CN109134371B

CN109134371B - Fluorescence ratio probe, synthetic method thereof and application of fluorescence ratio probe in detecting intracellular lysosome formaldehyde

Info

Publication number: CN109134371B
Application number: CN201811176194.5A
Authority: CN
Inventors: 陈帅; 贾叶; 于永亮
Original assignee: Northeastern University China
Current assignee: Northeastern University China
Priority date: 2018-10-10
Filing date: 2018-10-10
Publication date: 2020-07-10
Anticipated expiration: 2038-10-10
Also published as: CN109134371A

Abstract

Fluorescent ratiometric probe and synthesis method and detection thereofAn application for detecting lysosome formaldehyde in cells belongs to the field of analytical chemistry. The structural formula of the fluorescence ratio probe is as follows:

Description

Fluorescence ratio probe, synthetic method thereof and application of fluorescence ratio probe in detecting intracellular lysosome formaldehyde

Technical Field

The invention belongs to the technical field of analytical chemistry, and relates to a fluorescence ratio probe for detecting formaldehyde in cells; the invention also relates to a synthetic method of the fluorescence ratio probe; in addition, the invention also relates to the use of the fluorescence ratiometric probes. In particular to a fluorescence ratio probe, a synthetic method thereof and application in detecting lysosome formaldehyde in cells.

Background

Formaldehyde (FA) is a common carcinogenic and teratogenic substance that has been identified by the world health organization as a potential source of allergic reactions as a chemical contaminant, formaldehyde poisoning caused by formaldehyde in indoor air, atmospheric environment and food contamination, causing dizziness, headache or nausea, vomiting, severely causing memory loss and even death, but FA has been reported to be present in cells at higher concentrations under normal physiological conditions, up to 500 μm in certain organelles in most biological organisms, some amino acids and exogenous substances are metabolized under the catalytic action of demethylases or oxidases, and endogenous formaldehyde is also produced.

Due to the development of spectroscopic techniques and the development of fluorescent molecular probes, fluorescence imaging technology has become a powerful means for tracking biomolecules in biological systems. The existing fluorescent probe for detecting formaldehyde in organisms is basically an organic molecule. However, the water solubility of the compound is poor, so that the application of the compound is limited, and some organic molecules have the problems of complex synthesis process, fast photobleaching, short fluorescence life, long reaction time, low detection sensitivity, high construction ratio difficulty caused by solvent and the like. In order to solve these problems, Carbon Dots (CDs) are used as an effective fluorescent probe, and are used as a more effective fluorescent probe due to their simple synthesis, low cytotoxicity, good chemical inertness, high light stability, good water solubility, easy surface functionalization, etc., as compared to other imaging techniques. Organic molecules are combined with carbon dots, on one hand, the water solubility of the organic molecules is enhanced, so that the organic molecules can be operated and reacted in aqueous solution, on the other hand, the ratio detection can be formed, the detection sensitivity is improved, the ratio fluorescent probe can provide built-in environmental interference correction, and the fluctuation of the light excitation intensity and factors such as instrument efficiency and the interference of environmental conditions are eliminated.

Disclosure of Invention

Aiming at the current situations that the synthesis process is complex, the photobleaching is fast, the fluorescence service life is short, the water solubility is poor, the detection accuracy is easily interfered by the probe environment, the detection sensitivity is low and the like when the existing fluorescent probe detects formaldehyde, the invention synthesizes a ratio type formaldehyde fluorescent probe with double emission bands by combining nano carbon dots with good water solubility with organic micromolecules, which is abbreviated as CDs-NA; further provides a synthetic method and application of the CDs-NA. A carbon dot is combined with a molecule with a naphthalimide structure, the carbon dot is used as a background fluorescence signal, the naphthalimide is used as a reaction group, and a high-efficiency fluorescence ratio probe (CDs-Na) for detecting formaldehyde is constructed. The fluorescent ratiometric probe successfully realizes the detection of formaldehyde inside and outside cells, has good selectivity and detection sensitivity, and provides important theoretical significance for further applying the dual-emission fluorescent ratiometric probe to the fields of biological labeling and the like in the future.

The invention relates to a fluorescence ratio probe, which has a structural formula as follows:

the fluorescence ratiometric probe is a ratiometric fluorescent probe, and the excitation wavelength of the ratiometric fluorescent probe is 365 nm; the fluorescence peak value of the fluorescence ratio probe at 414nm is kept unchanged, and the fluorescence peak value at 535nm is gradually increased along with the increase of the concentration of formaldehyde; the ratio of the fluorescence intensity of the ratiometric probe at 535nm to the fluorescence intensity of the ratiometric probe at 414nm (F)₅₃₅/F₄₁₄) And increases with increasing formaldehyde concentration.

The fluorescence ratio probe, as time increased, showed the ratio (F) of the fluorescence intensity at 535nm to the fluorescence intensity at 414nm₅₃₅/F₄₁₄) Gradually become larger and reach a maximum at 30 minutes.

The fluorescent ratiometric probe can resist PBS, glyoxal, acetaldehyde, acetone, 4-nitrobenzaldehyde, chloral, NaClO and H₂O₂、CaCl₂、MgCl₂、NaNO₂、Na₂SO₃、NaHS、NaHSO₃Glutathione reduction, acetyl L cysteine, D L-cysteine, arginine, L-cysteine, phenylalanine arginine, L-cysteine and phenylalanine or the mixture of a plurality of compounds.

The invention discloses a method for synthesizing a fluorescence ratio probe, which comprises the following steps:

step 1: preparation of methionine carbon point solid powder

Adding methionine and citric acid into water, and dissolving uniformly to obtain a reaction solution; wherein, according to the mass ratio, the methionine: citric acid ═ (0.8-1.6): (1-2), adding water in an amount sufficient to dissolve methionine and citric acid;

reacting the reaction solution at 180-200 ℃ for 2-4 h, and cooling to room temperature after the reaction is finished to obtain a mixed solution;

centrifuging the mixed solution to obtain a supernatant for later use;

dialyzing the supernatant with dialysis bag to remove residual raw material molecules, freezing, and vacuum drying to obtain methionine carbon point solid powder;

step 2: preparation of fluorescent ratiometric Probe crude product

(1) Mixing 4-bromo-1, 8-naphthoic acid, 6-aminocaproic acid and absolute ethyl alcohol, stirring and refluxing for 1-3 h, carrying out rotary evaporation, and drying the obtained solid material in vacuum to obtain S1, wherein the solid-liquid ratio of (1.0-1.5) g of (0.5-0.8) g of (20-40) m L is (1.0-1.5) g of 4-bromo-1, 8-naphthoic acid, 6-aminocaproic acid and absolute ethyl alcohol;

dissolving S1 in dimethyl sulfoxide (DMSO) to obtain a DMSO solution of S1;

(2) dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 4-Dimethylaminopyridine (DMAP) in water, and uniformly stirring to obtain a catalyst solution; wherein, according to the mass ratio: edc.hcl: DMAP ═ 0.01 to 0.02: (0.001 to 0.003);

(3) mixing the DMSO solution of S1 and the catalyst solution, finally adding methionine carbon point solid powder, uniformly stirring, and stirring at room temperature in a dark place for 35-40 h to obtain a reaction solution of a fluorescent ratio probe crude product; wherein, according to the mass ratio: s1: catalyst: methionine carbon point solid powder (0.01-0.02): (0.01-0.02): (0.015 to 0.03);

and step 3: substitution reaction

Adding absolute ethyl alcohol and hydrazine hydrate with the mass concentration of 60-80% into a reaction liquid of a crude product of the fluorescence ratio probe, stirring and refluxing for 3-5 h at 70-80 ℃, carrying out rotary evaporation, dialyzing the residual solid material for 46-50 h through a dialysis bag, removing unreacted raw materials, carrying out rotary evaporation, and freeze-drying to obtain the fluorescence ratio probe, wherein the volume ratio of the reaction liquid of the crude product of the fluorescence ratio probe, the absolute ethyl alcohol, the hydrazine hydrate with the mass concentration of 60-80% (3-5) m L, (3-5) m L, (300-500) mu L is adopted.

In the step 1, the dialysis bag is a dialysis bag with the molecular weight cutoff of 500D, raw material molecules smaller than 500D are removed, the dialysis time is 10-15 hours, and water is changed every 1-3 hours.

In the step 1, the centrifugation is carried out at a centrifugation rotating speed of 10000-13000 r/min for 10-20 min.

In the step 2(1), the reflux temperature is 70-85 ℃.

In the step 3, the dialysis bag is a dialysis bag with the molecular weight cutoff of 500-1000D.

In the step 3, the reflux temperature is 70-90 ℃, and the reflux time is 1-3 h.

The application of the fluorescence rate probe is used for detecting formaldehyde, particularly formaldehyde in water environment and formaldehyde in biological samples.

The fluorescence ratiometric probe may be used to detect a biological sample, such as a living cell.

The application of the fluorescence ratio probe comprises the following specific application methods:

when detecting the formaldehyde in the water environment, detecting the formaldehyde in the water environment by adopting a fluorescence spectrometer through the fluorescence spectrum change of a fluorescence ratio probe;

when detecting formaldehyde in a biological sample, formaldehyde in the biological sample is detected by observing changes in a fluorescence image of the ratiometric probe using a confocal microscope.

The application of the fluorescence ratio probe adopts a fluorescence spectrometer, and the excitation wavelength is 365 nm; as the concentration of formaldehyde increases, the fluorescence peak of the fluorescence spectrum at 414nm gradually decreases, and a new emission band at 535nm is generated and the fluorescence peak gradually increases.

The fluorescence ratio probe is applied by adopting a confocal microscope, wherein an excitation light source is a mercury lamp, and blue channel fluorescence and yellow channel fluorescence are respectively collected; as the concentration of formaldehyde increased, the blue channel fluorescence remained constant while the yellow channel fluorescence gradually increased.

The use of said ratiometric fluorescent probes when detecting formaldehyde in a biological sample, the ratiometric fluorescent probes are predominantly distributed in lysosomes.

In the synthesis method of the fluorescence ratio probe, the process route is as follows:

(1) synthesis of CDs

(2) Synthesis of S1

(3) Synthesis of CDs-NA

The application of the fluorescence ratio probe of the invention comprises the following reaction process with formaldehyde:

the fluorescence ratio probe, the synthesis method and the application in detecting intracellular lysosome formaldehyde have the advantages that:

the fluorescence ratio probe has good water solubility, high accuracy, high sensitivity and high anti-interference capability, is not easily influenced by the concentration of the probe, the environment where the probe is positioned and a detection instrument, can detect the formaldehyde with the minimum concentration of 338 nmol/L, realizes high-selectivity detection of the formaldehyde in a water solution, can detect the formaldehyde in a water environment, can detect the formaldehyde in a cell environment, has good targeting property on lysosomes, realizes fluorescence imaging of the formaldehyde in a living cell level, and has potential practical application value.

Drawings

FIG. 1 is a three-dimensional fluorescence spectrum of the probe CDs-NA of example 1, in which FIG. 1(a) shows no formaldehyde addition and FIG. 1(b) shows an enhancement in fluorescence at emission at 535nm with 30. mu.M formaldehyde addition;

FIG. 2 shows the changes of fluorescence spectra of the probe CDs-NA in example 2 with the addition of different amounts of formaldehyde; from bottom to top, the fluorescence spectra of the probes in the order of 0,1,2,5,10,15,20,30,40,50,60,100,160 μ M formaldehyde concentration;

FIG. 3 is the ratio of fluorescence intensity of CDs-NA of the probe in example 3 (F)₅₃₅/F₄₁₄) Linear plots with different amounts of formaldehyde;

FIG. 4 is a graph of spot fluorescence data of the probe CDs-NA and formaldehyde as a function of time in example 4;

FIG. 5 is a bar graph of fluorescence data for the selectivity of the probe CDs-NA for different interfering analytes in example 5; in the figure, PBS, glyoxal, acetaldehyde, acetone, 4-nitrobenzaldehyde, chloral, formaldehyde, NaClO, H₂O₂、CaCl₂、MgCl₂、 NaNO₂、Na₂SO₃、NaHS、NaHSO₃Glutathione reduction, acetyl L cysteine, D L-cysteine, arginine, L-cysteine, phenylalanine;

FIG. 6 is a graph of fluorescence images of the probe CDs-NA and formaldehyde response in cells in example 6, in which fluorescence images of the probe CDs-NA added with 0.1mg/M L CDs-NA after 4h incubation, and different concentrations of formaldehyde (0, 200, 500, 1000. mu.M) were added to the probe CDs-NA to continue incubation with Hela cells for 1h fluorescence images, and the scale is 50 μ M.

FIG. 7 is a graph of the co-localization of CDs-NA and the lysosomal probe of example 7, wherein a is the light of the blue channel of the probe, b is the light of the green channel of the probe, c is the light of the red channel of the lysosomal probe, d is the overlay of a, b, and c, and e is the overlay coefficient.

FIG. 8 is a pathway for uptake of probes CDs-NA by cells in example 8 cells were pretreated with inhibitors and the pretreated cells were incubated with probes for fluorescence imaging (1) control (2) amiloride (3) methyl β -cyclodextrin (4) chlorpromazine (5)4 deg.C (6) genistein.

FIG. 9 is an IR spectrum of methionine carbon dot solid powder (CDs) and fluorescence ratio probe (CDs-NA) prepared in example 1 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1 Synthesis of Compound CDs-NA, comprising the following steps:

step 1: preparation of methionine carbon point solid powder

Weighing 0.8g of methionine and 1g of citric acid in a 20m L small beaker, slowly adding 5m L of secondary water, uniformly stirring by using a glass rod to dissolve the methionine and the citric acid, and obtaining a reaction solution after the secondary water is completely dissolved;

and (3) transferring all reaction liquid into a 25m L reaction kettle, packaging a steel sleeve, heating and reacting for 3 hours in a forced air drying oven at 200 ℃, and cooling to room temperature after the reaction is finished to obtain a mixed liquid.

Then, the mixture was aspirated and centrifuged at 12000r/min for 15 minutes to remove non-fluorescent large particles and obtain a supernatant of the carbon spots for use.

Then, the supernatant was dialyzed for 12 hours against a dialysis bag with a molecular weight cut-off of 500D, removing the residual molecules of the starting material, during which water was changed every 2 hours. The finally obtained carbon point aqueous solution is frozen and dried in vacuum to obtain methionine carbon point solid powder (CDs).

The prepared CDs were subjected to infrared analysis, and the obtained infrared spectrogram by KBr pellet method was shown in FIG. 9.

Step 2: preparation of fluorescent ratiometric Probe crude product

(1) 1.3824g of 4, bromo-1, 8-naphthalic anhydride, 0.6597g of 6-aminocaproic acid and 30m of L anhydrous ethanol are mixed, stirred and refluxed for 2 hours at 80 ℃, rotary steaming is carried out at 40 ℃, substances left after rotary steaming are transferred to a 50m L small beaker, and the substances are dried in a vacuum oven at 30 ℃ to obtain S1.

Weighing 0.0116g of S1, dissolving in 1m L DMSO, and performing ultrasonic dissolution to obtain a DMSO solution of S1;

(2) dissolving 0.0141g of EDC.HCl and 0.0023g of DMAP in 4m L water, and stirring for 1h to obtain a catalyst solution;

(3) and mixing the DMSO solution of S1 with the catalyst solution, adding 20mg of methionine carbon-point solid powder, wrapping tin paper in the dark, and stirring at room temperature for 36h to obtain a reaction solution of a crude fluorescence ratio probe product.

And step 3: substitution reaction

Adding 4m L absolute ethyl alcohol and 300 mu L80% hydrazine hydrate into the reaction solution of the crude product of the fluorescence ratio probe, heating and stirring at 80 ℃, refluxing for 4h, carrying out rotary evaporation to 4m L, dialyzing (500- & gt 1000D) the residual solid material for 48h, removing unreacted raw materials, carrying out rotary evaporation, and freeze-drying to obtain the final product, namely the fluorescence ratio probe (CDs-NA).

Performing infrared analysis on the prepared CDs-NA, and adopting KBr tabletting method to obtain infrared spectrogram shown in figure 9 of 3500-3200 cm^-1The wide vibration band of (A) is attributed to-OH and-NH₂Telescopic vibration at 1632cm^-1The strong vibration band (-C ═ O stretching vibration) at (b) confirmed the presence of-COOH groups on the surface of CDs. 2926,2856cm^-1Corresponding to CH₂and-CH₃Expansion and contraction of-CH₂1399cm of deformation^-1The bands of (A) demonstrate successful synthesis of a complex with-COOH and-NH₂CDs of groups. Compared with the infrared spectrum of CDs, the CDs-NA is 1463 and 1605cm^-1Where additional vibration bands are present, corresponding to the aromatic ring skeleton of naphthalene. At 2957cm^-1There is a reinforced vibration band and a small vibration band at 2852cm-1, representing-CH₂Is used to extend. 3431cm^-1，1627cm^-1And 1354cm^-1Indicates stretching vibration of-NH, C ═ O, C — N of amide bond, confirming coupling of CDs to naphthalimide units.

The structure is as follows

The three-dimensional fluorescence spectrum of the fluorescence ratio probe (CDs-NA) is shown in FIG. 1, in which FIG. 1(a) shows no formaldehyde addition and FIG. 1(b) shows 30. mu.M formaldehyde addition, with an increase in fluorescence at 535nm emission.

Example 2 fluorescence ratio Probe CDs-NA changes with the addition of different amounts of Formaldehyde

PBS (10mM, pH 7.4) was used as a solvent to prepare a concentration of 0.1mg m L^-1Adding 100 mu L of formaldehyde solutions with different concentrations (0,1,2,5,10,15,20,30,40,50,60,100 and 160 mu M) into a centrifuge tube filled with 400 mu L of PBS-Na solution in sequence, shaking for 30min, measuring the fluorescence emission spectrum of the sample by using a cuvette with an optical path of 0.5cm, a fixed excitation wavelength of 365nm and a photomultiplier voltage of 650V by using a Hitachi F-7000 type fluorescence spectrophotometer, and measuring different amounts of formazanThe fluorescence spectrum of the probe CDs-NA for aldehyde changes as shown in FIG. 2, and it can be seen from FIG. 2 that the fluorescence peak at 414nm remains unchanged and the fluorescence peak at 535nm gradually increases as the concentration of formaldehyde increases.

Example 3 calculation of the lower limit of Formaldehyde detection by Probe CDs-NA

According to the fluorescence spectrum of example 2, the ratio of the fluorescence intensity at 535nm to the fluorescence intensity at 414nm (F)₅₃₅/F₄₁₄) The concentration of formaldehyde is plotted against the corresponding concentration of formaldehyde, see FIG. 3. As can be seen from FIG. 3, the concentration of formaldehyde is 0-40. mu. mol/L, F₅₃₅/F₄₁₄Has good linearity with formaldehyde concentration. The lower limit of detection can be calculated to be 338nM according to the formula Dl-3 σ/k (Dl is the lower limit of detection, σ is the intercept standard deviation, and k is the slope).

Example 4 fluorescence Rate Probe CDs-NA plus variation in fluorescence intensity over time for different amounts of Formaldehyde

To a container with 400 μ L, 0.1mg m L^-1The PBS solution of CDs-NA was added to the centrifuge tube with 100 μ L0, 20 and 100 μ M concentration of formaldehyde standard solution, and the fluorescence spectrum of the probe in formaldehyde mother liquor as a function of time was measured by fluorescence spectrometer as shown in FIG. 4. the response equilibrium was reached within 30 minutes at 20 μ M concentration and the plateau was reached for 10 minutes when 100 μ M formaldehyde was used.

Example 5 Selective study of fluorescence ratiometric probes CDs-NA on different interfering analytes

21 numbered EP tubes of 1.5m L were taken, and 400. mu. L of CDs-Na (0.1mg m L) were added to each tube^-1) Then 100. mu. L of formaldehyde (150. mu.M), PBS, 250. mu.M of aldones (glyoxal, acetaldehyde, acetone, 4-nitrobenzaldehyde, chloral) and 500mM of active oxygen nitrogen (NaClO, H) were added in this order₂O₂、NaNO₂) 25mM zwitterion (CaCl)₂、MgCl₂、NaHS、Na₂SO₃、NaHS、NaHSO₃) 25mM amino acids (reduced glutathione, acetyl-L-cysteine, D L-cysteine arginine, L-cysteine, phenylalanine), shaking for 30min, using a cuvette with an optical path of 0.5cm, fixing the excitation wavelength at 365nm, setting the photomultiplier voltage at 650V, and using Hitachi F-7000 type fluorescenceThe fluorescence response spectrum of the dual emission fluorescence peak of the probe to each analyte measured by the spectrophotometer is shown in FIG. 5, and in FIG. 5, substances represented by each number on the abscissa are shown in the following table.

TABLE 1 substances indicated by the respective numbers on the abscissa in FIG. 5

Serial number	Representative substances
		1	PBS
2	Glyoxal
		3	Acetaldehyde
4	Acetone (II)
		5	4-nitrobenzaldehyde
6	Chloral
		7	Formaldehyde (I)
8	NaClO
		9	H₂O₂
10	CaCl₂
		11	MgCl ₂
12	NaNO₂
		13	Na₂SO₃
14	NaHS
		15	NaHSO ₃
16	Glutathione reduction
		17	Acetyl L cysteine
18	D L-cysteine
		19	Arginine
20	L-cysteine
		21	Phenylalanine

Example 6: fluorescence imaging of fluorescence ratiometric probes CDs-NA with formaldehyde in cells

Selecting cells in logarithmic growth phase, pouring out the culture solution in the cell bottle, adding 10mmol L^-1The PBS of (5) was washed 2 times, and each soaking was carried out for 1min, and a CDs-NA solution diluted with a DMEM high-sugar medium was added to the cell flask to a final concentration of 0.1mg/m L, and the flask was placed in CO₂Inoculating at 37 deg.C for 4h, pouring off the labeling solution, and inoculating with 10mmol L^-1PBS (pH 7.4) 3 washes. After further incubation for 1h with different concentrations of formaldehyde (0, 200, 500, 1000. mu.M) added to the HeLa cells, the cells were washed three times with PBS. The cells were observed under a confocal fluorescence microscope (Olympus, Japan) with the cell surface kept wet during the observation. In cell imaging experiments, 405nm was used as an excitation light source. (Blue in picture; Green Green; Bright brightfield; Overlay channel Overlay; Ratio scale) A40-fold objective lens was used in the cell imaging experiments. When observed, 2 channels were used, the excitation wavelength was 405nm, and the emission wavelengths were 420-460nm (channel 1) and 520-560nm (channel 2), respectively. A graph of the fluorescence image of the probe CDs-NA in response to formaldehyde in the cells is shown in FIG. 6.

Example 7: fluorescence ratio probe CDs-NA and lysosomal targeting in cells

The distribution of the CDs-NA in the cells is determined by adopting a fluorescent dye co-localization method, the cells and the CDs-NA (0.1mg/m L) are incubated for 4 hours, a culture solution is discarded, the cells are washed by PBS buffer solution, then the cells are stained by L ysoTracker-Red for 40 minutes, and residues are washed by the PBS buffer solution.

Example 8: cellular uptake of fluorescent ratiometric probes CDs-NA

HeLa cells were preincubated with various pathway inhibitors such as chlorpromazine (10 μm, clathrin-mediated endocytosis inhibitor), genistein (185 μm, caveolin-mediated endocytosis inhibitor), methyl β -cyclodextrin (5mm, lipid raft-mediated endocytosis inhibitor) and amiloride (100 μm, macrocytosis inhibitor) at 37 ℃ for 1h, and a group of cells were preincubated with 4 ℃ C for 1h, and the effect of energy on the process of cell internalization was observed, and a group of controls was set up, after which CDs-NA (0.1mg/m L) was co-incubated with cells for 4h, followed by 3 washes with PBS physiological buffer (10mm, pH7.40) and finally fluorescence imaging was performed (1) control group (control; (2) amiloride (MCI); (3) methyl β -cyclodextrin (4); (4) Chlorpromazine (CHP) (5) 4; (6) genistein pathway uptake by DNA-probe (GEN).

When the probe is incubated with chlorpromazine and genistein, the internalization rate of the probe is respectively reduced to 68% and 72%, which shows that the internalization is mainly carried out through clathrin-mediated endocytosis and caveolin-protein-mediated endocytosis.

Claims

1. A ratiometric fluorescent probe having the structural formula:

2. the ratiometric probe of claim 1, wherein the ratiometric probe is a ratiometric probe having an excitation wavelength of 365 nm; the fluorescence peak value of the fluorescence ratio probe at 414nm is kept unchanged, and the fluorescence peak value at 535nm is gradually increased along with the increase of the concentration of formaldehyde; the ratio of the fluorescence intensity of the ratiometric probe at 535nm to the fluorescence intensity at 414nm increases with increasing formaldehyde concentration.

3. The ratiometric probe of claim 1, wherein the ratio of the fluorescence intensity at 535nm to 414nm increases gradually with time and reaches a maximum at 30 minutes.

4. The ratiometric fluorescent probe of claim 1, wherein the ratiometric fluorescent probe is resistant to PBS, glyoxal, acetaldehyde, acetone, 4-nitrobenzaldehyde, chloral, NaClO, H₂O₂、CaCl₂、MgCl₂、NaNO₂、Na₂SO₃、NaHS、NaHSO₃Glutathione reduction, acetyl L cysteine, D L-cysteine, arginine, L-cysteine, phenylalanine arginine, L-cysteine and phenylalanine or the mixture of a plurality of compounds.

5. The method of synthesizing a fluorescence ratiometric probe of claim 1, comprising the steps of:

step 1: preparation of methionine carbon point solid powder

centrifuging the mixed solution to obtain a supernatant for later use;

step 2: preparation of fluorescent ratiometric Probe crude product

dissolving S1 in dimethyl sulfoxide (DMSO) to obtain a DMSO solution of S1;

and step 3: substitution reaction

6. The method for synthesizing a fluorescence rate probe according to claim 5, wherein in the step 1, the dialysis bag is a dialysis bag with a cut-off molecular weight of 500D, the molecules of the raw material less than 500D are removed, the dialysis time is 10-15 h, and the water is changed every 1-3 h.

7. The method for synthesizing a fluorescence ratiometric probe of claim 5, wherein in the step 1, the centrifugation is performed at a centrifugation speed of 10000 to 13000r/min for 10 to 20 min.

8. The method for synthesizing a fluorescence rate probe according to claim 5, wherein in the step 3, the dialysis bag is a dialysis bag with a molecular weight cut-off of 500-1000D;

9. Use of the fluorescence ratiometric probe of claim 1, for detecting formaldehyde in aqueous environments and for detecting formaldehyde in biological samples.

10. The use of a ratiometric fluorescent probe according to claim 9, wherein the ratiometric fluorescent probe is predominantly distributed in lysosomes when detecting formaldehyde in a biological sample.

11. The use of a ratiometric fluorescent probe according to claim 9, wherein the specific application method is:

12. Use of a ratiometric fluorescent probe according to claim 11, wherein the ratiometric fluorescent probe is used in a fluorescence spectrometer with an excitation wavelength of 365 nm; the fluorescence peak value of the fluorescence spectrum at 414nm is gradually reduced along with the increase of the concentration of formaldehyde, and a new emission band is generated at 535nm and the fluorescence peak value is gradually increased;