1. A polymer universal fluorescence platform for FRET fluorescence probe donor is characterized in that the name of the fluorescence platform is MS-1, and the specific structural formula is as follows:

2. a method for preparing a fluorescent platform of claim 1, wherein the method is prepared by the following steps:

the preparation steps of MS-1 are as follows:

s1. synthesis of MS-3: heating and refluxing p-hydroxybenzaldehyde, allyl chloride and potassium carbonate in acetonitrile solution for two hours to obtain a compound MS-3;

s2, synthesis of MS-2: heating methyl methacrylate and MS-3 in dimethylformamide under the initiation of azodiisobutyronitrile for 24 hours to polymerize to obtain a polymer MS-2;

s3, synthesis of MS-1: and the MS-2 is further reacted with coumarin hydrazine hydrate in a dimethylformamide solution for 24 hours under heating to obtain the polymer fluorescence platform MS-1.

3. The method of claim 2, wherein allyl chloride is added in the same molar amount as p-hydroxybenzaldehyde and potassium carbonate is added in an amount three times the molar amount of p-hydroxybenzaldehyde in step S1.

4. The method for preparing a universal fluorescence platform for a polymer used for a FRET fluorescent probe donor according to claim 2, wherein the acetonitrile is added in an amount of 1g p-hydroxybenzaldehyde to 15ml acetonitrile based on p-hydroxybenzaldehyde in step S1.

5. The method of claim 2, wherein the amount of MS-3 added in step S2 is 0.41 times the molar amount of methyl methacrylate, and the amount of azobisisobutyronitrile added is 0.4% by mass of methyl methacrylate.

6. The method of claim 2, wherein the amount of dimethylformamide added in step S2 is based on methylmethacrylate, and 4 ml of dimethylformamide is added to 1g of methylmethacrylate.

7. The method for preparing a universal fluorescence platform for a polymer used for a FRET fluorescent probe donor according to claim 2, wherein the amount of coumarin hydrazine hydrate added in step S3 is 9.4% by mass of MS-2.

8. The use of the universal fluorescent platform for polymers of claim 1 in fluorescence detection.

Background

Since the 21 st century, Optical fiber sensors have been of intense interest due to their great potential for use in industrial analysis, biochemistry, drug detection, and the like (Adv Optical mater.2021,9,2001913). Designing such a sensor requires a new functional material with high sensitivity and is not easy to implement. The polymer material of the fluorescent sensor has become a key direction for researchers to overcome due to excellent detection performance and easy preparation of the polymer fiber material. However, the fluorescence quenching easily occurs due to the excessive concentration of the fluorescence sensor, so that the detection sensitivity is greatly reduced, and how to maintain the sensing characteristic of the probe in the polymer matrix is a great challenge.

Compared with the method of simply relying on fluorescence intensity to identify target molecules, the proportional fluorescence probe detects the target molecules by using the double changes of the ratio of the fluorescence intensity of two wavelengths and the color, is suitable for visual observation and is more intuitive, and meanwhile, the detection errors caused by objective factors such as instrument loss, solvent, temperature change and the like are avoided, so that the detection sensitivity is greatly improved (Chem Soc Rev.2016,45,2976). However, it requires precise molecular design and complicated synthesis process to combine two fluorophores with different emission wavelengths into one molecule without affecting the ratio fluorescence recognition of target molecules. In contrast, single-wavelength fluorescent probes that specifically recognize targets are more synthetically accessible and have many mature molecular alternatives.

Therefore, the invention is expected to realize the development of a universal polymer blue light platform, and the blue light emitting fluorophore coumarin with excellent photophysical properties is combined into the polymer fiber material in a proper ratio, and the coumarin ratio is controlled to keep a proper distance in the polymer to avoid aggregation. In the coumarin polymer universal fluorescence platform, coumarin can be used as an energy donor for Fluorescence Resonance Energy Transfer (FRET) and can also be used as a fluorescence internal standard. When the target molecule is identified, the dual-wavelength fluorescence detection of the target molecule can be realized only by selecting a mature fluorescent probe which can be well energy-matched with coumarin and combining the mature fluorescent probe with a polymer general fluorescence platform in a proper proportion, so that the defect that the single-wavelength fluorescence detection is easy to interfere is avoided, and the difficulty of molecule design is reduced. The development of the polymer universal fluorescent platform also makes the later preparation of the fluorescent probe into the flexible nano fabric possible, which greatly expands the application field.

Disclosure of Invention

The invention aims to combine a simple and easily obtained blue light emitting group coumarin with a prepared polymer molecule with an aldehyde group active site through Schiff base reaction to prepare a polymer universal fluorescent platform capable of emitting blue light, and further combine the platform with a fluorescent probe for identifying mercury ions through pure fluorescence enhancement, so that high-sensitivity ratio fluorescence detection of the mercury ions can be realized, and external interference on the pure fluorescence enhancement is avoided. The polymer universal fluorescence platform is beneficial to the later-stage molding processing of the fluorescent probe (such as flexible film preparation through electrostatic spinning), and can control the concentration of aldehyde group active sites on polymer molecules to enable the concentration of coumarin in the polymer to be appropriate, so that fluorescence quenching caused by coumarin aggregation in solid materials can be avoided.

The technical scheme adopted by the invention for solving the technical problems is as follows: methyl methacrylate and 4-allyl oxy benzaldehyde are polymerized in a reasonable ratio, and a coumarin fluorophore is further introduced through a high-efficiency Schiff base reaction to prepare the universal polymer blue light platform MS-1, wherein the specific structural formula is as follows:

in order to further examine the application effect, the polymer universal fluorescence platform and the published rhodamine fluorescence probe MS-4 are further combined to cooperatively realize the dual-wavelength ratio fluorescence detection of target molecules.

The invention is carried out by the following specific steps: p-hydroxybenzaldehyde and equimolar allyl chloride and three times molar amount of potassium carbonate are heated and refluxed in acetonitrile (1g of p-hydroxybenzaldehyde/15 ml of acetonitrile) solution for two hours to obtain a compound MS-3, methyl methacrylate and 0.41 times molar amount of MS-3 are heated and polymerized in Dimethylformamide (DMF) (1g of methyl methacrylate/4 ml) under the initiation of azobisisobutyronitrile (0.4%) for 24 hours to obtain a polymer MS-2, and the MS-2 is further heated and reacted with coumarin hydrazine hydrate (9.4% of the mass of MS-2) in Dimethylformamide (DMF) solution for 24 hours to obtain a polymer fluorescence platform MS-1.

The preparation reaction route of the polymer fluorescent platform MS-1 with the structure is as follows:

the main absorption peak in the ultraviolet absorption spectrum of the polymer universal fluorescence platform MS-1 comes from the absorption peak of coumarin composing the polymer universal fluorescence platform MS-1 at 439 nanometers, and the emission wavelength is blue fluorescence of the coumarin at 478 nanometers. When combined with MS-4, a new absorption peak belonging to rhodamine appears in the absorption spectrum with the addition of mercury ions, and the two peaks appear simultaneously in the ground state. After the mercury ions are added, a new fluorescence peak which belongs to the ring opening of rhodamine and is formed at 568 nm appears in a fluorescence spectrum, and meanwhile, the fluorescence peak at 478 nm of coumarin has a descending trend. When the mercury ion reaches a certain concentration, the fluorescence does not change any further. The general fluorescence platform for the polymer provides blue light wavelength as an internal standard for the detection system, and can realize dual-wavelength fluorescence detection of mercury ions.

The invention has the advantages that the raw materials of the polymer universal fluorescent platform prepared by using the coumarin fluorophore are easy to obtain, the synthesis is simple and feasible, and the cost is low. When combined with mature long-wavelength fluorescent probes, the fluorescent probe can realize proportional fluorescent detection of target molecules, and even can realize visual detection by naked eyes through colors. The development of the polymer platform also makes possible its later application to flexible nanomaterials.

Drawings

FIG. 1 shows the CDCL of MS-3 in example 2 of the present invention₃A nuclear magnetic hydrocarbon spectrum;

FIG. 2 shows the CDCL of MS-5 in example 2 of the present invention₃Hydrogen spectrum of nuclear magnetism;

FIG. 3 shows the CDCL of MS-2 in example 3 of the present invention₃Hydrogen spectrum of nuclear magnetism;

FIG. 4 shows the CDCL of MS-1 in example 4 of the present invention₃Hydrogen spectrum of nuclear magnetism;

FIG. 5 is a graph of a UV-visible spectral titration of MS1-Hg in example 5 of the present invention;

FIG. 6 is a graph showing the trend of UV absorbance change at 537nm of MS1-Hg in example 5 of the present invention; .

FIG. 7 is a graph showing a fluorescence spectrum titration of MS1-Hg in example 5 of the present invention;

FIG. 8 is a graph of MS1-Hg vs. Hg (NO) for example 5 of the present invention₃)₂When the multiple increases F₅₆₄/F₄₇₈Change in the ratio of fluorescence intensities of;

FIG. 9 shows the addition of Hg to MS1-Hg at various pH values in example 5 of the present invention²⁺Measurement of fluorescence spectra before and after.

Detailed Description

In order to more clearly illustrate the present invention, specific examples are described below, which do not limit the scope of the present invention.

Example 1

Synthesis of MS-4: adding phenyl isothiocyanate (94 mu l and 0.69 mu mol) with the molar mass being 1.5 times that of rhodamine 6g hydrazine hydrate (200mg and 0.46mmol) in dimethylformamide (about 3mL), gradually heating to 60 ℃, heating and stirring for four hours, extracting a reaction liquid by using dichloromethane after TLC (thin layer chromatography) confirms that the reaction is finished, carrying out reduced pressure rotary evaporation on an organic phase, and carrying out solid column chromatography separation to obtain a pink target product with the yield of 50%.

Example 2

Synthesis of MS-3: 1g of p-hydroxybenzaldehyde, an equimolar amount of allyl chloride and a three-fold molar amount of potassium carbonate were placed in a 50 ml flask, and 15ml of acetonitrile was added. The reaction solution is heated and refluxed for two hours, cooled and filtered, and the filtrate is decompressed and dried by spinning to obtain white solid powder (MS-3 p-allyl-oxygen benzaldehyde) with the yield of 70 percent. The product was used in the next reaction without purification.

Synthesis of coumarin hydrazine hydrate: coumarin ester (1g, 3.46mmol) is dissolved in 15ml of methanol solution, 2ml of hydrazine hydrate is slowly dropped, the reaction solution is stirred for 30 minutes at normal temperature, TLC confirms that after the reaction is finished, precipitated solid is filtered to obtain yellow solid powder 0.54g, and the yield is 56%. The product was used in the next reaction without purification.

Synthesis of MS-5: 500 mg of coumarin hydrazine hydrate and 1.5 times of the molar mass of MS-3 are placed in a 50 ml flask, 25 ml of ethanol is added, heating reflux stirring is carried out, after four hours of reaction, cooling and filtering are carried out, and a solid product is obtained, wherein the yield is 30%.

Example 3

Synthesis of MS-2: 5g of methyl methacrylate (0.0499mol) and 3.3687g (0.0207mol) of MS-3 were mixed, 0.04g of azobisisobutyronitrile (0.0002mol) as an initiator and 20mL of dimethylformamide were added, and the oxygen in the solution was removed by bubbling nitrogen for 10 minutes. The reaction solution was placed in a constant temperature oil bath and heated at 70 ℃ for 24 hours to give a transparent polymer. The reaction mixture was then poured into methanol (200ml) and the polymer precipitated from methanol, sonicated, filtered, washed several times with methanol to remove a solid portion of unreacted monomer, and the polymer obtained by the filtration was dried in a vacuum oven at 30 ℃ for 24 hours to constant weight. Yield: 4.1557 g.

Example 4

Synthesis of MS-1: 536mg MS-2 and 50.32mg (0.0207mol) coumarin hydrazine are mixed in 5mL dimethylformamide, 20. mu.l glacial acetic acid is added, and the reaction is heated at 70 ℃ for 24 hours. The reaction mixture was poured into methanol (200ml), the polymer precipitated in methanol, sonicated, filtered, washed with methanol several times to remove the solid portion of unreacted monomer, and the polymer MS-1 obtained by the filtration was dried in a vacuum oven at 30 ℃ for 24 hours to constant weight. Yield: 481.6 mg.

Example 5 preparation of Mercury ion detection solution MS1-Hg

MS-1 and MS-4 are respectively prepared into 10^-3A solution of mol/L in methylene chloride. Respectively taking 1 microliter and 100 microliter, placing into a 10 milliliter volumetric flask, blowing the solvent dry, and adding ethanol-water (volume ratio is 8: 2) mixed solution to prepare a detection solution MS 1-Hg.

Example 6 ultraviolet absorption Spectroscopy

The ultraviolet absorption spectrum of the detection solution MS1-Hg is measured, and the test result is shown in figure 5. Without addition of Hg²⁺Then, a distinct absorption peak at 439 nm was observed, which is characteristic of the coumarin fluorophore, while there was almost no absorption at 538 nm, indicating that the rhodamine fluorophore is in a closed loop state. With Hg²⁺Gradually adding the mercury ions into the solution, and a new absorption peak appears at 538 nm, wherein the absorption peak gradually increases along with the addition of the mercury ions when Hg is added²⁺The absorbance peak at 538 nm reached equilibrium at 4 times the molar concentration of MS-4 in solution (as shown in FIG. 6). While the absorption peak at 439 nm does not follow Hg²⁺The addition was varied. This indicates that after the addition of mercury ions, the rhodamine fluorophore moiety gradually opens the ring, but coumarin fluorescesThe group and rhodamine fluorophore did not interact in the ground state.

Example 7 fluorescence Spectroscopy determination of detection solution MS1-Hg

The fluorescence emission spectrum of the detection solution MS1-Hg is measured by the invention, and the test result is shown in FIG. 7. Before adding mercury ions, only an emission peak of coumarin at 478 nm can be seen at an excitation wavelength of 440 nm. With the addition of mercury ions, the fluorescence peak at 478 nm gradually weakens, and the fluorescence emission peak at 564 nm gradually strengthens, which is the fluorescence emission peak after the ring opening of rhodamine 6G. When the mercury ions were added four times, the fluorescence intensity ratios at 564 nm and 478 nm tended to level off (as shown in FIG. 8). This shows that when the mercury ions are added, the emission spectrum of the coumarin fluorophore and the absorption spectrum of the open-ring rhodamine fluorophore are crossed due to the ring opening of the rhodamine derivative, and thus fluorescence resonance energy transfer between the coumarin donor and the rhodamine acceptor occurs. We can therefore observe a decrease in coumarin fluorophore at 478 nm and an increase in rhodamine fluorophore at 564 nm. When the mercury ions are added to a certain amount, the rhodamine is completely opened, and the fluorescence is not obviously changed after the mercury ions are added.

EXAMPLE 8 addition of Hg at various pH values²⁺Measurement of fluorescence spectra before and after

Fluorescence spectra of the detection solution MS1-Hg at different pH values were measured, and the results are shown in FIG. 9. It can be seen that the fluorescence of the detection solution before adding mercury ions is relatively stable between pH 4.5-10, and the fluorescence is obviously enhanced only when the pH is less than 4, which is mainly caused by the ring-opening reaction of rhodamine part under acidic condition. After mercury ions are added, the fluorescence of the detection solution is relatively stable between pH 4.5 and pH 10. The detection solution MS1-Hg in the patent can realize the detection of mercury ions under normal environmental and physiological conditions without being influenced by pH change.

The foregoing examples are provided for illustration and description of the invention only and are not intended to limit the invention to the scope of the described examples. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that many variations and modifications may be made in accordance with the teachings of the present invention, which variations and modifications are within the scope of the present invention as claimed.