A fluorescent probe and detection method for detecting pyrophosphate

Abstract

The invention relates to the field of detection technology and discloses a method for detecting a polycyclotriphosphazene-curcumin (PC) fluorescent microsphere and a Cu 2+ The strong coordination of PC‑Cu 2+ The probe can detect pyrophosphate PPi. The fluorescent probe and detection method show high sensitivity to pyrophosphate, with a minimum detection limit as low as 18nM. The detection process is simple, has strong anti-interference ability, is fast and sensitive, and the detection results are accurate, which has development and application prospects.

Description

Technical Field

[0001] The invention relates to the field of detection and materials, and specifically discloses a fluorescent probe and a method for detecting pyrophosphate. Background Art

[0002] Pyrophosphate (PPi), as an important biological functional anion, plays a very important role in life sciences, environmental sciences, pharmaceutical fields, and chemical processes. PPi is a product of the hydrolysis of adenosine triphosphate (ATP) under cellular conditions, and its stable metabolic process ensures the normal functioning of the organism. In addition, the detection and quantification of PPi is of great significance in the fields of cancer and various disease research. Excessive or low levels of PPi in biological systems may lead to a variety of diseases, including calcium pyrophosphate deposition disease (CPPD), cartilage fractures, and painful arthritis. Therefore, the development of a rapid, sensitive, and specific method for the detection of PPi is of great research significance.

[0003] So far, several techniques have been developed to detect PPi, such as electro-optical, colorimetric, and fluorescent methods, and fluorescent probes stand out among many methods due to their advantages such as simple operation and high sensitivity. Therefore, the method of designing fluorescent probes for rapid detection of PPi can meet the requirements of simple, rapid, and sensitive detection. Summary of the invention

[0004] The present invention provides a method and application for fluorescent detection of PPi, which solves the problems of difficult detection of PPi and low detection sensitivity.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A fluorescent probe for detecting pyrophosphate is a mixture of a copolymer formed by curcumin and hexachlorocyclotriphosphazene and copper ions. Preferably, the copolymer formed by curcumin and hexachlorocyclotriphosphazene is a fluorescent microsphere with a particle size of 0.5-2 microns.

[0007] The copolymer formed by curcumin and hexachlorocyclotriphosphazene is prepared by the following method:

[0008] (1) cyclotriphosphazene and curcumin are dissolved in an organic solvent;

[0009] (2) Add triethylamine and react for 4-12 hours.

[0010] The hydroxyl groups of curcumin condense with the chlorine of hexachlorocyclotriphosphazene to form a copolymer.

[0011] The organic solvent in step (1) is acetonitrile, toluene, tetrahydrofuran or DMF, preferably acetonitrile.

[0012] In step (1), the molar ratio of hexachlorocyclotriphosphazene to curcumin is 1:1.5-5, preferably 1:2-4. In a preferred embodiment of the present invention, the molar ratio of hexachlorocyclotriphosphazene to curcumin is 1:3.

[0013] In step (2), the molar ratio of hexachlorocyclotriphosphazene is 1:50-150, preferably 1:80-100. In a preferred embodiment of the present invention, the molar ratio of hexachlorocyclotriphosphazene to triethylamine is 1:90.

[0014] After the reaction in step (2) is completed, the precipitate is collected (by centrifugation or filtration, suction filtration), washed with water and ethanol, and dried.

[0015] Preferably, the copolymer formed by curcumin and hexachlorocyclotriphosphazene and copper ions are used in a ratio of 1g:0.3-1mol.

[0016] The fluorescent microspheres PC were dissolved in DMSO and then mixed with copper salt solution to form PC-Cu 2+ Fluorescent probe: The copper salt is copper chloride, copper nitrate or copper sulfate, preferably copper chloride.

[0017] The above fluorescent probes are excited at 430nm due to Cu 2+ As a quencher, the probe PC-Cu 2+ It has no obvious fluorescence brightness by itself; after adding pyrophosphate or its derivatives, it emits strong yellow-green fluorescence at 555nm. Therefore, the above fluorescent probe material can be used to detect pyrophosphate compounds.

[0018] The pyrophosphate compounds include compounds containing pyrophosphate or pyrophosphate esters and derivatives.

[0019] Based on the above fluorescent probe materials, pyrophosphate compounds can be detected both qualitatively and quantitatively.

[0020] A method for detecting pyrophosphate compounds comprises the following steps: mixing a fluorescent probe material with a sample to be detected, and detecting the fluorescence emission intensity at 450nm-750nm.

[0021] If the fluorescence emission intensity at 555nm is very obvious under the condition of 430nm excitation, that is, bright yellow-green fluorescence is produced, it means that pyrophosphate compounds are contained. 2+ The probe has no obvious fluorescence absorption due to the quenching effect. However, when pyrophosphate compounds are present, as their concentration increases, at 430nm, the probe will undergo fluorescence onset, that is, from no fluorescence to bright yellow-green.

[0022] When using the fluorescence method for detection, quantitative detection can be performed based on the fluorescence intensity at 555nm. With 430nm as the excitation wavelength, the fluorescence emission intensity at 450nm-750nm is measured on a fluorescence spectrometer, with the concentration of pyrophosphate as the horizontal axis and the fluorescence intensity ratio at 555nm as the vertical axis, a working curve of pyrophosphate concentration can be obtained for qualitative and quantitative detection.

[0023] Preferably, the fluorescent probe material is mixed with the sample to be tested, ethanol is added, and then the fluorescence emission intensity at 450nm-750nm is detected.

[0024] PC-Cu 2+ The working principle of the probe is that the curcumin part is used as the fluorophore, Cu 2+ The photoinduced electron transfer (PET) effect is used as a quencher to detect pyrophosphate compounds through fluorescence enhancement (on-type) probes.

[0025] PC emits yellow-green fluorescence, PC-Cu 2+ Cur and Cu in the probe 2+ The strong coordination effect of PC-Cu leads to photoinduced electron transfer, which quenches the fluorescence of PC. 2+ A certain amount of PPi is added to the system, and the O atoms on the PO bonds in the PPi structure interact with the Cu 2 + Strong complexation occurs, and the PPi in the solution will react with the Cur on the PC. 2+ A competitive reaction occurs in the coordination, thereby inhibiting the photoinduced electron transfer effect, causing partial recovery of fluorescence, and the system shows obvious fluorescence enhancement, thereby achieving effective detection of PPi. As the concentration of PPi increases, under ultraviolet light (365nm), the probe changes from very weak fluorescence to bright yellow-green, thereby achieving a rapid response to PPi and realizing rapid visual detection.

[0026] The above-mentioned probe and detection method can achieve a rapid response to pyrophosphate. The minimum detection limit of pyrophosphate detected by fluorescence method is 18nM. It has high sensitivity, strong anti-interference ability, rapid recognition and accurate detection results.

[0027] The beneficial effects of the present invention are:

[0028] (1) The fluorescent probe obtained by the present invention can be used for PPi detection. It is a fluorescence-enhanced probe with good specificity, anti-interference and high sensitivity. The present invention utilizes the good coordination ability of PPi with metal ions. After the probe combines with PPi, it releases strong fluorescence, and the fluorescence of the solution system is significantly enhanced, thereby realizing the detection of PPi.

[0029] (2) The present invention can quantitatively detect PPi by fluorescence method and can achieve rapid response to PPi in a relatively short time; at the same time, the PPi concentration of the probe at 0 to 10 μM is significantly linearly correlated with the fluorescence intensity at 555 nm, and the minimum detection limit for PPi is as low as 18 nM.

[0030] (3) The detection process of the present invention is simple, has strong anti-interference ability, is rapid, sensitive, and has accurate detection results, and can be used for the detection of PPi. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Example 2 is based on the fluorescent probe PC-Cu of the present invention 2+ Detection of fluorescence intensity effect of PPi.

[0032] Figure 2 Example 2 is based on the fluorescent probe PC-Cu of the present invention 2+ UV-visible absorbance effect diagram for detecting PPi.

[0033] Figure 3 The fluorescent probe PC-Cu of Example 3 of the present invention 2+ Minimum detection limit diagram for identifying PPi, excitation wavelength 430nm, emission wavelength 555nm;

[0034] Figure 4 The fluorescent probe PC-Cu of Example 4 of the present invention 2+ Fluorescence titration diagram for identification of PPi (PPi concentration 0-10 μmol / L), excitation wavelength 430 nm;

[0035] Figure 5 The fluorescent probe PC-Cu of Example 5 of the present invention 2+ Fluorescence selectivity map, excitation wavelength 430nm;

[0036] Figure 6 The fluorescent probe PC-Cu of Example 6 of the present invention 2+ Anti-interference diagram for identifying PPi, excitation wavelength 430nm, emission wavelength 555nm;

[0037] Figure 7 The fluorescent probe PC-Cu of Example 7 of the present invention 2+ Stability studies of identified PPi. DETAILED DESCRIPTION

[0038] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The fluorescent probe PC-Cu is prepared in the present invention. 2+ The chemical reagents and solvents used in the process were purchased from Exploration Company, and the metal ions were purchased from Aladdin Reagent Company. The fluorescence spectrum was recorded using the Edinburgh FS-5 fluorescence analyzer from the United Kingdom.

[0040] Example 1 Fluorescent probe PC-Cu 2+ Preparation

[0041] (1) Preparation of fluorescent microspheres (PC)

[0042] In a 250mL round-bottom flask, 0.10g (0.30mmol) of hexachlorocyclotriphosphazene (HCCP) and 0.32g (0.90mmol) of curcumin were dissolved in 50mL of acetonitrile. After the hexachlorocyclotriphosphazene and curcumin were completely dissolved in acetonitrile, 2mL of triethylamine (about 27mmol) was added and stirred at room temperature for 8h. After the reaction was complete, the lower precipitate was obtained by centrifugation and washed with ethanol and purified water until the supernatant was clear. The obtained precipitate was vacuum dried at 50℃ for 12h to obtain a light yellow powder, which was PC fluorescent microspheres. The particle size was 0.5-2 microns.

[0043] PC fluorescent microspheres have strong fluorescence at 450-750nm under 430nm excitation wavelength, and the fluorescence intensity is highest around 555nm. Figure 1 .

[0044] The technical route is:

[0045]

[0046] (2) Fluorescent probe (PC-Cu 2+ )

[0047] Use DMSO to prepare a PC solution with a concentration of 2 mg / mL; use purified water to prepare a CuCl2 aqueous solution with a concentration of 2 mM.

[0048] Add 20 μL of PC solution and 10 μL of CuCl2 solution to 2 mL of ethanol and mix well to prepare the probe. This fluorescent probe was used to detect pyrophosphate, and its selectivity, anti-interference, stability and sensitivity were studied.

[0049] Example 2 Response of fluorescent probe to PPi

[0050] Prepare 2 mg / mL PC DMSO solution, 2 mM CuCl2 aqueous solution and 2 mM PPi aqueous solution. Add 2 mL of ethanol solution, 20 μL PC and 10 μL CuCl2 aqueous solution to a clean cuvette and test on UV-visible spectrophotometer and fluorescence spectrum. The absorption peak of the single probe is observed at 430 nm. 2+ The solution exhibited very weak fluorescence emission.

[0051] However, adding PPi to the probe solution resulted in a significant increase in the fluorescence intensity at 555 nm, e.g. Figure 1 The probe alone has an absorption peak at 430 nm. 2+ or Cu 2+ and PPi, the absorbance decreases, e.g. Figure 2 shown.

[0052] Example 3 Minimum detection limit experiment

[0053] A good detection limit is one of the criteria for testing whether a probe molecule has application value. Prepare 2mg / mL PC DMSO solution, 2mM CuCl2 aqueous solution and 2mM PPi aqueous solution. Add 2mL of ethanol solution, 20μL PC and 10μL CuCl2 aqueous solution to a clean fluorescence cuvette, and measure its response intensity to different concentrations of PPi. As the PPi concentration increases, the system fluorescence emission intensity at 555nm continues to increase, and the working curve of PPi concentration is obtained. The linear regression equation is: Y = 79892.98595x-64147.234 (R 2 =0.98), and the detection limit of the probe molecule for PPi is 18 nM after calculation (3σ / k). Figure 3 As shown, there is good linearity.

[0054] Example 4 Probe Recognition PPi Concentration Titration

[0055] A 2 mg / mL DMSO solution of PC, a 2 mM CuCl2 aqueous solution, and a 2 mM PPi aqueous solution were prepared. 2 mL of ethanol, 20 μL of PC, and 10 μL of CuCl2 aqueous solution were added to a clean cuvette, and the probe PC-Cu was observed using a fluorescence spectrometer. 2+ For the change of fluorescence intensity of different concentrations of PPi, the concentrations of PPi were gradually added to 0, 2μM, 4μM, 6μM, 8μM, and 10μM, respectively. As the concentration of PPi increased, the fluorescence intensity at 555nm gradually increased, such as Figure 4 shown.

[0056] The results showed that the fluorescence intensity varied with the PPi concentration and could be quantitatively detected.

[0057] Example 5 Fluorescence Specific Selectivity

[0058] Prepare a 2 mg / mL DMSO solution of PC, a 2 mM aqueous solution of CuCl2, and a 2 mM aqueous solution of PPi.

[0059] 2 mL of ethanol solution, 20 μL of PC solution and 10 μL of CuCl2 solution were added to a clean cuvette. The probe PC-Cu 2+ The selectivity of pyrophosphate (PPi) in ethanol solution. Figure 5 As shown, under the condition of excitation at 430 nm, the probe PC-Cu 2+ In ethanol solution, it has a weak fluorescence emission intensity at 555nm. When PPi is added, the fluorescence emission intensity at 555nm is significantly enhanced. However, when other substances are added, the fluorescence emission intensity of the solution system does not change significantly compared with the fluorescence emission intensity of the single probe system. - The fluorescence intensity of β-actin is slightly higher than that of other substances, but still significantly different from that of PPi.

[0060] The above experimental results show that the probe PC-Cu 2+ It has a better response to PPi in ethanol solution.

[0061] Example 6 Anti-interference of fluorescent probe

[0062] Prepare 2 mg / mL PC in DMSO, 2 mM CuCl2 in water, and 2 mM PPi in water. Add 2 mL of ethanol solution, 20 μL of PC solution, and 10 μL of CuCl2 in 10 clean fluorescent cuvettes. 2+ solution, and then 10 μM of other analytes (Cl-, CO3 2- ,SO4 2- ,NO3 - ,F - ,Br - ,I - ,AcO - ,H2PO4 - ) and 10 μM PPi, detected on a fluorescence spectrometer, and a histogram of the fluorescence intensity at 555 nm corresponding to different analytes was drawn to obtain a fluorescence emission histogram, such as Figure 6 shown.

[0063] Experiments have shown that the probe PC-Cu 2+ The recognition of PPi in ethanol solution is not interfered by the other analytes mentioned above and has good anti-interference ability.

[0064] Example 7 Stability of fluorescent probe

[0065] Prepare 2mg / mL PC in DMSO, 20mM CuCl2 in water and 10mM PPi in water. Add 2mL ethanol, 20μL PC and 2μL CuCl2 in a cuvette and keep it for 0-20min to measure the detection effect on PPi. Figure 7 As shown, the probe PC-Cu 2+ Fluorescence and probe PC-Cu 2+ The fluorescence intensity of PPi detection hardly changed within 20 min.

[0066] The above experimental results show that the probe PC-Cu 2+ It exhibits good stability in ethanol solution system.

Claims

1. A fluorescent probe for detecting pyrophosphate compounds, characterized in that: It is a mixture of a copolymer of curcumin and hexachlorocyclotriphosphazene and copper ions.

2. The fluorescent probe for detecting pyrophosphate compounds according to claim 1, characterized in that: The copolymer formed by curcumin and hexachlorocyclotriphosphazene is prepared by the following method: (1) Cyclotriphosphazene and curcumin are dissolved in an organic solvent; (2) Add triethylamine and react for 4-12 hours.

3. The fluorescent probe for detecting pyrophosphate compounds according to claim 1, characterized in that: The usage ratio of the copolymer formed by curcumin and hexachlorocyclotriphosphazene to copper ions is 1g:0.3-1mol.

4. Use of the fluorescent probe according to any one of claims 1 to 3 in the preparation of reagents and kits for detecting pyrophosphate compounds.

5. A method for detecting pyrophosphate compounds, characterized in that: The fluorescent probe according to any one of claims 1 to 3 is mixed with a sample to be tested, and the change in fluorescence emission intensity is detected.

6. The method for detecting pyrophosphate compounds according to claim 5, characterized in that: The fluorescence intensity was measured at 450-750 nm.

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