obtained Cy5-M in pyridine, then dropwise adding phosphorus oxychloride into the mixed solution, stirring and reacting for 0.5-1 h at room temperature, adding water, and continuously stirring and reacting for 0.5-1 h;

s4: after the reaction was completed, the solvent was removed, and the obtained crude product was extracted in a mixture of dichloromethane and water, and the aqueous phase was retained and collected under reduced pressure. And purifying the product by a reverse phase silica gel C18 chromatographic column (75-80% methanol/water) to obtain the probe CyP. The probe CyP prepared by the invention has high selectivity, can specifically respond to ALP through pH value and is not interfered by ACP. In the study of the photophysical properties of the probe CyP, the probe possessed a strong UV absorption peak at 550nm and a strong fluorescence intensity at 570nm after responding to ALP. The ALP activity can be quantitatively detected, and the fluorescence intensity after probe response and the ALP activity have good linear relation in the ALP activity range of 0-150U/L. The probes still show good selectivity after reaction with other enzymes and proteins. The fluorescent probe has good light stability, can be suitable for single ALP detection in different environments, and has high specificity, sensitivity and selectivity.

Preferably, the structural formula of the prepared fluorescent probe is as follows:

preferably, the mass ratio of 4-hydroxy-benzo [ b ] thiophene-7-carboxaldehyde to ethylbenzindole in step S1 is 1: 1.8 to 2.2.

Preferably, in the step S1, a mixed solution of n-butanol and toluene is used in the reaction process, and the volume ratio of the two is 7: 2-4; the liquid-solid ratio of the mixed solution to 4-hydroxy-benzo [ b ] thiophene-7-carboxaldehyde is 3-4 mL: 1 mg; the heating reflux reaction time is 3-4 h.

Preferably, in the step S3, the solid-to-liquid ratio of the fluorescent compound Cy5-M to the phosphorus oxychloride is 1.6-1.8 mg: 1 μ L.

Preferably, the synthetic route of the fluorescent probe is as follows:

the invention also discloses application of the fluorescent probe in detecting the activity of alkaline phosphatase in the environment.

A method for fluorescence detection of alkaline phosphatase activity in an environment comprising:

s1: mixing the fluorescent probe with alkaline phosphatase standard solutions with different known activities in MgCl₂Mixing and incubating in Tris-HCl buffer solution, and performing fluorescence spectrum test;

s2: drawing a standard curve by utilizing the linear relation between the activity of the alkaline phosphatase standard solution and the fluorescence spectrum intensity;

s3: placing the fluorescent probe and the alkaline phosphatase solution to be detected in MgCl₂Mixing and incubating the Tris-HCl buffer solution, and performing fluorescence spectrum test to obtain the to-be-tested sampleThe intensity of the fluorescence spectrum of the alkaline phosphatase solution;

s4: and calculating to obtain the activity of the alkaline phosphatase to be detected by using the standard curve of the step S2 and the fluorescence spectrum intensity measured in the step S3. The fluorescence detection method based on the prepared fluorescent probe can quantitatively detect the ALP activity, and the fluorescent intensity after the probe responds has a good linear relation with the ALP activity within the ALP activity range of 0-150U/L; compared with the prior art, the method has lower detection limit which can reach 0.093U/L. The fluorescence detection method provided by the invention has high sensitivity and selectivity for detecting ALP activity in the environment, and can be used for analyzing the influence of various metal ions on the ALP of chlorella and escherichia coli. Wherein, the transition metal ion Zn²⁺、Cu²⁺、Hg²⁺、Pb²⁺And Cd²⁺Has effect in inhibiting ALP of Chlorella and Escherichia coli, and has Mn inhibiting effect² ⁺、Co²⁺And alkaline earth metal ions have an effect of promoting ALP activity.

Preferably, the activity of the alkaline phosphatase standard solution in step S1 is 0-150U/L.

Preferably, the concentration of the fluorescent probe in the step S1 is 18-23 μ M.

Preferably, the concentration of the Tris-HCl buffer solution in the step S1 is 45-55 mM, and the pH value is 7.5-8.5; MgCl₂The concentration of (B) is 0.5 to 1.5 mM.

Preferably, the incubation temperature in steps S1 and S3 is 35-40 ℃ for 10-30 min.

Preferably, the above fluorescence detection method is used to study the effect of metal ions in seawater under heavy metal contamination on ALP activity.

Compared with the prior art, the invention has the following beneficial effects:

the probe CyP prepared by the invention has high selectivity, can specifically respond to ALP through pH value and is not interfered by ACP. And the probe still shows good selectivity after reacting with other enzymes and proteins. The fluorescence detection method based on the prepared fluorescent probe can quantitatively detect ALP activity, and the fluorescent intensity and the ALP activity after the probe responds have good linearity in the ALP activity range of 0-150U/LA relationship; compared with the prior art, the detection limit is lower and can reach 0.093U/L. The fluorescence detection method provided by the invention has high sensitivity and selectivity for detecting ALP activity in the environment, and can be used for analyzing the influence of various metal ions on the ALP of chlorella and escherichia coli. Wherein, the transition metal ion Zn²⁺、Cu² ⁺、Hg²⁺、Pb²⁺And Cd²⁺Has effect in inhibiting ALP of Chlorella and Escherichia coli, and has Mn inhibiting effect²⁺、Co²⁺And alkaline earth metal ions have an effect of promoting ALP activity.

Therefore, the invention provides a fluorescent compound, a preparation method and an application thereof, the fluorescent compound has sharp signal response in a wider pH range, and the prepared fluorescent probe has higher selectivity, specificity and sensitivity for ALP detection.

Drawings

FIG. 1 is a schematic diagram showing the detection mechanism of the probe in example 1 of the present invention;

FIG. 2 is a graph showing the results of the UV absorption test in example 1 of the present invention;

FIG. 3 is a graph showing a fluorescence spectrum at 550nm in example 1 of the present invention;

FIG. 4 is a graph showing the results of the UV absorption test in example 1 of the present invention;

FIG. 5 is a graph showing a fluorescence spectrum at 550nm in example 1 of the present invention;

FIG. 6 is a graph showing the change of fluorescence intensity with time of different concentrations of CyP on ALP in example 1 of the present invention;

FIG. 7 is a graph of a linear fit in example 1 of the present invention;

FIG. 8 shows the result of the selectivity test of CyP probe in example 2 of the present invention;

FIG. 9 shows the results of the response of the probe CyP to the ACP activity in example 2 of the present invention;

FIG. 10 is a linear fit curve for detection of ALP activity in example 3 of the present invention;

FIG. 11 is a result of examining the influence of alkaline earth metal ions on the ALP activity of chlorella in example 4 of the present invention;

FIG. 12 is a result of examining the influence of alkaline earth metal ions on the ALP activity of Escherichia coli in example 4 of the present invention;

FIG. 13 is a result of examining the influence of transition metal ions on the ALP activity of chlorella in example 4 of the present invention;

FIG. 14 shows the results of examining the influence of alkali transition metal ions on the ALP activity of Escherichia coli in example 4 of the present invention;

FIG. 15 shows the measurement results of the influence of other metal ions on the ALP activity of chlorella in example 4 of the present invention;

FIG. 16 is a graph showing the results of examining the influence of other metal ions on the ALP activity of Escherichia coli in example 4 of the present invention.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:

n-butyl alcohol (n-butyl alcohol), toluene (toluene) and dichloromethane (CH) used in the examples of the present invention₂Cl₂) Ethyl acetate (EtOAc), methanol (CH)₃OH), pyridine (pyridine), phosphorus oxychloride (phosphorus oxychloride) are all available from national institute of chemical agents, Inc.

Chlorella vulgaris (Chlorella vulgaris Beij.) is provided by the Marine biologies institute of tobacco terrace university. The microalgae are cultured by using an F/2 marine algae nutrient medium. The seawater is collected from the surface of the first sea water bath of the smoke bench at high tide, boiled, filtered and disinfected for use. The microalgae are cultured in an illumination incubator with the temperature of 25 ℃, the illumination intensity of 6000LX and the light and shade of 12h respectively.

Extraction of microalgal proteins protein extraction kit (Minute) for thick-walled microorganisms was chosen^TMTotal Protein Extraction Kit for Microbes with Thick Cell Walls). For the quantification of microalgal proteins, protein quantification kit (BCA kit), siemer feishel technologies, inc.

Ultrapure water (18.2M Ω cm) was further filtered from the Onhaha water. During the chromatographic column separation, the silica gel P60(200-300 mesh) used is produced by Qingdao sea wave silica gel dryer, Inc., and the analytical pure quartz sand (silica, 10-20 mesh) is produced by Tianjin Kemiou chemical reagent, Inc. The reaction progress is monitored, and a thin-layer chromatography silica gel plate is produced by the research institute of chemical industry in cigarette end. Commonly used glassware was purchased from synthware instruments ltd. The heating instrument is an air electric heating sleeve, and the solvent removing instrument is a rotary evaporator. Because the synthesis and phosphorylation processes of the cyanine need to avoid the interference of water, the cyanine is synthesized by using a new opened Chinese medicine reagent, and further water removal operation is avoided.

Establishing a chlorella ALP model:

extraction and quantification of microalgal proteins

Selecting Chlorella vulgaris (Chlorella vulgaris Beij.) with good growth situation, placing into 50mL centrifugal tube, centrifuging at 1500r/min for 3 times, each time centrifuging for 5min, adding deionized water and loading on the machine after each centrifugation to remove culture medium. Breaking the wall of the obtained centrifugal chlorella by using a protein extraction kit, and cracking cells. And measuring the content of the extracted protein by using a BCA kit. The obtained protein extract is stored in a refrigerator at-80 ℃ in a dark place.

Quantification of microalgae ALP

10 μ L of the protein extract was removed and diluted 100-fold with 50mm tris-HCl buffer (pH 8.0) to a final volume of 1 mL. The mixed solution was shaken on a vortex shaker for 5s to ensure thorough mixing. After standing for 5 minutes, 20. mu.L of LCyP was added to the mixed solution, and the fluorescence intensity was measured after 5 minutes of reaction. Lambda [ alpha ]_ex/λ_em＝550/570nm。

In the test of the effect of different metal ions on the ALP activity in the present example, the probe and the protein extract were both maintained at the above concentrations, and the metal ions at different concentrations were previously mixed with 50mm tris-HCl buffer (pH 8.0).

Example 1:

preparation of a fluorescent probe:

s1: 4-hydroxy-benzo [ b ] thiophene-7-carboxaldehyde (40mg, 0.33mmol) and ethylbenzindole (80.5mg, 0.33mmol) were added to a 250mL three-necked flask, and 150mL of a mixed solution of n-butanol/toluene (7: 3, V/V) was added and dissolved with stirring. The reaction system is heated and refluxed for 3.5h at the temperature of 255 ℃, and the progress of the reaction is monitored by thin-layer chromatography: (ii) a

S2: after the reaction is finished, the solvent is removed in vacuum, the crude product is kept still in a mixed system of water and dichloromethane for layering, an organic phase is reserved, and the operation is repeated for three times. The crude product was further purified by column chromatography on silica gel eluting with ethyl acetate/methanol (3: 1, V/V). The final product, Cy5-M, was an orange solid in 87.1% yield;

s3: the resulting Cy5-M (17.2mg, 5mmol) was dissolved in 15mL of pyridine. Phosphorus oxychloride (10uL, 5mmol) was then added dropwise to the mixed solution. Stirring for 0.5h at room temperature, adding 5mL of water into the reaction system, and continuously stirring for 0.5 h;

s4: after the reaction was completed, the solvent was removed by a rotary evaporator, and the obtained crude product was extracted in a mixed solution of dichloromethane and water, and the aqueous phase was retained and collected under reduced pressure and concentrated. The product was purified by reverse phase silica gel C18 chromatography (75% methanol/water). The resulting probe CyP was a yellow oil in 71.8% yield.

Research on detection mechanism and dynamics of probe CyP

1. Mechanism of detection

As shown in FIG. 1, CyP is non-fluorescent, and since the hydroxyl group of hemicyanine Cy5-M is blocked by a phosphate group, the substituted phosphate group has strong electron-withdrawing ability, which hinders the Intramolecular Charge Transfer (ICT) process in hemicyanine, resulting in quenching of fluorescence. After ALP addition, the phosphoester bond is broken and the blocked hydroxyl group is reformed through H₂And (4) participation of O. Thus, recovery of ICT within hemicyanine results in the turn-on of fluorescence. The unique pH response of hemicyanine Cy5-M, in turn, gave good selectivity for ALP but no response to ACP.

2. Effect of different pH values on Probe CyP

To investigate the effect of different pH values on the CyP probe, the UV absorption and fluorescence spectra (. lamda.M) of CyP (10. mu.M) were measured after 1h in buffer solutions of different pH values_ex＝550nm,λ_em560-750 nm). As shown in fig. 2 and 3, it can be seen that the uv absorption and fluorescence spectra do not respond under any pH conditions.

3. Probe CyP Property test

The spectral properties of probe CyP were studied at a temperature of 37 ℃ under simulated optimized physiological conditions. Escherichia Coli Alkaline Phosphatase (ECAP) was selected as the ALP model. The working solution was 50mM Tris-HCl buffer (pH 8.0) and 1mM MgCl was added₂To ensure that the enzyme is at maximum activity. The results are shown in fig. 4, probe CyP exhibited almost no significant absorption of light; whereas, when ALP was added (100U/L) for 10min, there was a visually observed change in yellow fading, at which time a strong absorption peak appeared at 550nm, which is an absorption spectrum of the typical fluorescent compound Cy 5-M. Its fluorescence spectrum (. lamda.)_ex＝550nm、λ_em560 to 750nm) also gave the same results, as shown in fig. 5. Thus, it was confirmed that CyP was a probe having an OFF-ON property.

4. Study of kinetics

Fluorescence spectroscopy (lambda) was performed by exposing 100U/L ALP to CyP prepared at a concentration of 0.5, 1,2, 5, 10. mu.M_ex/λ_em550/570nm), the fluorescence intensity was recorded as a function of time, as shown in fig. 6. The change in fluorescence intensity within 5min was chosen as the initial rate of reaction, the fitted Lineweaver Burke equation 1/V₀＝K_M/Vmax[S]+1/V_maxShown in FIG. 7, the linear fit equation is 1/V₀(μΜ^-1min)＝150.56/[QcyP]+9.43, r-0.9977. The Michaelis constant (KM) was calculated to be 15.97 μm.

Example 2:

selectivity of the Probe CyP

Adding potential interfering enzyme or protein including lysozyme, esterase, bovine serum albumin, avidin, trypsin, phosphodiesterase and acidic phosphoric acid into fluorescent probe solution with concentration of 10 μ M, incubating at 37 deg.C for 30min, and performing fluorescence spectrum test (λ:)_ex/λ_em550/570 nm). The concentration of ALP and potential interfering enzyme to be detected was 0.17. mu.g/mL, and the final concentration of potential interfering protein was 100. mu.g/mL, as shown in FIG. 8. The experimental result proves that the probe CyP can be used for lysozyme, esterase and bovine serumThe albumin, the avidin, the trypsin, the phosphodiesterase and the acidic phosphate have no obvious response, and the probe prepared by the invention has good detection specificity on the activity of the alkaline phosphatase.

To further verify the response of the probe CyP to the ACP activity, 10mM citric acid-hydrochloric acid-sodium hydroxide buffer solution (pH 4.5) was used as a diluted solution of ACP; 50mM Tris-HCl buffer (pH 8.0) and 1mM MgCl₂As a dilute solution for ALP; wherein the concentration of ALP and ACP is 100U/L, reacting at 37 deg.C for 10min, and performing fluorescence spectrum test (lambda)_ex/λ_em550/570 nm). As a result, as shown in fig. 9, ACP did not respond to the probe in the environment of pH 4.5, because ACP responded to the probe CyP under acidic conditions, but the fluorescence intensity of Cy5-M obtained after the reaction was low. It can be seen that the probe CyP designed by the present invention is a probe with high selectivity for ALP.

Example 3:

s1: mu.L of CyP prepared in example 1 was mixed with 0, 1, 10, 15, 25, 50, 75, 100, 125, 150U/L of active alkaline phosphatase standard solution in the presence of 1mM MgCl₂Mixed and incubated at 37 ℃ for 30min in 50mM Tris-HCl buffer (pH 8.0) solution, and subjected to fluorescence spectroscopy (lambda)_ex/λ_em＝550/570nm)；

S2: drawing a standard curve by utilizing the linear relation between the activity of the alkaline phosphatase standard solution and the fluorescence spectrum intensity; in the measurement of ALP activity, 20. mu.M CyP was used since it was ensured that the substrate concentration was higher than K_MThe value is obtained. As shown in FIG. 10, the fluorescence intensity of CyP is linearly related to ALP activity in the range of 0-150U/L. The linear fit equation is F₅₇₀ _nm＝59847.4[ALP]–74396.3(U/L)，r²0.9953; the detection limit of the detection method is calculated to be 0.093U/L according to 3 sigma/K.

S3: mu.L of CyP and alkaline phosphatase to be tested in a solution containing 1mM MgCl₂In 50mM Tris-HCl buffer (pH 8.0) at 37 deg.CIncubation for 30min, fluorescence spectroscopy (lambda)_ex/λ_em＝550/570nm)；

S4: and calculating to obtain the activity of the alkaline phosphatase to be detected by using the standard curve of the step S2 and the fluorescence spectrum intensity measured in the step S3.

Example 4:

detection of the Effect of different Metal ions on ALP Activity

The main ALP in seawater comes from algae and bacteria, for which, we chose Escherichia Coli Alkaline Phosphatase (ECAP) as representative of bacteria ALP, and chlorella vulgaris (Chlorella vulgaris Beij.) ALP as representative of algae ALP to measure the influence of different metal ions on the activity of seawater ALP. It has been shown that metal ions have various degrees of promotion and inhibition effects on ALP activity (Journal of Environmental Sciences,2007,19 (7): 834-428; Clinical Chemistry,1980,26 (3): 423-428).

Influence of monovalent 4.1 Metal ions

To 50mM Tris-HCl buffer solution (pH 8.0), inorganic salts LiCl and Na were added at different concentrations₂SO₄、KNO₃To examine the influence of the metal ions on the activity of ALP, the method was the same as in example 3. The results showed that the activity of ALP was not changed even at a concentration of 10 mM. Experiments prove that univalent metal ions Li⁺、Na⁺、K⁺There was neither promotion nor inhibition of ALP activity. Anion Cl^-、SO₄ ^2-And NO₃ ^-There was also no effect on ALP activity.

4.2 Effect of alkaline earth metals on ALP Activity

To 50mM Tris-HCl buffer solution (pH 8.0) was added Mg-containing solutions of various concentrations²⁺、Ca²⁺And Ba²⁺The effect of the metal ion on the activity of ALP was examined in the same manner as in example 3.

Effect of alkaline earth metals on ALP Activity of Chlorella and Escherichia coli, the results are shown in FIG. 11 and FIG. 12, and the alkaline earth metals have an accelerating effect on the enhancement of ALP activity, and the accelerating effect is Mg²⁺>Ca²⁺>Ba²⁺. Wherein, Mg²⁺The effect of improving ALP activity is most obvious. The same experimental phenomenon occurs in other kinds of alkaline phosphatase, including chlorella and E.coli (proceedings of Xiamen university: Nature science edition, 1986 (6): 87-93; Proc. Aquaculture, 2001,25 (4); Sichuan animal, 2009,28 (6): 835-838).

4.3 Effect of transition Metal ions on ALP Activity

Selecting alkaline earth metal ions MnCl with different concentrations₂、ZnSO₄、CoCl₂、CuSO₄The procedure was as in 4.2 for the study. As a result, as shown in FIGS. 13 and 14, Mn contained in chlorella and Escherichia coli²⁺、Co²⁺Has different degrees of promotion effects on the enzyme activity improvement, and Zn²⁺、Cu²⁺Inhibition was shown and a similar experimental phenomenon was found to be on the effect of metal ions on the alkaline phosphatase of Wenchang fish (proceedings of Xiamen university: Nature science edition, 1986 (6): 87-93).

4.4 Effect of other Metal ions on ALP viability

Selecting HgCl₂、CdCl₂And Pb (NO)₃)₂As a donor of the ions, the test procedure is as in 4.2, where PbCl is responsible₂Precipitation was carried out at room temperature, using Tricine-NaOH buffer (pH 8.0) instead of Tris-HCl buffer. As a result, Hg was observed in FIGS. 15 and 16²⁺、Pb²⁺And Cd²⁺Has different degrees of inhibition effects on ALP of chlorella and escherichia coli, and the inhibition effects are reduced in sequence. Similarly, Hg²⁺、Pb²⁺And Cd²⁺Similar inhibitory effects on other alkaline phosphatases were also observed (proceedings of Xiamen university: Nature science edition, 1986 (6): 87-93; Sichuan animal, 2007,26 (3): 641-643).

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A method for preparing a fluorescent probe, comprising:

s1: according to the mass ratio of 1: 1.8-2.2, adding 4-hydroxy-benzo [ b ] thiophene-7-carboxaldehyde and ethyl benzindole into a mixture with a volume ratio of 7: stirring and dissolving 2-4 of a mixed solution of n-butyl alcohol and toluene, and heating and refluxing for reaction for 3-4 hours;

s4: and after the reaction is finished, removing the solvent, extracting in a mixed solution of dichloromethane and water, reserving a water phase, and carrying out reduced pressure concentration and purification to obtain the fluorescent probe CyP.

2. The method for preparing a fluorescent probe according to claim 1, wherein: the solid-to-liquid ratio of Cy5-M to phosphorus oxychloride in the step S3 is 1.6-1.8 mg: 1 μ L.

3. The method for preparing a fluorescent probe according to claim 1, wherein: the synthetic route of the fluorescent probe is as follows:

4. use of the fluorescent probe of claim 1 to detect alkaline phosphatase activity in an environment.

5. A method for fluorescence detection of alkaline phosphatase activity in an environment comprising:

s1: combining the fluorescent probe of claim 1 with standard solutions of alkaline phosphatase of different known activities in a solution containing MgCl₂Mixing and incubating in Tris-HCl buffer solution, and performing fluorescence spectrum test;

s3: placing the fluorescent probe and the alkaline phosphatase solution to be detected in MgCl₂Mixing and incubating the Tris-HCl buffer solution, and performing fluorescence spectrum test to obtain the fluorescence spectrum intensity of the alkaline phosphatase solution to be tested;

6. The method of claim 5 for fluorescence detection of alkaline phosphatase activity in an environment, wherein: the activity of the alkaline phosphatase standard solution in the step S1 is 0-300U/L.

7. The method of claim 5 for fluorescence detection of alkaline phosphatase activity in an environment, wherein: the concentration of the fluorescent probe in the step S1 is 18-23 mu M.

8. The method of claim 5 for fluorescence detection of alkaline phosphatase activity in an environment, wherein: the concentration of the Tris-HCl buffer solution in the step S1 is 45-55 mM, and the pH value is 7.5-8.5; said MgCl₂The concentration of (B) is 0.5 to 1.5 mM.

9. The method of claim 5 for fluorescence detection of alkaline phosphatase activity in an environment, wherein: and the incubation temperature in the steps S1 and S3 is 35-40 ℃ for 10-30 min.

10. The method of claim 5 for fluorescence detection of alkaline phosphatase activity in an environment, wherein: the fluorescence detection method is used for researching the influence of metal ions in the seawater polluted by the heavy metals on ALP activity.